NUEVASMETODOLOGASEN

ANLISISDEALIMENTOSCONEL
USODENANOPARTCULAS

NEWMETHODOLOGIESINFOOD
ANALYSISUSINGNANOPARTICLES

TesisDoctoral
JuanGodoyNavajas
Marzo2014

TITULO: Nuevas metodologas en anlisis de alimentos con el uso de

nanopartculas. New metodologies in food analysis using nanoparticles

AUTOR: Juan Godoy Navajas

Edita: Servicio de Publicaciones de la Universidad de Crdoba. 2014
Campus de Rabanales
Ctra. Nacional IV, Km. 396 A
14071 Crdoba
www.uco.es/publicaciones
publicaciones@uco.es

Mediante la defensa de esta Memoria se pretende optar a

la obtencin de la Mencin Doctorado Internacional habida
cuentadequeeldoctorandorenelosrequisitosexigidosparatal
mencin:

1. Cuenta con los informes favorables de dos doctores

pertenecientes a Instituciones de Enseanza Superior de
paseseuropeosdistintosaEspaa.

2. Uno de los miembros del tribunal que ha de evaluar la

Tesisperteneceauncentrodeenseanzasuperiordeotro
paseuropeo.

3. PartedeladefensadelaMemoriaserealizarenlalengua
oficialdeotropaseuropeo.

4. El doctorando ha realizado una estancia en el

Departamento of Biotecnologa de la Universidad de
Turku, Finlandia, de tres meses de duracin, que ha
contribuido a su formacin y permitido desarrollar parte
deltrabajoexperimentaldeestaMemoria.

Quisiera mostrar mi ms sincero agradecimiento a mis directoras

deTesisDoctoral,TinayMariPaz.

GraciasTinapordarmelaoportunidaddetrabajarcontigodurante

estosaosyporverenmialgoquenadiehabavistoanteriormente.

MariPaz,graciasporvolcartodostusconocimientosconmigo,por

sacarlomejordemyporensearmelofascinantequepuedellegaraserel
mundodelainvestigacin.

Aambasosestareternamenteagradecido.

A Juan Manuel Fernndez Romero que siempre ha estado ah

cuandolohenecesitado.

Graciasamiscompaerasdelaboratorio.Todashabisconseguido

que mi da a da sea mucho ms ameno. Siempre es mucho ms fcil

trabajarrodeadodesonrisasyalegra.

A mis compaeros y amigos del departamento de Qumica

Analtica (Paco, Mara, Isa Montesinos, Mara Jos, Jose, Noelia Luque,
Noelia Caballero, Antoito, Carmen, Ana Ballesteros, Laura Soriano,
Guille, Lola, Nani, Isa Mrquez) pero especialmente a mi grandsimo
amigo JuanMa Jimnez. A veces la vida pone en mitad de tu camino
pruebas muy difciles de superar. Pero en otras ocasiones, coloca a gente
maravillosaqueteayudaasuperarestaspruebas.Muchasgraciasportodo
ynuncaosolvidar.

Nopuedoolvidarmedevosotros,demiscompaerosdepisoyde

carrera.Estecaminolocomenzamosjuntoshaceyamsdeunadcadaylo

estamos finalizando juntos. Muchas gracias de todo corazn a Jess,

Alejandro, Pepe, Puri, Kaquisco, Jose, Trcoli, Carlos, Cristel, porque
gracias a vosotros tengo la gran suerte de recordar todos estos aos con
unasonrisaenloslabios.

Graciasamifamilia,especialmenteamishermanos:Puri,Carmen

yJose.Fuielpequeodeunafamilianumerosapero,sobretodo,mesent
siempre el ms querido. Hemos vivido muchos momentos buenos y
algunosmuymalos,perosiemprehemosestadotodosunidos.Muchsimas
graciasportodovuestroapoyoincondicional.

Hedejadoparaelfinalalaspersonasmsimportantesenmivida.

Mispadres.Vosotroshabissidolospilaresdemivida,demieducaciny
formacin.Graciasavosotros,hoysoyloquesoy.Vosotroshabisluchado
durante toda vuestra vida para que pudisemos llegar a lo ms. No hay
palabras para agradecer todo lo que habis hecho por m y por mis
hermanos.EstaTesisDoctoralesvuestra.

IwouldliketothankProf.TeroSoukkaforofferingmethe
opportunity to work with him in the Department of
BiotechnologyoftheUniversityofTurku(Finland).

Special thanks should be given to PhD. Terhi Riuttamki

for her support and unconditional help in both academic and
personalfields,andforthetimedevotedtome.

I also thank to other colleagues (Timo, Sami, Ari and

Rikka) for their valuable help and kindness during my stay in
Finland.

Agradezco a la Consejera de Economa, Innovacin, Ciencia y

EmpleodelaJuntadeAndalucalaconcesindeunabecapredoctoral
adscrita al Proyecto de Excelencia P09FQM4933 que me ha permitido
dedicarestosltimos4aosaldesarrollodeestaTesis.

INDICE

NDICE

Pgina

Objeto/Aim

Introduccin

Captulo1

HerramientasAnalticas
Materialesyreactivos
Instrumentacin
Programasinformticos

77
80
85
86

Captulo2

Sntesis y caracterizacin de nanopartculas

de silice con fluorescencia a larga longitud
de onda y su aplicacin al anlisis de
alimentos
Synthesis and characterization of
oxazinedoped silica nanoparticles
for their potential use as stable
fluorescentreagents
Heterogeneousimmunoassayforsoy
protein determination using nile
bluedoped silica nanoparticles as
labels and frontsurface long
wavelengthfluorimetry
Determination of monensin in milk
samples by frontsurface long
wavelength
fluoroimmunoassay
using nile bluedoped silica
nanoparticlesaslabels

87

95

123

145

Captulo3

Nuevas aportaciones para la determinacin 169

deantioxidantesenalimentos

Longwavelength
fluorimetric 197
determination of food antioxidant
capacitybyusingnileblueasreagent

Automatic
determination
of
polyphenols in wines using laccase 219
andterbiumoxidenanoparticles

Captulo4

Innovaciones en ensayos de afinidad 245

mediante el uso de upconverting
phosphors y fenmenos de transferencia
deenergaresonanteluminiscente

Evaluation of different donor 255

acceptorpairsforthedevelopmentof
homogeneous bioaffinity assays
using upconversion luminescence
resonanceenergytransfer

Captulo5

Discusinderesultados
Introduccin
1 Nanopartculas de slice en anlisis
dealimentos
2 Nuevas
estrategias
para
la
determinacin de antioxidantes en
alimentos
3 Nuevas investigaciones en el uso de
upconverting
phosphors
en
sistemas de transferencia de energa
resonanteluminiscente
Discussionoftheresults

Introduction

1 Silicananoparticlesinfoodanalysis
2 Newstrategiesforthedetermination
ofantioxidants
3 New investigations in the use of
upconverting
phosphors
in
luminescence resonance energy
transfer
Conclusiones /Conclusions
Anexo/Annex

283
285
285

306

324

343
343
343
362

378

397
407

OBJETO
AIM

Objeto

El objetivo genrico de las investigaciones que constituyen esta

Memoria ha sido el desarrollo de mtodos rpidos para el anlisis de
alimentos utilizando, bsicamente, las especiales propiedades que
presentan distintos nanomateriales. Los estudios realizados han
pretendido expandir la aplicabilidad analtica de la Nanotecnologa para
abrirnuevasvas,alternativasalasyaestablecidas,quemejorenelcontrol
de la calidad alimentaria. Para alcanzar este objetivo se han realizado las
siguientesinvestigaciones:

Sntesis y caracterizacin de nanopartculas de slice dopadas con

los fluorforos de larga longitud de onda azul nilo y violeta de
cresilo y su utilizacin para formar marcadores aplicables a la
determinacin de macromolculas y molculas pequeas en
alimentosmedianteinmunoensayoheterogneo.

Desarrollo

de

nuevas

metodologas

analticas

para

la

determinacin de parmetros globales en alimentos, tales como la

capacidad antioxidante y contenido de polifenoles, utilizando
fluorimetra de larga longitud de onda y nanopartculas de xido
deterbio,respectivamente.

Estudiosistemticodelautilidaddelfenmenodetransferenciade
energa de resonancia luminiscente (LRET) entre nanocristales de
iones lantnidos, que presentan luminiscencia antiStokes
(upconverting phosphors), y fluorforos orgnicos para la
determinacin de biotina mediante ensayos de afinidad en medio
homogneo.

Aim

ThegeneralaimoftheinvestigationsincludedinthisDissertation
hasbeenthedevelopmentoffastmethodsforfoodanalysismainlyusing
theespecialpropertiesofnanomaterials.Thestudiesperformedhavetried
to expand the application field of Nanotechnology and open new
possibilitiesbydevelopinganalyticalmethodsalternativetothosealready
established in order to improve food quality control processes. The
investigationsperformedtoachievethisgoalaredescribedbelow:

Synthesis and characterization of silica nanoparticles doped with

the longwavelength fluorophores cresyl violet and nile blue and
theirfurtherusetoobtaintracerstobeappliedtothedetermination
of either macromolecules or haptens in foods by heterogeneous
immunoassay.

Development of new analytical methodologies to estimate global

parameters in foods, such as the antioxidant capacity and total
polyphenol content, using long wavelength fluorometry and
terbiumoxidenanoparticles,respectively.

Studyofthepotentialusefulnessofluminescentresonanceenergy
transfer(LRET)betweennanocrystalsoflanthanideionsthatshow
antiStokes luminescence (upconverting phosphors) and organic
fluorophorestodevelophomogeneousaffinityassaysforbiotin.

INTRODUCCIN

Introduccin

Las investigaciones que se presentan en esta Memoria han dado

lugar a diversos mtodos determinativos orientados principalmente a
ampliar el campo de aplicacin de la Nanotecnologa en anlisis de
alimentos. Como prembulo a estas investigaciones se describen a
continuacin algunos aspectos relacionados con las metodologas
desarrolladas,incidiendoenlostrestiposdenanomaterialesutilizadosen
esta Memoria, en los fluorforos de larga longitud de onda y en el
inmunoensayo. Al inicio de cada captulo se describirn aspectos ms
concretos relacionados con su contenido. Esta introduccin general se ha
divididoencuatroapartados:1)nanopartculasdeslice,2)nanomateriales
basadosenlantnidos,principalmenteupconvertingphosphorsyxidos
de lantnidos, 3) fluorforos de larga longitud de onda, y 4) tcnicas de
inmunoensayoysuusoenanlisisdealimentos.

1. Nanopartculasdeslice
Las numerosas investigaciones que se estn desarrollando en el
campo de la Nanotecnologa, y los procesos involucrados en la sntesis,
manipulacinydesarrollodenanomateriales,estndandolugaranuevas
herramientasmetodolgicaseinstrumentalesconaplicacionesendistintas
reas analticas [1]. Entre la variedad de los nanomateriales actualmente
disponibles, el uso de nanopartculas de slice (SiO2NPs) constituye una
opcinmuytilcomosedescribeacontinuacin.
El inters que presenta la utilizacin de SiO2NPs con fines
analticos es atribuible a sus especiales caractersticas. No solo muestran

Introduccin

ausencia de toxicidad, elevada solubilidad en agua y estabilidad,

principalmente en medio acuoso, sino tambin otras caractersticas tales
como su fcil funcionalizacin y enlace a biomleculas, la posibilidad de
controlarsuporosidad,sutransparenciaalaradiacin,subajocosteysu
capacidad para utilizarlas como portadores de una amplia variedad de
reactivos [2]. Estas propiedades hacen de las SiO2NPs un material muy
adecuadoparasuusoenbioensayos.
Los fluorforos convencionalmente utilizados como marcadores
moleculares estn siendo sustituidos por SiO2NPs dopadas con dichos
fluorforosyaquestasposeenpropiedadespticassuperiores,unamayor
estabilidad qumica y una menor fotodescomposicin. Adems, se han
desarrollado NPs dopadas con especies electroquimioluminiscentes con
fines bioanalticos por su gran estabilidad y mayor seal luminiscente,
siendo fcilmente aplicables en sistemas miniaturizados [3]. Tambin se
han

sintetizado

otros

tipos

de

SiO2NPs

usando

materiales

semiconductores, metlicos, magnticos e incluso orgnicos. Adems, la

elevadaconcentracindegrupossilanolesqueexisteenlasuperficiedelas
SiO2NPs facilita una gran variedad de reacciones de funcionalizacin y
uninabiomolculascomosonanticuerposyotrasprotenas,ADNuotras
molculas para ensayos de bioafinidad, como son los sistemas biotina
avidinaybiotinaestreptavidina.

1.1 Sntesisdenanopartculasdeslice

Existen dos mtodos principales para la sntesis de SiO2NPs: el

mtodoStberyelmtododemicroemulsindemicelasinversas.

10

Introduccin

El mtodo Stber es el proceso tradicionalmente utilizado, con el

queseobtienenSiO2NPsdedimetromedioinferiora100nm.Elproceso
implicalahidrlisisdeunprecursoralcxidodeslice(comopuedeserel
tetraetoxisilano, TEOS) en una mezcla de etanol e hdroxido amnico.
Duranteestahidrlisistienelugarlageneracindecidosilcicoy,cuando
la concentracin de ste supera su solubilidad en etanol, se produce la
nucleacin, dando lugar a la formacin de las NPs. El dimetro de estas
partculas puede ser controlado mediante modificacin de diversas
variables experimentales como las concentraciones de los diferentes
reactivos y la temperatura de la reaccin [4]. El dimetro de las NPs
obtenidaspuedeoscilarentre10nmy1m,loquedalugaraunconjunto
bastante heterogneo. La incorporacin de fluorforos orgnicos a estas
NPs puede realizarse mediante enlace covalente para lo que es necesario
modificarlosfluorforoscongruposfuncionalescomoelisocianato,elcual
seuneagruposaminosprocedentesdealgnprecursordeslice,comoel
3aminopropiltrietoxisilano, APTES, por lo que los reactivos TEOS y
APTESseincluyensimultneamenteenelprocesodesntesis.
El mtodo de microemulsin de micelas inversas, tambin
conocidocomomtododemicroemulsindeaguaenaceite(W/O),origina
agregados termodinmicamente estables a partir de un surfactante
anfiflico.Lascabezashidroflicasseorientandeformaqueaslanmultitud
de gotas de agua de tamao nanomtrico, mientras que las colas
hidrofbicas quedan orientadas hacia el disolvente orgnico. Estas
nanogotasaisladas del medioorgnico actancomo nanoreactores donde
sellevaacabolaformacindelasNPs.AligualqueenelmtodoStber,

11

Introduccin

las NPs se forman por hidrlisis del precursor de slice y el dimetro de

stas puede ser controlado variando la proporcin entre el agua y el
surfactante (W0) ya que la reaccin tiene lugar en el ncleo acuoso. En
comparacin con el mtodo Stber, este proceso de sntesis necesita un
mayor tiempo de reaccin, pero las NPs obtenidas son ms esfricas,
presentanmayordispersinentreellasyunadistribucindetamaosms
homognea.Estemtododemicroemulsindemicelasinversaspuedeser
utilizado para preparar SiO2NPs dopadas con fluorforos o materiales
metlicostalescomoncleosmagnticososemiconductorestipoquantum
dots(QDs)[5].

1.2 Nanopartculasdopadas

En los ltimos aos se han desarrollado numerosos mtodos

analticos en los que se utilizan marcadores formados por SiO2NPs

dopadas con flurroforos orgnicos, QDs, partculas magnticas o
partculas activas a la radiacin Raman, aprovechando las ventajas que
proporciona el uso de estos materiales encapsulados en la matriz de la
slice, mencionadas anteriormente. A continuacin se describen algunos
ejemplos:
1.2.1

Nanopartculasdopadasconfluorforosorgnicos

Las molculas marcadas con fluorforos han sido ampliamente

utilizadas en bioanlisis. Los fluorforos convencionales, tales como el

isotiocianato de fluorescena, rodaminas o cianinas, presentan ciertas
desventajascomosonsuelevadainestabilidad,sensibilidadaprocesosde
fotodescomposicin y estrecho desplazamiento Stokes. Adems,

12

Introduccin

normalmente, slo un nmero limitado de fluorforos puede unirse a las

biomolculas debido a problemas de impedimento estrico y a enlaces
inespecficosconpotencialesinterferentes.

Porelcontrario,lasSiO2NPsdopadasconmolculasfluorescentes

presentan varias ventajas. Poseen una elevada fluorescencia, buena

estabilidad, gracias a la proteccin que confiere la slice, y amplia
aplicabilidad en el campo del bioanlisis mediante la modificacin de su
superficie para enlazarse a diversos tipos de molculas. Tanto reactivos
orgnicoscomoinorgnicospuedenserincorporadosalinteriordelaslice
usando diferentes mtodos de sntesis, obteniendo NPs que contienen un
elevado nmero de estos fluorforos y que son especialmente tiles para
formar marcadores en ensayos ultrasensibles. El isotiocianato de
fluorescena ha sido ampliamente utilizado para obtener SiO2NPs
fluorescentesaplicablesaladeterminacindebiomolculas.Tambinseha
descrito

la

utilizacin

de

SiO2NPs

dopadas

con

tris(2,2

bipiridil)diclororutenio(II) hexahidratado (Ru(bpy)32+) para desarrollar

ensayos con deteccin electroquimioluminiscente (unidas a un electrodo
paradisearunsensor,enmicroplacasoensistemasmicrofludicos)para
determinar marcadores tumorales [6 9], ADN [10] y otras biomolculas
[11,12].Recientemente,estasNPsdopadasconRu(bpy)32+sehanutilizado
como marcadores quimioluminiscentes para determinar cidos nucleicos
en ensayos de flujo lateral [13] as como para la determinacin de iones
metlicos [14]. Otro derivado fluorescente de rutenio(II), diclorotris(1,10
fenantrolin)rutenio(II) hidratado [Ru(phen)32+], se ha utilizado para
sintetizar NPs fluorescentes y disear un nuevo mtodo para la

13

Introduccin

determinacindeozonobasadoenelfenmenodetransferenciadeenerga
resonante electroquimioluminiscente (ECRET). En este caso, las NPs
dopadasconRu(phen)32+(RuSiNPs)transfierenlaenergaaotrofluorforo
queactacomoaceptor[15].
1.2.2

Nanopartculasdeslicedopadasconquelatosdeiones
lantnidos

Las NPs dopadas con quelatos de iones lantnidos poseen

propiedades luminiscentes nicas tales como un elevado rendimiento

cuntico, estrechas bandas de absorcin y emisin, y una excelente
fotoestabilidad.Estosquelatosdeioneslantnidosunidosalasuperficiede
SiO2NPs se han utilizado en un ensayo inmunofluorimtrico de tiempo
resuelto para la determinacin de la hormona estimulante del tiroides
humano[16].TambinsehanencapsuladoenelinteriordelasNPspara,
por ejemplo, aplicaciones en imagen celular mediante luminiscencia de
tiemporesuelto[17].
1.2.3

Nanopartculasdeslicedopadasconquantumdots(QDs)

Se han encapsulado diversos tipos de quantum dots (QDs) en el

interior de SiO2NPs para formar marcadores y utilizarlos en bioanlisis.

Por

ejemplo,

se

ha

descrito

un

nuevo

inmunosensor

electroquimioluminiscente que emite en el infrarrojo cercano para la

determinacindeprotenashaciendousodeestosQDsencapsuladosenla
matriz de slice [18]. Este tipo de nanomaterial dopado tambin se ha
utilizado en ensayos basados en el fenmeno de transferencia de energa
resonante de fluorescencia (FRET) donde el QD acta como dador del

14

Introduccin

sistemaparaladeterminacindeionesmercurio[19]odemelamina[20].
Otra combinacin de los QDs con las SiO2NPs ha sido la unin de estos
nanocristales a la superficie de la slice mediante enlaces covalentes para
serutilizadosenladeterminacindeclulasymarcadorestumorales[21
23], anticuerpos y otras protenas [24, 25], as como molculas de menor
tamaocomoeltrinitrotolueno[26].
1.2.4

NanopartculasdesliceactivasalaradiacinRaman

Las seales generadas mediante dispersin Raman contienen una

elevada informacin aunque suelen ser poco sensibles. Esta limitacin

puede evitarse en parte inmovilizando la molcula de inters sobre una
superficiemetlica(especialmentesobremetalesnoblestalescomoplatau
oro).Estaprimeraetapadalugaraunincrementoenlaintensidaddeseal
devariosrdenesdemagnitud.ParaconseguirmetodologasRamanms
robustas,estosmetalespuedensercombinadosconSiO2NPs.Porejemplo,
sehadesarrolladounmtodosimpleparaprepararmarcadoresRamancon
nanopartculasdeplata(AgNPs),enlazadasacido4mercaptobenzoicoy
encapsuladasenelinteriordeSiO2NPs[27].Paralasntesissehautilizado
tantoelmtododemicroemulsindemicelasinversas[28]comoelmtodo
Stber [29]. Tambin se ha descrito un mtodo para sintetizar
nanopartculas hbridasde plata y slice utilizando un polmero orgnico,
como el etilenglicol, el cual acta como soporte entre las AgNPs y la
superficiedeslicepara,combinarlasposteriormenteconxidodegrafeno.
Utilizando este procedimiento se ha descrito un nuevo biosensor para la
determinacindeglucosaenmuestrasdeorinaysuero[30].

15

Introduccin

Las NPs de oro (AuNPs), al igual que las AgNPs, pueden

incorporarseenelinteriordelasSiO2NPsduranteelprocesodesntesiso
bienunirsealasuperficie.Porejemplo,sehanutilizadoAuNPs@SiO2NPs
para disear un biosensor basado en la dispersin Raman superficial
donde las NPs actan como marcador mediante su enlace a un aptmero
para la determinacin de adenosina trifosfato [31] o a anticuerpos para
determinar la bacteria del clera utilizando un inmunoensayo [32]. Otra
opcineslacombinacindelasAuNPsylasSiO2NPsconotrosmateriales
para conseguir una amplificacin de la seal. Por ejemplo, utilizando
xidosmetlicos,comoelZrO2,sehadesarrolladouninmunosensorpara
la determinacin del virus de la hepatitis C [33] y, utilizando xido de
grafenoreducidoyAuNPsretenidasenunamatrizdeSiO2/quitosn,seha
descritounbiosensorelectroqumicoparaladeterminacindedopaminay
cido rico [34]. Tambin se han combinado SiO2NPs dopadas con oro y
enzimasysehanutilizadoparacatalizarunareaccinquimioluminiscente
para la determinacin de residuos de estreptomicina [35]. Otra aplicacin
descritaparaestasNPshasidosucombinacinconnanotubosdecarbono
(CNTs) y xido de grafeno para el diseo de un inmunosensor
electrquimicoparaladeterminacindegonadotropinacorinicahumana
(hCG) [36], as como la utilizacin de AuNPs y acetilcolinesterasa en una
matriz solgel y CNTs de pared mltiple (MWCNTs) para modificar un
electrododeplatinoyaplicarloaladeterminacindeacetilcolina[37].
1.2.5

Nanopartculasmagnticas

Lasnanopartculasmagnticas(MNPs)tienenungranatractivoen

el mbito cientfico debido a su elevado potencial en diversos campos,

16

Introduccin

como es el suministro y liberacin controlados de frmacos, diagnstico

por imagen en resonancia magntica (MRI), terapia tumoral mediante
hipertermia magntica, biomarcadores y bioseparacin. Estasaplicaciones
normalmente requieren estabilidad qumica y fcil dispersin en medio
lquido, por lo que las SiO2NPs con ncleo magntico son una buena
opcin debido a la estabilidad que presenta la slice en medio acuoso, su
biocompatibilidadyfcilfuncionalizacinquepermitesuuninaespecies
biolgicamente activas. En Qumica Analtica, las MNPs han sido
utilizadas con diferentes fines. Por ejemplo, las SiO2NPs dopadas con
materiales magnticos se han usado como medio de separacin en la
determinacin simultnea de dos marcadores tumorales (fetoprotena y
antgeno carcinoembrinico) mediante una reaccin electroquimio
luminiscente[38].Enotraaplicacin,sehancombinadoconAuNPsparala
determinacin de trazas de protena C reactiva mediante un
immunosensor piezoelctrico utilizando un anticuerpo y peroxidasa de
rbanoinmovilizadosenlasAuNPs[39].

1.3 Mtodosparamodificarlasuperficiedelasnanopartculasdeslice
El uso de SiO2NPs en aplicaciones analticas se ha extendido
gracias a la relativa facilidad para modificar su superficie. Existen
precursores de slice que permiten introducir grupos funcionales como
aminas, carboxilos, y tioles, creando as puntos de unin a biomolculas.
Porejemplo,losoligonucletidospuedenserinmovilizadosenlasuperficie
de las SiO2NPs usando la reaccin de acoplamiento del disulfuro, donde
las SiO2NPs se funcionalizan con 3mercaptopropiltrimetoxisilano. Una
reaccin de intercambio tioldisulfuro permite la conjugacin de las

17

Introduccin

SiO2NPs modificadas con grupos tioles con los oligonucletidos

modificados con grupos disulfuro sin ningn tipo de reaccin adicional.
Un protocolo comnmente utilizado para la unin de enzimas y
anticuerpos es la unin va glutaraldehido. Para ello, se funcionalizan las
NPs utilizando el 3aminopropiltrietoxisilano o el N[3(trimetoxisilil)
propil]dietilentriaminoylosgruposaminodelasuperficiefuncionalizada
se unen a los grupos amino de estas biomolculas mediante
entrecruzamientoconglutaraldehidoodisuccinidimidilglutarato.Porotro
lado,lasSiO2NPspuedenmodificarsetambinconcarbonatosdicoocon
gruposOCNmediantereaccinconbromurodeciangenoenacetonitrilo
[3].
Como se ha indicado anteriormente, las SiO2NPs son un soporte
adecuado debido a su superficie qumicamente inerte y su fcil
modificacin con grupos funcionales. Cuando estas NPs son utilizadas
como medios de transporte deben presentar una buena dispersin y una
morfologa superficial homognea para conseguir una concentracin de
especies similar entre las diferentes nanoesferas, requisito necesario para
conseguir buena sensibilidad, reproducibilidad y otras propiedades
analticas deseables. Diferentes molculas fluorescentes pueden ser
inmovilizadas en la superficie de las NPs. El fluorforo Ru(bpy)32+ se ha
utilizado para el diseo de un sensor para la determinacin de
inmunoglobulina G mediante la combinacin de estas NPs con CNTs
proporcionando una seal quimioluminiscente [40]. En otros casos, la
superficiedelasNPssehamodificadoconmaterialeselectroactivos,como

18

Introduccin

elcidofosfnico,paraladeterminacindeglucosaenmuestrasrealesen
presenciadeglucosaoxidasa,midindoselacorrienteelctrica[41].
Las biomolculas ms utilizadas para inmovilizarlas en la
superficie de las SiO2NPs son las enzimas y los anticuerpos, debido a su
elevadaselectividad,peroesimprescindiblelageneracindealgntipode
sealquepuedamedirseyrelacionarseconlaconcentracindeanalito.Por
ejemplo, se han utilizado enzimas unidas a la superficie de SiO2NPs que
puedencatalizarreaccionesquimioluminiscentes.Sehadescritoelusode
SiO2NPs marcadas con anticuerpos antistaphylococcal enterotoxin B y
peroxidasa de rbano (HRP) para obtener una seal luminiscente en
presencia de perxido de hidrgeno y luminol [42]. Tambin se han
utilizadoespeciesmagnticascomomtododeseparacin.Porejemplo,en
una separacin basada en una inmunoreaccin tipo sndwich para
microcistinaLR, se ha marcado uno de los anticuerpos con NPs
ferromagnticas, mientras que el segundo anticuerpo se ha enlazado a
SiO2NPs sobre las que se ha inmovilizado previamente la HRP. En
presencia del analito, ambos conjugados quedan unidos y se separan del
resto de la matriz de la muestra utilizando un imn, debido a las
propiedades magnticas de uno de los conjugados. A continuacin, se
adiciona un precursor quimioluminiscente que acta como sustrato de la
enzimayseregistralasealquimioluminiscente[43].

La confinacin fsica de biomolculas en la superficie de las

SiO2NPs,porejemplo,medianteatrapamientoenunamatrizporosadeeste
material, puede ser una alternativa til en ciertas tcnicas biomdicas,
biotecnolgicas y bioanalticas. Utilizando las caractersticas de la matriz

19

Introduccin

de slice se ha desarrollado un sensor combinando MWCNTs y SiO2NPs

para la determinacin de epinefrina [44]. Una tecnologa similar se ha
usado para desarrollar varios tipos de biosensores para la determinacin
indirecta de dibutilftalato utilizando materiales de reconocimiento
molecularySiO2NPs,dondesellevaacabolapolimerizacin[45],oparael
reconomiento

de

tertbutilhidroquinona

utilizando

un

sensor

electroqumicodeimpresinmolecular[46].

1.4.Usodenanopartculasdesliceenanlisisdealimentos

En lo que respecta al anlisis de alimentos haciendo uso de las

SiO2NPs,sehandescritodiversasaplicacionesinteresantes,aunquesuuso
no est generalizado. Estos nanomateriales se han utilizado como nuevos
soportes slidos en mtodos de extraccin y separacin del analito de la
muestra, previos a la etapa de determinacin. Una opcin descrita en la
queseutilizanSiO2NPsparalaseparacinhaconsistidoensudopajecon
un ncleo magntico y su posterior funcionalizacin con un anticuerpo.
Utilizando estas NPs como medio de extraccin, se ha determinado
Salmonella en muestras de zumo de limn y leche pasteurizada va PCR
[47].

Como se ha descrito anteriormente, las SiO2NPs se han utilizado

para formar marcadores y desarrollar nuevas metodologas aplicables al

anlisis de alimentos. Por ejemplo, se han aplicado a la determinacin de
enterotoxinaBenleche[42]ytertbutilhidroquinonaenaceites[46].

Los mtodos de screening o tests de respuesta rpida

desempean una funcin destacable en anlisis de alimentos,

20

Introduccin

concretamente en el control de sustancias prohibidas, gracias a la

posibilidad de realizar dichos ensayos in situ sin necesidad de
instrumentacin costosa y sofisticada. Las SiO2NPs se han utilizado en
estos mtodos de screening dopndolas con algn fluorforo y
funcionalizndolas con un anticuerpo. Estas NPs se han usado como
marcadores en el desarrollo de inmunocromatografas utilizando tiras
reactivas para la determinacin rpida de enrofloxacina en muestras de
carnedepollo[48]oresiduosdeagonistasenorinadecerdo[49].

En resumen, aunque la aplicacin de las SiO2NPs al anlisis de

alimentos ha sido relativamente limitada, sus especiales caractersticas

ofrecennuevasposibilidadesdeutilidadenestereaanaltica.

2. Nanomaterialesbasadosenlantnidos
Esta seccin se centrar en la descripcin de las principales
caractersticas y aplicaciones de los nanomateriales basados en lantnidos
utilizadosenlasinvestigacionesincluidasenestaMemoria,talescomolos
upconverting phosphors y las nanopartculas de xidos de lantnidos.
Aunqueexistenotrosmaterialesutilizadosconfinesanalticos,talescomo
lasNPsdeslicedopadasconquelatosdeioneslantnidos,sudescripcin
se realiz en el apartado anterior de esta Introduccin por lo que no se
incluyeenestaseccin.

21

Introduccin

2.1 Upconvertingphosphors
Debido a que en el Captulo IV de esta Memoria se describen las
investigaciones realizadas con un tipo especial de nanomateriales como
son los upconverting phosphors, se dedica una parte de esta
Introduccin a la descripcin de sus caractersticas y de algunas
aplicacionesanalticas.
Elfenmenodeupconversionesunprocesoenelcualsegenera
unaintensaemisindeenergaapartirdeunaradiacinmenosenergtica.
Este incremento de energa se debe a la absorcin de varios fotones,
normalmente dos o tres, por cada fotn emitido. La transicin del estado
electrnico excitado superior al estado electrnico ms bajo, o a otros
niveles menos energticos, da lugar a una emisin luminiscente a
longitudesdeondamscortasquelalongituddeondadeexcitacin.Este
proceso ptico no lineal, tambin conocido como fotoluminiscencia anti
Stokes,implicaestadosdeenergaintermedios,imprescindiblesparallevar
acaboelsaltoelectrnicoylaemisindeenerga[50].
A diferencia de los fenmenos de fluorescencia basados en
desplazamientoStokes,dondesellevaacabolaexcitacinalongitudesde
ondamscortasylaemisintienelugaraunalongituddeondamayor,el
fenmeno de upconversion requiere una menor energa de excitacin
que los fenmenos Stokes (Figura 1). Por lo tanto, este proceso permite
irradiar la muestra sin el riesgo de descomposicin fotoqumica de la
misma. Adems, se minimizan las interferencias espectrales procedentes
dedichamatriz,comosecomentarposteriormente.

22

Introduccin

Figura1.Representacinesquemticadelosfenmenosdefluorescencia
condesplazamientoStokes(a)yantiStokes(b).

Las aplicaciones analticas de la luminiscencia antiStokes se han

incrementado en las ltimas dcadas debido a la disponibilidad de
nanomateriales inorgnicos capaces de llevar a cabo este fenmeno
luminiscente, denominados upconverting phosphors (UCPs). Estos
materialesestncompuestosporunaestructurainorgnica,siendoelms
utilizadoelNaYF4,aunquetambinsepuedeusarGdyLaenlugardeY,y
se suelen dopar con iones, normalmente lantnidos, modelando as sus
caractersticas espectrales. Los diferentes mtodos utilizados para la
sntesis de UCPs con fines analticos son optimizados para que estos
nanomateriales presenten las siguientes caractersticas: monodispersos,
formahomognea,fcildispersinenaguayunaestructuracristalogrfica
puraconuntamaouniforme(preferiblementeconundimetroinferiora
50nm),ascomoelevadaluminiscencia.LosUCPsdetamaonanomtrico
se han sintetizado usando varios procesos, los cuales van desde el ms

23

Introduccin

comnmente utilizado proceso de coprecipitacin, descomposicin

trmica y cristalizacin en disolvente orgnico por encima del punto de
ebullicinhastalosmtodosdesntesishidroysolvotermal[51].
Los iones dopantes desempean una funcin crucial en lo que
respecta a la absorcin y emisin de fotones. Estos iones determinan, por
ejemplo, la longitud de onda de la radiacin emitida. Algunos lantnidos
trivalentes poseen estados electrnicos intermedios metaestables muy
tiles para la generacin de la emisin antiStokes. Las propiedades
fotoluminiscentes vienen definidas por sus electrones 4f, los cuales estn
bienprotegidosporloselectronesdemenorenerga5sy5p,localizadosen
orbitales ms externos [52]. Los saltos energticos de los niveles
electrnicosdelosionesEr(III),Tm(III)yHo(III),sonmuyapropiadospara
utilizarlos como iones dopantes en materiales cuya finalidad es la
produccin del fenmeno antiStokes, mientras que los iones Pr(III),
Nd(III)yDy(III),presentannivelesenergticosmenosadecuados.
Con el fin de aumentar la eficiencia del proceso de emisin de
fluorescencia

con

desplazamiento

antiStokes,

estos

UCPs

son

normalmente codopados con el ion Yb(III), el cual presenta una elevada

capacidad de absorcin [53]. Adems, los iones Yb(III) son menos
susceptibles que otros lantnidos al fenmeno de inhibicin por
concentracin, es decir, a la prdida de fluorescencia cuando se utilizan
concentraciones elevadas. Por lo tanto, se pueden usar elevadas
concentracionesdeYb(III)paraaumentarlaprobabilidaddeexcitacinde
losionesdopantes[54].Adems,losniveleselectrnicosdelYb(III)yde
losionesdopantesEr(III),Ho(III)yTm(III)responsablesdelatransferencia

24

Introduccin

electrnica y del fenmeno de fluorescencia, son energticamente muy

similares, lo que facilita el salto de los electrones de unos iones a otros,
obtenindoseasemisionesfotoluminiscentesantiStokesmuyintensas.
Los

UCPs

son

cristales

inorgnicos

hidrofbicos

que,

originalmente, no disponen de grupos funcionales. Por lo tanto, la

modificacindesusuperficieesunaspectofundamentalparatransformar
estaspartculasenunmaterialmshidroflicoycongruposfuncionalesen
su superficie para utilizarlos con fines analticos. Con este fin, estos se
recubren con slice y, posteriormente, se funcionalizan mediante los
mtodosdescritosanteriormente[4,5].

Los UCPs estn emergiendo en el campo de la Qumica Analtica

como una alternativa a los biomarcadores fluorescentes tradicionales, los
cualessebasanenlaemisindefluorescenciacondesplazamientoStokes.
Suusoparaprepararmarcadorespresentaunaseriedeventajas.Muestran
un alto rendimiento cuntico, estrechas bandas de emisin, gran
desplazamiento antiStokes, baja toxicidad y buena estabilidad
fotoqumica. Adems, la excitacin de los UCPs se lleva a cabo a una
longitud de onda situada en el infrarrojo cercano, por lo que la relacin
sealruidoesmayorquealongitudesdeondamsbajas,mejorandoasla
sensibilidad gracias a la disminucin del fenmeno de autofluorescencia.
LasexcelentescaractersticasdelosUCPsloshacenmuytilesparasuuso
enbioanlisisgraciasaladisminucindelasealdefondo,obtenindose
lmitesdedeteccinmsbajos.

25

Introduccin

LacombinacindelosUCPsconbiomolculasselectivas,comoson
las implicadas en las interacciones antgenoanticuerpo o biotina
estreptavidina, constituyen una excelente alternativa para su uso en
aplicaciones bioanalticas. Tanto la biomolcula como el UCP, deben
presentar grupos funcionales adecuados para poder unirse mediante
enlaces covalentes. La adsorcin fsica de las biomolculas sobre la
superficie de los UCPs ha sido estudiada aunque la inestabilidad que
presentanlashaceinapropiadasparalosbioensayos[55].
Sehandesarrolladovariasaplicacionesbioanalticasutilizandolos
UCPs como marcadores en ensayos homogneos [56 59] y heterogneos
[60 62], en ensayos de flujo lateral [63 66] y como sensores [67, 68]
basndose algunas de estas aplicaciones, especialmente los ensayos
homogneos, en sistemas basados en transferencia de energa resonante
luminiscente (LRET). En el captulo IV correspondiente a las
investigaciones realizadas con el uso de upconverting phosphors se
abordar con ms profundidad la utilidad de estos nanocristales en el
desarrollodesistemasLRET.
2.2.Nanopartculasdexidoslantnidos
Lasnanopartculasdexidoslantnidos,ymsespecficamentelas
nanopartculas de xido de terbio y de europio, a pesar de haberse
utilizado en numerosas aplicaciones industriales, tales como en el
desarrollodedispositivosdeiluminacinenestadoslido,hanpresentado
hastalafechaescasasaplicacionesanalticas.Sehadescritorecientemente
elusodelasnanopartculasdeEu2O3paraladeterminacindelantibitico

26

Introduccin

tetraciclinaenmuestrasdeorinaanimalymiel[69].Elmtodoestbasado
en la interaccin directa de dicho antibitico con las Eu2O3NPs, para
producir una intensa luminiscencia sensibilizada. Esta emisin
luminiscentepresentacaractersticassimilaresalaluminiscenciaobservada
conquelatosdeioneseuropio,talescomoestrechasbandasdeemisin,un
amplio desplazamiento Stokes y una larga duracin de la luminiscencia
[70]. Esta ltima caracterstica posibilita la realizacin de medidas en el
mododetiemporesuelto,quepermiteeliminarlainterferenciadeseales
fluorescentes de ms corta duracin. Las nanopartculas de Eu2O3 se han
utilizadotambinparaeldesarrollodeinmunoensayosenfaseslida[71,
72]. Estas NPs no tienen grupos funcionales en su superficie, por lo que
sta ha de modificarse para poder ser utilizadas como marcador, lo que
puede conducir a la inhibicin de su luminiscencia por la accin de los
reactivos requeridos para la activacin y conjugacin. Para evitar esto, se
recurreasuencapsulamientoenmatricesdesliceoalmina,comoseha
descrito para un inmunoensayo para la determinacin de atrazina, en el
quelasEu2O3NPssehanrecubiertodesliceparaformarelmarcador[72].
La luminiscencia sensibilizada de las nanopartculas de xido de
terbio(III,IV),tambindenominadoterbia(Tb4O7),porsalicilatoylasalocid
se ha estudiado de forma sistemtica, desarrollndose finalmente un
mtodoparaladeterminacindelasalocidenmuestrasdeagua,depienso
y de huevos [73]. La selectividad del mtodo propuesto ha permitido la
determinacin de dicho antibitico con un tratamiento sencillo de las
muestras.EnlasinvestigacionesincluidasenestaMemorianoseabordar
elusodelaluminiscenciasensibilizadadeestasnanopartculas,sinoquese

27

Introduccin

describirunanuevapropiedadcomoactivadordelaenzimalaccasapara
ladeterminacindepolifenolesenvinos.
3. Fluorforosdelargalongituddeonda
Elusodefluorforosdelargalongituddeonda(LWFs)enQumica
Analtica es una alternativa til para mejorar la selectividad espectral de
lasmedidasfluorescentesfrentealosfluorforostradicionales.Laemisin
de fluorescencia a larga longitud de onda tiene lugar en una zona del
espectroelectromagntico(>600nm)dondeprcticamentenoseproducela
absorcin o emisin de posibles interferentes presentes en la matriz de la
muestra. Adems, debido a la baja energa utilizada para excitar el
fluorforo,elriesgodedegradacindelamuestraesbajomientrasquelas
interferencias que originan seales Raman tambin se reducen
considerablemente [74]. Por otro lado, la posibilidad de sufrir fenmenos
de inhibicin es muy reducida ya que los LWFs muestran un tiempo de
vida corto. La utilidad de este tipo de fluorforos se ha demostrado
ampliamente, especialmente en anlisis biolgico, donde la seal
procedente de la matriz de la muestra puede ser una fuente de
interferenciasmuyimportante.

Laversatilidaddeestoscompuestossehapuestodemanifiestoen

eldesarrollodenuevossustratosenzimticos,actuandocomomarcadores
eninmunoensayos,ensecuenciacindecidosnucleicosycomoreactivos
derivatizantes en electroforesis capilar (CE) y cromatografa de lquidos
(LC). Adems, estos fluorforos se han utilizado para el desarrollo de

28

Introduccin

metodologasbasadasensistemasFRETydenuevossensores,ascomoen
metodologascinticas[75].

3.1 Tiposypropiedadesdelosfluorforosdelargalongituddeonda
Enlosltimosaossehanutilizadodiferentestiposdefluorforos
delargalongituddeondacomoreactivos,entrelosquesepuedendestacar
tres grupos bien diferenciados: materiales inorgnicos, fluorforos
orgnicosycompuestosorganometlicos(Figura2).

Figura2.Clasificacindelosfluorforosdelargalongituddeonda.

29

Introduccin

Dentrodelosmaterialesinorgnicos,sepuedendestacardostipos,
los quantum dots (QDs) [76] y los upconverting phosphors (UCPs) [77],
estos ltimos descritos en el apartado anterior. Los QDs suelen estar
formados por una estructura sintetizada a partir de materiales
semiconductores como CdSe, CdTe, PbS, o materiales similares, y cuya
composicin y tamao inciden en las longitudes de onda mximas de
excitacinyemisinquepresentanestosmateriales.
Aunque el nmero de fluorforos orgnicos de larga longitud de
ondaesmenorqueeldelosfluorforosconvencionales,sehasintetizado
una gran variedad de compuestos de este tipo en los ltimos aos, los
cuales se han utilizado en muchos casos en el diseo de lseres de
colorantes. Un LWF debe presentar una estructura rgida con enlaces
conjugadosoanillosaromticoscondensados.Estesistemadeconjugacin
puede favorecer la inestabilidad del reactivo as como procesos de foto
descomposicin cuando es excitado. Tambin pueden mostrar otros
inconvenientes tales como estrechos desplazamientos Stokes, baja
solubilidad, y procesos de fotooxidacin. Sin embargo, las ventajas
anteriormente indicadas justifican su aplicacin en Qumica Analtica [78,
79],describindoseacontinuacinalgunascaractersticasdelosLWFsms
utilizados.
Losfluorforosdelafamiliadelascianinasconstituyenunodelos
principalesgruposdeLWFs.Suestructurapresentadosanillosaromticos
y heterocclicos unidos mediante una cadena de polimetina con dobles
enlaces carbonocarbono conjugados (Figura 3). Estos fluorforos tienen
mximos de excitacin en el intervalo 600900 nm, obtenindose amplios

30

Introduccin

desplazamientosespectralesbatocrmicosconlaadicindegruposviniloa
la cadena de polimetina. Las cianinas presentan algunas limitaciones: no
tienen grupos reactivos para unirlos a los analitos, su vida media en el
estado excitado es muy corta, bajo rendimiento cuntico y tienden a
agregarseendisolucin,loqueoriginaunrpidodescensodelaintensidad
defluorescencia[80].Sinembargo,lafotofsicadeestoscompuestospuede
mejorarseaadiendomacromolculasodisolventesorgnicosalmedio.La
adicindegrupossulfonatoalaestructuradelfluorforopuedemejorarsu
solubilidadenagua,elrendimientocunticoysuestabilidadfotoqumica.
Adems, la adicin de grupos funcionales, como el isotiocianato, permite
su uso como marcador ya que puede unirse a otras molculas. Los
derivados indolio de estos colorantes muestran una buena estabilidad
fotoqumica, la cual puede incluso mejorarse incluyendo una estructura
anularalacadenadepolimetina[81].
Dosfluorforoscianinaampliamenteutilizadosconfinesanalticos
sonCy5yverdedeindocianina(ICG),denominadotambinIR125(Figura
3). Se han descrito numerosas aplicaciones analticas para Cy5,
principalmenteenCE[82]ysensores[83].ElICGesunfluorforocargado
negativamente,solubleenagua,queseutilizinicialmenteparatcnicasde
diagnstico mdico ya que no es txico para el organismo humano [84].
AunqueelICGporssolopresentaunabajaintensidaddefluorescenciaen
disolucin acuosa, sta aumenta despus de su unin a algunos
compuestos,talescomoprotenas[85].

31

Introduccin

+
N

X,Y=O,S,C(CH3 )2 oC2 H2 ;n=4,R:(CH2 )SO3

a)

H3C

CH3

HO3S

SO3H

H3C

CH3
+
N

N
(CH2)5COOH

CH2CH3

b)

CH3

H3C

CH3

H3C

+
N

CH2

CH2

(CH2)3

(CH2)3

SO3Na

SO3Na

c)
Figura3.Estructuradelosfluorforoscianina:a)Estructurabsica,b)Cy5,
c)verdedeindocianina.

32

Introduccin

Los fluorforos oxacina constituyen otro grupo de LWFs que

presentanmejorestabilidadfotoqumicaquelosfluorforosanteriores.Las
estructurasdeestoscompuestossemuestranenlaFigura4,dondepuede
observarse que son ms compactos que los colorantes cianina. Se han
descritoalgunasaplicacionesanalticasconvioletadecresilo[86,87],azul
nilo[8891]yOxacina750[92].

N
+

H2N

a)

C2H5HN

N
R

b)

Figura4.Estructuradeloscolorantesoxacina:a)violetadecresilo(R:H2)
yazulnilo(R:C2H5),b)Oxacina750.

El tercer grupo de LWF de tipo orgnico son los derivados de la

rodamina.Existenalgunosejemplosdelautilidadanalticadedosdesus
derivados, Rodamina 800 [93 97] y Rojo Texas [98 100] (Figura 5). El
primero de ellos se ha utilizado recientemente para la determinacin de
metales pesados en muestras acuosas y como sensor de pH intracelular
[95]. El Rojo Texas tambin se ha utilizado en diversas metodologas
analticas. Por ejemplo, este fluorforo se ha encapsulado junto con una
enzima(peroxidasaderbano)enelinteriordeNPsparaeldiseodeun
sensorparaladeterminacindeperxidodehidrgeno[98].

33

Introduccin

SO2Cl

SO3

CN

a)

N Cl

b)

Figura5.Estructurade:a)Rodamina800,b)RojoTexas.

Los colorantes BODIPY son una familia de compuestos que

respondenalafrmulageneral4,4difluoro4bora3a,4adiazasindaceno
(Figura 6). La sustitucin en distintas posiciones del sistema de anillos
heterocclicos origina diferencias en el comportamiento luminiscente de
estos compuestos. Los primeros colorantes sintetizados, que incluan
fundamentalmente sustituyentes alqulicos en su estructura, presentaban
algunos inconvenientes, como la emisin en la zona verde del espectro
electromagntico(entre400600cm1)ycortosdesplazamientosStokes.No
obstante,sehansintetizadorecientementealgunosderivadosqueemitena
longitudesdeondamslargas[101103].Estoscolorantesincluyengrupos
fenlicosonaftlicosenlaposicin8desuestructurayotrossustituyentes
en las posiciones 3 y 5, que contribuyen a aumentar la conjugacin con
respecto a la de la estructura bsica.La fluorescencia de algunos de estos
colorantes aumenta en medio cido, por lo que se ha propuesto su uso
comosensoresdepH[101].

34

Introduccin

7
6

N
5

1
2

N
F

Figura 6. Estructura bsica y numeracin de las posiciones de los anillos

heterocclicosenloscolorantesBODIPY.

Los fluorforos Alexa Fluor son otro tipo de molculas orgnicas

que han sido utilizados como marcadores de biomolculas en diferentes

ensayos analticos. La fotoestabilidad es una de las principales
caractersticasdeestetipodecolorantes,porloquesehanutilizadopara
mejorar las tcnicas de captura de imagen celular y en ensayos analticos
condetectoresfluorescentes[104,105].

Los fluorforos ATTO son otra familia de compuestos que

presentan fluorescencia en una amplia regin del espectro, en la que

algunos compuestos como el ATTO 647 presentan fluorescencia a larga
longituddeonda.Algunasdesuscaractersticassonsuelevadacapacidad
deabsorcin,altorendimientocuntico,unabuenasolubilidadenaguay
un comportamiento fluorescente independiente del pH en un amplio
intervalo,enalgunoscasos,de2a11.Unalimitacindeestoscoloranteses
su estrecho desplazamiento Stokes, lo que podra originar fenmenos de
dispersin de la radiacin. La principal utilidad de estos fluorforos est
orientadaaldesarrollodemtodosbasadosenFRET[106].

35

Introduccin

Por ltimo, cabe destacar otro grupo de LWFs muy utilizados en

QumicaAnaltica,comosonloscomplejosderutenio,loscualesmuestran
una luminiscencia con un tiempo de vida similar a los fluorforos
orgnicos. La principal ventaja que presentan es la posibilidad de usar
diferentes ligandos que permiten modificar las propiedades qumicas y
espectroscpicasdeestoscomplejos,mostrandomximosdeemisinentre
610 y 650 nm. El uso analtico de estos compuestos se ha orientado
principalmenteparaeldesarrollodenuevossensores[107109].
En los ltimos aos, los LWFs han sido bastante utilizados en
anlisis de alimentos con matrices complejas debido, principalmente, a la
selectividadespectral que presentan.Por ejemplo, se han empleado como
reactivosderivatizantesparaladeterminacindemolculasendgenasde
los alimentos, como son los flavonoides, mediante cromatografa [110], o
bien para la determinacin de protenas de soja en bebidas mediante
inmunoextraccin [111]. Tambin se ha descrito el uso combinado de
AuNPs,laenzimalaccasayverdedeindocianinaparaladeterminacinde
compuestospolifenlicosenmuestrasdezumoytmediantelatcnicade
mezcladeflujodetenido[112].
Ademsdelosfluorforosdelargalongituddeondaindicados,se
handescritonuevospolmeroscapacesdeemitirenlazonadelinfrarrojo
cercano, caracterstica que ha sido fundamental para el desarrollo de
nuevos sensores, como es el caso de un sensor para la determinacin de
Cu(II) y cuya aplicabilidad se ha demostrado mediante el anlisis de t
[113]. Tambin se han propuesto nanomateriales que emiten a longitudes
deondarelativamentelargasparautilizarlosenanlisisdealimentos.Por

36

Introduccin

ejemplo, los QDs descritos anteriormente se han utilizado para la

determinacindevitaminaB1ensuplementosalimenticios[114].

4. Tcnicasdeinmunoensayoysuusoenanlisisdealimentos
Los inmunoensayos son tcnicas analticas basadas en la elevada
selectividad que presentan las reacciones antgenoanticuerpo, siendo
ampliamente utilizados en anlisis clnico, ambiental y agroalimentario.
Desdeunpuntodevistageneral,losinmunoensayospuedendividirseen
dosgrandesgrupos,directoseindirectosoconreactivosmarcados.
Los inmunoensayos directos se basan en la medida de una
propiedadfsicoqumicadelmedioquesemodificaalreaccionarelanalito
con el inmunoreactivo. Dentro de los inmunoensayos directos, los ms
utilizadoshansidolainmunoturbidimetraylainmunonefelometra[115,
116],basadasenlamedidadeladispersindelaradiacinalformarseel
inmunocomplejo. Estas tcnicas se utilizan para la determinacin de
macromolculas, principalmente protenas. Su principal limitacin es la
posiblesealdefondodebidaalacapacidaddispersantedecomponentes
de la muestra, como pueden ser lipoprotenas y agregados de protenas,
dandolugaralmitesdedeteccinrelativamentealtos.
Los inmunoensayos indirectos ofrecen mayor versatilidad que los
anteriores y se basan en el uso de un reactivo adicional, denominado
marcadorotrazador.Estereactivoesunantgenoounanticuerpo,segnel
tipodeinmunoensayo,unidoaunasustancia,denominadalabel(L)en
laterminologainglesa,quepresentaunapropiedadcomoradioactividad,

37

Introduccin

fluorescenciaoactividadenzimtica,entreotras,cuyamedidaserelaciona
conlaconcentracindelanalito.Estosinmunoensayosseclasificanendos
grandes grupos en funcin del formato que utilicen, homogneos y
heterogneos[117].
En el inmunoensayo homogneo, la unin del antgeno y el
anticuerpo da lugar a un cambio en la propiedad del marcador que se
relaciona con la concentracin de analito. En este caso no es necesario
eliminar la matriz de la muestra para realizar la medida, por lo que el
ensayo es muy rpido, pero la presencia de la seal de la matriz de la
muestra da lugar a lmites de deteccin mayores que el inmunoensayo
heterogneo.
En el inmunoensayo heterogneo el marcador no modifica su
propiedad al intervenir en la inmunoreaccin, por lo que es necesario
inmovilizar algn reactante en un soporte slido para conseguir la
separacin de las fracciones enlazada y libre del marcador. Este
inmunoensayo es ms verstil que el homogneo, obtenindose adems,
como se ha indicado, mejores lmites de deteccin. Sin embargo, se
requiere un mayor nmero de etapas por lo que los ensayos son ms
lentos. A continuacin se describensucintamente algunos de los distintos
tiposincluidosencadaformato.

4.1 Inmunoensayohomogneo
Normalmente es competitivo, es decir, se considera que el
anticuerpo no distingue entre el analito y el marcador y ambos compiten

38

Introduccin

por enlazarse al anticuerpo, el cual se encuentra en defecto. Existen dos

posibilidades:
1) Lapropiedadquepresentaelmarcadorseanulaalenlazarseal
anticuerpo, por lo que la seal medida slo corresponde al
marcadorquequedalibre,siendodirectamenteproporcionala
laconcentracindeanalito.
2) El marcador slo presenta la propiedad medida cuando se
enlaza al anticuerpo, de forma que la seal disminuye al
aumentarlaconcentracindeanalito.
Estos ensayos se aplican bsicamente a la determinacin de
molculas pequeas tales como frmacos, drogas y plaguicidas. Como
ejemplos de estos ensayos se describen a continuacin los ms
representativos:

EMIT(Enzymemultipliedimmunoassaytechnique):Fueelprimer

enzimoinmunoensayo que se describi mediante el uso de una enzima

unida al hapteno como marcador. Este marcador presenta actividad
enzimtica y la pierde al enlazarse al anticuerpo, por lo que se mide la
actividad del marcador libre mediante la adicin del sustrato adecuado
[118].

CEDIA(Clonedenzymedonorimmunoassay):Esuntipoespecial

deensayohomogneo,patentadoporMicrogenics.Comosemuestraenel
Figura 7, utiliza dos fragmentos inactivos de la enzima galactosidasa, a
los que se les denomina dador (ED) y aceptor (EA) enzimticos, y que al
unirse forman la enzima activa. El ensayo se realiza en una etapa

39

Introduccin

mezclandolamuestraquecontieneelanalitocondosreactivos:1)Reactivo
1,formadoporelanticuerpoyelfragmentoEAdelaenzima,y2)Reactivo
2,quecontienealmarcadorformadoporelfragmentoEDunidoalhapteno
y el sustrato de la enzima. Despus de incubar, slo el fragmento ED del
marcador libre puede unirse al fragmento EA formando la enzima,
mientrasquestanoseformaconelfragmentoEDdelmarcadorunidoal
anticuerpo. Por tanto, la seal originada por el producto de la reaccin
enzimticaserdebidaalmarcadorlibreydirectamenteproporcionalala
concentracindeanalito[119].

AceptorEnzimtico
Antgeno()

(Analito)
Anticuerpo

FormaInactiva

Antgeno(+)
(Analito)

FormaActiva

DadorEnzimtico

Figura7.EsquemadelformatoCEDIA.

FETI (Fluorescence resonance energy transfer immunoassay): Se

dise como una alternativa a los inmunoensayos homogneos

tradicionalesysebasaenelprocesodetransferenciadeenergaquetiene

40

Introduccin

lugar al unirse el antgeno y el anticuerpo, ambos marcados con las

molculasimplicadasenesteproceso.Puededarlugaraunaemisinpor
parte del inmunocomplejo a una longitud de onda diferente a la de
excitacin, o bien, a una inhibicin de fluorescencia al producirse la
reaccininmunoqumica.Aunqueestetipodeinmunoensayohomogneo
anseutiliza,latendenciaactualeselusodenanomaterialesmediantelos
formatoshomogneooheterogneo[120].

FPIA(Fluorescencepolarizationimmunoassay):Enestesistemase

utiliza un hapteno unido a un fluorforo como marcador y radiacin

polarizada para la excitacin [121, 122]. Esta radiacin excita a las
molculascuyosdipolosdeabsorcinvibranenlamismadireccinqueel
vector elctrico de dicha radiacin. Es decir, si la radiacin excitante est
polarizada verticalmente, se excitarn las molculas de marcador que
vibran paralelamente a esa direccin. La emisin de radiacin polarizada
vaadependerdelavelocidadderotacindelasmolculasydelintervalo
detiempoquetranscurreentrelaexcitacinylaemisin.Comomuestrala
Figura8,existendossituaciones:
1) Elpequeotamaodelmarcadorlibrepermitequesumovimiento
rotacional sea ms rpido que la duracin del estado excitado, de
forma que cuando se produce la emisin de fluorescencia ha
cambiado su posicin por lo que sta se produce en todas las
direcciones.
2) El marcador unido al anticuerpo tiene un movimiento rotacional
mslento,debidoalelevadotamaodeste,porloquelaemisin
de fluorescencia se produce cuando su posicin prcticamente no

41

Introduccin

hacambiado.Portanto,sisehaexcitadoconradiacinpolarizada
verticalmente,emitirradiacinpolarizadaenlamismadireccin.

Figura8.EsquemadelformatoFPIA.

4.2 Inmunoensayoheterogneo
Estos inmunoensayos tienen mayor versatilidad ya que mediante
formatoscompetitivosynocompetitivososndwichpuedendeterminarse
haptenos,antgenosmacromolecularesyanticuerpos.
Puesto que, como se ha indicado anteriormente, la propiedad del
marcador no se modifica al producirse la reaccin inmunoqumica, en
cualquier inmunoensayo heterogneo es imprescindible la inmovilizacin
dealgnreactanteenunsoporteslidoparaconseguirlaseparacindelas
fracciones enlazada y libre del marcador. Este tipo de inmunoensayo ha
sufridounacontinuaevolucinalolargodeltiempo.Latendenciaactual

42

Introduccin

secentraenelusodenanomateriales,yaqueofrecenunagransuperficie
til para la inmovilizacin, aumentando la eficacia de las interacciones
entre analitos y reactivos, y, con ello, la sensibilidad de los diferentes
mtodos. Adems, la utilizacin de nanopartculas magnticas facilita su
separacinmedianteunimn.
Losdiferentesinmunoensayosheterogneospuedenclasificarsea
su vez en dos formatos bsicos en funcin del diseo del ensayo,
competitivoynocompetitivoosndwich:

Inmunoensayo heterogneo competitivo (Figura 9): Este formato

puede ser directo con captura de antgeno o indirecto con captura de

anticuerpo. En el primer caso, el formato es similar al del ensayo
homogneo, pero el anticuerpo es inmovilizado en un soporte slido. El
analitoyelmarcadorcompitenporenlazarsealanticuerpoy,despusde
incubar y lavar, se mide la seal del marcador enlazado, la cual ser
inversamenteproporcionalalaconcentracindeanalito.Normalmente,se
utiliza paradeterminar haptenos yaque slo senecesitaundeterminante
antignico.

43

Introduccin

Figura 9. Formatos competitivos: A) con captura de antgeno y B) con

capturadeanticuerpo.
En el ensayo indirecto con captura de anticuerpo se inmoviliza el
antgeno y se adiciona el analito y el anticuerpo. Se incuba, se lava y se
adiciona el marcador formado por un anticuerpo inespecfico frente al
anticuerpo primario (anticuerpo secundario). Es decir, si el anticuerpo
primariosehaobtenidodeunindividuodeunaespecieanimaldada,por
ejemplounratn,elanticuerposecundariosehaobtenidoinmunizandoun

44

Introduccin

individuo de otra especie animal, por ejemplo una oveja, con suero de
ratn. Este marcador puede utilizarse como reactivo general para
determinardistintosanalitossiemprequeloscorrespondientesanticuerpos
primarios procedan de la misma especie animal. Como alternativa a este
marcador, podra usarse directamente el anticuerpo del analito marcado,
reduciendo as el nmero de etapas del ensayo, pero se necesitara un
marcador distinto para cada determinacin, aumentado su coste. Este
formato se aplica preferentemente a analitos macromoleculares, ya que
deben disponer de grupos adecuados para enlazarse al soporte y al
anticuerpo.

Inmunoensayo heterogneo no competitivo: Estos ensayos,

denominados tambin sndwich, son ms adecuados para determinar

especies macromoleculares, ya sean antgenos o anticuerpos. La
determinacin de antgenos, esquematizada en la Figura 10, implica la
inmovilizacindelanticuerpo,denominadoprimario,elcualseencuentra
en cantidad suficiente para que reaccione todo el analito. Despus de
incubar y lavar, existen dos posibilidades segn se utilice el formato
directooelindirecto.Enelprimercaso,seadicionaelmarcadorformado
porunanticuerpo,denominadosecundario,frentealanalito.Enelformato
indirecto,seadicionaelanticuerposecundarioyelmarcadorformadopor
unanticuerpoinespecficofrenteaesteanticuerposecundario,aligualque
sehadescritoanteriormente.

45

Introduccin

Figura10.Formatosnocompetitivos,A)directoyB)indirecto,concaptura
deantgeno.
El formato sndwich implica que el analito debe tener, al menos,
dosdeterminantesantignicosparaqueseenlacenaloscorrespondientes
anticuerpos.Teniendoencuentaquelosanticuerpossonmacromolculas,
espreferiblequeelanalitotambinseamacromolecular,deformaqueno
exista impedimento estrico y puedan enlazarse adecuadamente los dos
anticuerpos. Este formato, utilizado normalmente para determinar
protenas antignicas, ofrece mayor selectividad que el formato
competitivo,yaqueseusandosanticuerposfrentealanalito.
Elensayosndwichtambinseutilizaparadeterminaranticuerpos
frente a un antgeno y, al igual que en el caso anterior, la determinacin
puede realizarse de dos formas. Como muestra la Figura 11, se puede
46

Introduccin

llevaracaboinmovilizandoelantgenoounanticuerpoinespecficofrente
al anticuerpo a determinar. En el primer caso, si la muestra contiene el
anticuerpo que se va a determinar, ste quedar retenido en la superficie
slida. Despus de lavar, se adiciona el marcador formado por otro
anticuerpo.Enelsegundocaso,seinmovilizaunanticuerposecundarioen
la superficie slida y se adiciona la muestra. Tras incubar y lavar, se
adiciona el antgeno especfico frente al anticuerpo a determinar y,
finalmente, se aade el marcador, formado por un anticuerpo especfico
frentealantgeno.

Figura11.Formatosnocompetitivosparaladeterminacindeanticuerpos:
A)conantgenoinmovilizadoyB)conanticuerpoinmovilizado.

47

Introduccin

Los diferentes inmunoensayos heterogneos tambin pueden ser

clasificados en funcin de la propiedad que se mide del marcador. Entre
ellos,cabecitar:

RIA (Radioimmunoassay): Fue el primero en desarrollarse

medianteelusodeistoposradiactivosunidosabiomolculasparaformar
el marcador. Este ensayo presenta gran versatilidad, bajos lmites de
deteccin,elevadaprecisinyausenciadeinterferenciasbienambientales,
talescomolasdebidasalpH,temperaturaofuerzainica,obiendebidasa
la matriz de la muestra. Sin embargo, tambin presenta limitaciones,
siendoelprincipalinconvenienteelusodematerialesradioactivosconsus
posibles riesgos para la salud, necesidad de permisos y recintos
exclusivamente preparados para ello, as como la gestin especial de los
residuos, lo que encarece el ensayo y limita su uso a laboratorios de
referencia[123125].

FIA (Fluoroimmunoassay): Es una alternativa al uso de material

radiactivo en los inmunoensayos heterogneos. El procedimiento es muy

similar al utilizado en RIA pero se utilizan molculas fluorescentes para
formarelmarcador.Dentrodeestosfluoroinmunoensayos(FIA)cabecitar
el sistema DELFIA (DissociationEnhanced Lanthanide Fluorescent
Immunoassay) [126, 127]. Se trata deun tipo particulardeFIAdetiempo
resuelto, que utiliza quelatos de europio, samario o terbio y se basa en el
principiodeintensificacindisociativadelafluorescencia(Figura12).Uno
de los aspectos clave de este ensayo es el uso de una disolucin cida
compuesta por una diketona (por ejemplo, 2naftoiltrifluoroacetona,
NTA), xido de trinoctilfosfina (TOPO) y Tritn X100. El agente

48

Introduccin

quelatante (NTA) forma un complejo muy fluorescente con el ion

lantnido, el TOPO elimina las molculas de agua de la esfera de
coordinacin mientras que el Tritn X100 crea un medio micelar que
protege al complejo. Este tipo de ensayos presentan lmites de deteccin
similaresalRIAsinelinconvenientedelusodematerialradiactivo.

Figura12.EsquemadelsistemaDELFIA.

ELISA(Enzymelinkedimmunosorbentassay):Puedeconsiderarse

elinmunoensayoheterogneomsutilizadoenanlisisclnico,ambientaly
agroalimentario. En este caso, el marcador es sintetizado a partir de una
enzima que, en presencia del sustrato correspondiente, cataliza una
reaccin, dando lugar a cambios medibles normalmente mediante
fotometra o fluorimetra. Las enzimas ms utilizadas para formar el
marcador son galactosidasa, fosfatasa alcalina y peroxidasa de rbano,
las cuales necesitan su correspondiente sustrato para llevar a cabo la
deteccin[128131].

49

Introduccin

Inmunosensores: Constituyen un tipo de sensores muy selectivos

basadosenlainmovilizacindeunantgenoounanticuerpoenlazonade
reconomiento del sensor. Hasta hace unos aos, estos inmunosensores
presentabanproblemasdesensibilidad,yaqueloscambiosdesealapenas
podan ser recogidos por el transductor. En cambio, la utilizacin de los
nanomateriales ha provocado una revolucin en estos dispositivos. La
inmovilizacin de NPs sobre la superficie del sensor proporciona un
aumento de la superficie especfica del mismo y, en consecuencia, de la
cantidad de reactivo inmovilizado, consiguiendo mayores cambios de
sealyunamejoradelasensibilidaddelmtodo.
Los inmunosensores pueden ser clasificados en diferentes grupos
en funcin de la seal originada al unirse el analito a la superficie del
sensor(Figura13).
Potenciomtricos
Amperomtricos
Impedimtricos
Conductimtricos
Capacitivos

ELECTROQUMICOS

INMUNOSENSORES

PTICOS
PIEZOELCTRICOS

Quimioluminiscentes
Electroquimioluminiscentes
Resonanciadelplasmn superficial

Figura13.Clasificacindelosinmunosensores.

50

Introduccin

Acontinuacin,sedescribensucintamentelosdistintostipos:
Los inmunosensores electroqumicos se caracterizan por presentar

buenasensibilidad(desdelainclusindelosnanomateriales),bajocostey
fcil automatizacin. Como se muestra en la clasificacin, los
inmunosensores electroqumicos estn divididos en varios subgrupos, en
funcindeltipodesealqueseorigina.As,lospotenciomtricosregistran
el cambio de potencial en la superficie del sensor, los amperomtricos
recogen la corriente elctrica generada, los conductimtricos miden los
cambios en la conductividad, los sensores de impedancia miden la
resistenciaalpasodecorrienteelctricaylossensoresdecapacitanciase
basan en los cambios de la constante dielctrica del sensor al unirse el
analitoasusuperficie[132146].

Losinmunosensorespticossebasanenlamedidadelaabsorcin

oemisinproducidatrasllevarseacabolainmunoreaccinenlasuperficie
delsensor.Estosinmunosensoressonmuyutilizadosenbioanlisisgracias
a las ventajas que presentan: modo de trabajo no destructivo y rpida
generacin de la seal. Al igual que ocurre con los inmunosensores
electroqumicos, los inmunosensores pticos han experimentado una
revolucingraciasalusodenanopartculas.Losnanomaterialesutilizados
vandesdeSiO2NPsdopadasconfluorforos,AuNPs,QDs,CNTs,hastalos
recientemente utilizados UCPs. Estos inmunosensores pticos, como
muestra el esquema anterior, se pueden dividir en sensores
quimioluminiscentes (se recoge la intensidad luminiscente generada por
unareaccinqumica),electroquimioluminiscentes(implicanlaformacin
de especies que, tras una transferencia electrnica, emiten radiacin

51

Introduccin

luminiscente) e inmunosensores de resonancia de plasmn superficial

(fenmeno producido al interaccionar la radiacin con los electrones
superficialesdefinaspelculasmetlicas)[147153].

Otro

tipo

de

inmunosensores

son

los

inmunosensores

piezoelctricos, basados en el uso de la microbalanza de cristal de cuarzo

(QCM) que registra cambios en la frecuencia de resonancia de un
osciloscopioacopladoalsistema,lacualesdirectamenteproporcionalala
masa que se une a la superficie. Aunque esta tcnica presenta una buena
sensibilidad, su utilizacin es ms restringida debido a problemas de
selectividad,yaquecualquiermolculaqueseunaalasuperficiedeforma
noespecfica,originarunarespuestaeneldetector[154,155].
Untipoespecialdeinmunosensoresloconstituyenlosbasadosen
ensayos de flujo lateral o inmunocromatografa. Esta tcnica utiliza un
dispositivo muy simple, porttil y no requiere un sistema de deteccin
costoso ya que en ciertos casos, es visual (cualitativo) y, en otros, solo se
necesitaunrefractmetroounfluormetro(cuantitativo).Elejemplotpico
de este sistema es el test de embarazo comercialmente asequible. Este
dispositivo est fabricado con un material poroso capaz de transportar la
muestralquidaporcapilaridadalolargodelasdiferentesseccionesquelo
componen.ComopuedeobservarseenlaFigura14,sobrealgunasdeestas
seccionesseinmovilizanantgenosoanticuerposmarcadosparaquesean
arrastradosporlamuestralquidayproporcionendossealesvisibles,una
lneadecontrolyotradeensayo.Lalneadecontrolseformaporcaptura
del marcador libre, comprobando as que el ensayo se ha realizado
correctamente. En cambio, la lnea de ensayo se forma por el enlace del

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Introduccin

marcadorunidoalanalito,indicandolapresenciadelanalitoenlamuestra
[156160].

Figura14.Esquemadeensayodeflujolateral.

4.3.Usodelinmunoensayoenanlisisdealimentos
Aunque las tcnicas de inmunoensayo tienen su principal campo
deaplicacinenanlisisclnico,suusosehaidoconsolidandotambinen
anlisisdealimentos,comolodemuestraelnmerorelativamenteelevado
de kits de ensayos comerciales actualmente disponibles. La Figura 15
muestra la distribucin de los artculos publicados en los ltimos aos
sobre esta rea. Puede observarse que, aunque la mayora de las tcnicas
anteriormentedescritassehanaplicadoalanlisisdealimentos,ELISAha
sido y actualmente sigue siendo la ms utilizada. Algunas de las razones
quejustificansueleccinincidenenlosbajoslmitesdedeteccin,fciluso
y coste relativamente bajo que presentan los mtodos basados en esta
tcnica.

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Introduccin

Figura 15. Distribucin del nmero de artculos dedicados al

inmunoensayoenanlisisdealimentossegnlatcnicautilizada(basede
datos:Scopus).

EnlaTabla1serecogenalgunosejemplosdelosmtodosdescritos
utilizando distintos inmunoensayos [161 171]. Como puede observarse,
se han desarrollado determinaciones para diversos analitos tales como
micotoxinas,plaguicidas,residuosdeantibiticosyhormonasaplicablesal
anlisisdealimentosdestinadosalconsumohumanoydealimentospara
animales. Cabe destacar que, aunque ELISA sigue siendo la tcnica ms
extendida en esta rea, existe una clara tendencia al desarrollo de
inmunosensores, principalmente electroqumicos y pticos [172], aunque
sucomercializacinhastalafechahasidoprcticamentenula.

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Tabla1.Ejemplosrepresentativosdeinmunoensayosaplicadosalanlisis
dealimentos
Tipode

Fundamento

Analito

Muestra

Ref.

FREThomogneo

Micotoxinas

Cebada

[161]

[162]

Inmunoensayo
Homogneo

porfluorescencia
intrnsecadelos
anticuerpos
Homogneo

Dispersindela

Protenasde

Zumode

radiacincon

soja

frutasyyogur
consoja

AuNPs
Homogneo

FPIA

Micotoxinas

Grano

[163]

Homogneo

FPIA

Plaguicidas

Vegetablesy

[164]

rgano

agua

fosforados

ambiental

Heterogneo
Heterogneo

ELISA
ELISA

Fluoro

Pescadosy

quinolonas

mariscos

[165]

Dodecilciclo

Ternera

[166]
[167]

butanona
Heterogneo

ELISA

Heterogneo

DELFIA

Heterogneo

Ensayoflujolateral

Fenil

Piensosy

etanolaminaA

carne

Estradiol

Suerobovino

[168]

Residuosde

Orinadecerdo

[169]

Leche

[170]

Carnedecerdo

[171]

clembuteroly
raptomina
Inmunosensor

Sensor

Residuosde

amperomtrico

sulfonamiday
tetraciclina

Inmunosensor

Sensor

Clembuterol

electroquimio
luminiscente

55

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L. Zhang, M. Wang, C. Wang, X. Hu, G. Wang. Labelfree

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C. Zong, J. Wu, C. Wang, H. Ju, F. Yan. Chemiluminescence

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Y. R. Kim, H. J. Seo, J. W. Oh, H. Lim, T. H. Kim, H. Kim.

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J. Zhang, Y. Sun, B. Xu, H. Zhang, Y. Gao, H. Zhang, D. Song. A

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75

CAPTULO 1

Herramientas analticas

HerramientasAnalticas

EneldesarrolloexperimentaldelapresenteTesisDoctoralsehan
empleadodiferentesherramientasanalticasparallevaracabolasdistintas
investigaciones realizadas. En este captulo de la Memoria de la Tesis
Doctoral se enumeran dichas herramientas y se profundiza en sus
caractersticasyaspectosmsrelevantesdesuutilizacin.

79

Captulo1

Materialesyreactivos
1 Materialesnanoestructurados
EnlasiguienteTablaserelacionanlosdiferentesnanomaterialesno
sintetizados en el laboratorio y necesarios para desarrollar los trabajos
experimentales descritos en esta Memoria, sus dimetros medios y la
fuentedesuministro.
TipodeNanomaterial
NaYF4:Yb(III),Er(III)

Tamao

Distribuidor

1020nm

DepartamentodeQumicade
losMaterialesyAnlisis
Qumico.UniversidaddeTurku
(Finlandia)

NaYF4:Yb(III),Tm(III)

1020nm

DepartamentodeQumicade
losMaterialesyAnlisis
Qumico.UniversidaddeTurku
(Finlandia)

NaYF4:Yb(III),Ho(III)

1020nm

DepartamentodeQumicade
losMaterialesyAnlisis
Qumico.UniversidaddeTurku
(Finlandia)

Tb4O7

<100nm

Aldrich

Eu2O3

<150nm

Aldrich

Diamante

<10nm

Aldrich

10nm

Aldrich

Ag

80

HerramientasAnalticas

2 Precursoresdeslice
En la siguiente Tabla se relacionan los diferentes precursores de
slice utilizados para la sntesis de nanopartculas de slice y el
recubrimientodelosupconvertingphosphors,ascomolafuncionalizacin
delasuperficiedeambosmateriales.
Precursordeslice
Tetraetoxisilano(TEOS)
(3Aminopropil)trietoxisilano

Funcin

Distribuidor

Estructuradeslice

Aldrich

Introduccingrupos

Aldrich

(APS)

amino

3(Trihidroxisilil)propil

Introduccingrupos

metilfosfonato(THPMP)

fosfonato

(N(3(trimetoxisilil)propil)etilen

Introduccingrupos

diamino

Aldrich

Aldrich

amino

3 Fluorforos
En la siguiente Tabla se relacionan los diferentes fluorforos, as
como sus respectivas longitudes de onda de excitacin y emisin,
utilizadosenlasdiferentesinvestigacionespresentadasenestaMemoria.

81

Captulo1

excitacin

emisin

(nm)

(nm)

Violetadecresilo

585

620

Sigma

Azulnilo

620

680

Sigma

Fluorescenasdica

485

520

Sigma

cido8(hidroxipireno1,3,6

455

510

SigmaAldrich

AzureA

632

645

Sigma

AzureB

648

662

Sigma

2[4(Dimetilamino)styryl]1

465

505

Aldrich

Styryl7

565

620

SigmaAldrich

Azuldetoluidina

620

638

Sigma

ficoeritrina(BPE)

550

575

Cyanotech

Fluorforo

Distribuidor

trisulfnico(HPTS)

metilpiridinioyoduro
(2Di1ASP)

Corp
Rficoeritrina(RPE)

565

575

Cyanotech
Corp

AlexaFluor488

495

520

Molecular
Probes
Invitrogen
Paysley

AlexaFluor546

556

570

Molecular
Probes
Invitrogen
Paysley

AlexaFluor680

680

702

Molecular
Probes
Invitrogen
Paysley

ATTO495

495

82

525

SpaBioSpa

HerramientasAnalticas

4 Tensoactivos
Se han utilizado los siguientes tensoactivos neutros, aninicos y
catinicos:TritnX100(Sigma),Tween20(Sigma),dodecilsulfatosdico
(SDS) (SigmaAldrich) y bromuro de hexadeciltrimetilamonio (CTAB)
(Fluka).
5 Disolventesorgnicos
Se han utilizado diferentes disolventes orgnicos como medio de
reaccin,eliminacindereactantesypreparacindedisolucionesdeetanol
(Panreac), acetona (Panreac), ciclohexano (Panreac), 1hexanol (Merck),
metanol (Panreac), dimetilformamida (SigmaAldrich) y acetonitrilo
(Panreac).
6 Anticuerpos
Sehanutilizadovariostiposdeanticuerposparallevaracabolos
diferentes formatos de inmunoensayo de esta tesis: Anticuerpos
policlonales antiprotena de soja producidos en conejo (Sigma),
anticuerpos IgG anticonejo producidos en cabra (SigmaAldrich),
anticuerpos policlonales antimonensn producidos en oveja (Abcam) y
anticuerposIgGantiovejaproducidosenconejo(SigmaAldrich).
7 Estndares
EnlasiguienteTablaserelacionanloscompuestosqumicosutilizados
para preparar estndaresde los analitos y de lospotenciales interferentes
utilizados en las diferentes investigaciones experimentales recogidas en

83

Captulo1

esta Tesis Doctoral, su pureza y las casas comerciales que los han
suministrado.
Analito

Pureza

Distribuidor

Protenadesoja

90%

Doscadesa

9095%

Sigma

97%

SigmaAldrich

cidocafeico

98%

Sigma

cidomlico

99,5%

Merck

cidoglico

97,5102,5%

Sigma

99,8%

SigmaAldrich

cidoascrbico

99%

SigmaAldrich

Sacarosa

99%

Fluka

cidoctrico

99,5%

Sigma

Sulfitosdico

98%

Merck

Biotina

99%

SigmaAldrich

Monensn
Trolox

Glucosa

8 Otrosreactivos
Para llevar a cabo las investigaciones realizadas a lo largo de esta
Memoriasehanusadodisolucionesreguladoraspreparadasapartirdesus
correspondientes sales, cidos y bases, as como otros reactivos los cuales
se relacionan a continuacin junto con la casa comercial donde fueron
adquiridos: Hidrxido amnico (Panreac), albmina de suero bovino
(SigmaAldrich), borohidruro sdico (SigmaAldrich), acetato sdico
(SigmaAldrich), cido sulfrico (Panreac), additol (Surface Specialities),
carbonato sdico (SigmaAldrich), tetraborato sdico (Panreac), anhdrido

84

HerramientasAnalticas

glutrico (Sigma), monohidrgeno fosfato potsico (Merck), vainillina

(Aldrich), metaperyodato sdico (Scharlau), cido clorhdrico (Merck), N
(3dimetilaminopropil)Netilcarbodiimida

(EDAC)

(SigmaAldrich),

diclorhidrato2,2azobis(2metilpropionamida)(AAPH)(Aldrich),piridina
(Sigma),

sulfoNhidroxisuccinimida

(sulfoNHS)

(SigmaAldrich),

glutaraldehido (Sigma), disolucin DELFIA (Perkin Elmer life and

Analytical Sciences, enzima laccasa (Sigma), nitrato de terbio (Aldrich),
tampnensayoparareaccionesdebioafinidadydisolucindelavadopara
placas deensayos de bioafinidad (Innotrac diagnostic Oy), cloruro sdico
(SigmaAldrich)yreactivodeFolinCiocalteau(SigmaAldrich).

Instrumentacin

En el desarrollo experimental de esta Memoria se ha utilizado la

siguienteinstrumentacin:

Lector de microplacas 1420 Multilabel Counter Victor 3V (Perkin

ElmerandAnalyticalSciences,WallacOy,Turku,Finlandia).

Espectrofluormetro SLM Aminco modelo 8100 (Urbana, IL, USA)

equipado con una lmpara de arco de xenn de 450 W y un tubo
fotomultiplicadorR928.

Plate Chameleon (Hidex Oy, Turku) equipped with a 200mW

infrared laser module (Roithner Lasertechnik) and RG850 nm
longpass excitation filter (Andover Corp., Salem, NH). The
photomultiplier tube in the plate fluorometer was replaced with
HamamatsuR4632(HamamatsuPhotonicsK.K.).

85

Captulo1

Microscopio electrnico de transmisin Philips CM10, resolucin

0,5nmx0,34nm,yequipadoconunacmaradigitalmegaviewIII.
Rejillas de cobre (200CFC) recubiertas con unas pelcula de
carbono Formvar 200 mesh, suministradas por Aname (Madrid,
Espaa).

Espectrofotmetro Lambda 35 UV/VIS (Perkin Elmer, Reino

Unido).

Programasinformticos

Se emplearon diferentes programas informticos para el clculo

estadsticoyelaboracindelasdiferentesrepresentacionesgrficas:Origin
7,0(ORIGINLAB),Statgraphics5.1,MicrosoftExcelySigmaPlot7.0.

86

CAPTULO 2
CHAPTER 2

Sntesis y caracterizacin de
nanopartculas de slice con
fluorescencia a larga longitud de
onda y su aplicacin al anlisis de
alimentos
Synthesis and characterization of
longwavelength fluorescent silica
nanoparticles and their application
to food analysis

Sntesisdenanopartculasdesliceysuaplicacin

Estecaptulorecogelasinvestigacionesrealizadasparadesarrollar
nuevosmtodosdeanlisisutilizandoconjuntamentelasprestacionesque
ofrecen la nanotecnologa y la fluorimetra de larga longitud de onda. Se
describelasntesisycaracterizacindenanopartculasdeslice(SiO2NPs)
dopadasconoxazinasysedemuestrasuutilidadenanlisisdealimentos
mediante inmunoensayo heterogneo. Los estudios realizados han dado
lugaralossiguientesartculos:
-

Synthesis

and

characterization

of

oxazinedoped

silica

nanoparticlesfortheirpotentialuseasstablefluorescentreagents.
J.GodoyNavajas,M.P.AguilarCaballos,A.GmezHens,Journal
ofFluorescence,20(2010)171180.

Heterogeneous immunoassay for soy protein determination using

nile bluedoped silica nanoparticles as labels and frontsurface
longwavelength fluorimetry. J. GodoyNavajas, M.P. Aguilar
Caballos,A.GmezHens.AnalyticaChimicaActa,701(2011)194
199.

Determinationofmonensininmilksamplesbyfrontsurfacelong
wavelength fluoroimmunoassay using nile bluedoped silica
nanoparticles as labels. J. GodoyNavajas, M. P. AguilarCaballos,
A.GmezHens,Talanta,94(2012)195200.

Cabe indicar que las investigaciones desarrolladas han tenido dos

objetivos: 1) Estudiar la potencial versatilidad de estas NPs con

89

Captulo2

fluorescencia a larga longitud de onda para aplicarlas a la determinacin

de macromolculas y molculas pequeas; y 2) Demostrar que los
marcadores basados en SiO2NPs dopadas con oxacinas pueden constituir
una alternativa til frente a los marcadores enzimticos. Para alcanzar
estos objetivos se han seleccionado las protenas de soja y el antibitico
monensin debido a que un porcentaje elevado de los mtodos descritos
para la determinacin de estos analitos en alimentos utiliza tcnicas de
inmunoensayo, principalmente enzimoinmunoensayo [1,2]. Aunque
posteriormente se discutirn los resultados obtenidos, se ha demostrado
que las nuevas NPs pueden utilizarse satisfactoriamente para determinar
ambos tipos de analitos y que los correspondientes inmunoensayos
desarrolladospresentanmenoreslmitesdedeteccinquelosbasadosenel
usodemarcadoresenzimticos,siendotambinmenorladuracindelos
ensayos.
Otro aspecto a destacar es la contribucin de las investigaciones
realizadas a expandir el uso de las SiO2NPs dopadas con fluorforos en
anlisis de alimentos, poniendo de manifiesto la utilidad del efecto
protector de las NPs en las propiedades fluorescentes del reactivo
encapsulado. Como se ha indicado en la Introduccin general de esta
Memoria, dentro de los escasos antecedentes descritos, cabe citar el uso
reciente de SiO2NPs dopadas con un quelato de rutenio para la
determinacin de residuos del antibitico enrofloxacina en carne de pollo
utilizando

inmunocromatografa

en

nitrocelulosa[3].

90

membranas

desechables

de

Sntesisdenanopartculasdesliceysuaplicacin

BIBLIOGRAFA
(1)

D.L. Brandon, M. Friedman. Immunoassays of soy proteins. J.

Agric.FoodChem.(2002)50,66356642.

(2)

A.C. Huet, M. BienenmannPloum, U. Vincent, P. Delahaut.

Screening methods and recent developments in the detection of
anticoccidials.Anal.Bioanal.Chem.(2013)405,77337751.

(3)

X. Huang, Z. P. Aguilar, H. Li, W. Lai, H. Wei, H. Xu, Y. Xiong.

Fluorescent Ru(phen)32+doped silica nanoparticlesbased ICTS
sensorforquantitativedetectionofenrofloxacinresiduesinchicken
meat.Anal.Chem.(2013)85,51205128.

91

Chapter2

Thischaptercontainstheinvestigationsperformedtodevelopnew
analyticalmethodsbycombiningthebenefitsofnanotechnologyandlong
wavelength fluorometry. The synthesis and characterization of silica
nanoparticles (SiO2NPs) doped with oxazine dyes is described and their
usefulness in food analysis by the development of heterogeneous
immunoassays is shown. These investigations have given rise to the
followingarticles:
-

Synthesis

and

characterization

of

oxazinedoped

silica

nanoparticlesfortheirpotentialuseasstablefluorescentreagents.
J.GodoyNavajas,M.P.AguilarCaballos,A.GmezHens,Journal
ofFluorescence,20(2010)171180.

Heterogeneous immunoassay for soy protein determination using

nile bluedoped silica nanoparticles as labels and frontsurface
longwavelength fluorimetry. J. GodoyNavajas, M.P. Aguilar
Caballos,A.GmezHens.AnalyticaChimicaActa,701(2011)194
199.

Determinationofmonensininmilksamplesbyfrontsurfacelong
wavelength fluoroimmunoassay using nile bluedoped silica
nanoparticles as labels. J. GodoyNavajas, M. P. AguilarCaballos,
A.GmezHens,Talanta,94(2012)195200.

Theinvestigationsperformedhadtwomainobjectives:1)Tostudy
the potential versatility of the synthesized longwavelength fluorescent

92

Synthesisofsilicananoparticlesandtheirapplication

NPs to be applied to the determination of macromolecules and low

molecular weight compounds; and 2) To demonstrate that the tracers
based on oxazinedoped SiO2NPs can be a useful alternative to enzyme
tracers. To achieve these goals, soy proteins and the antibiotic monensin
havebeenselectedasanalytesowingtothefactthatahighpercentageof
analytical methods reported for their determination are based on
immunoassays, and more specifically, on enzyme immunoassays [1,2].
Although the results obtained will be discussed below, it is worth to
mention that the new NPs can be satisfactorily used to determine both
typesofanalytesandtheimmunoassaysdevelopedfeaturelowerdetection
limits and shorter analysis times than those based on the use of enzyme
tracers.
Another salient aspect of these investigations is their contribution
to expand the use of SiO2NPs doped with fluorophores in food analysis,
where the protecting role of NPs on the fluorescent properties of the
encapsulatedreagenthasbeenshown.Asithasbeenalreadymentionedin
the Introduction of this Dissertation, there are a scarce number of
applicationsoftheseNPsinfoodanalysis.Arecentexampleistheuseof
SiO2NPs doped with a ruthenium chelate to determine enrofloxacin
antibiotic residues in chicken muscle using immunochromatography on
disposablenitrocellulosemembranes[3].

93

Chapter2

LITERATURE
(1)

D.L. Brandon, M. Friedman. Immunoassays of soy proteins. J.

Agric.FoodChem.(2002)50,66356642.

(2)

A.C. Huet, M. BienenmannPloum, U. Vincent, P. Delahaut.

Screening methods and recent developments in the detection of
anticoccidials.Anal.Bioanal.Chem.(2013)405,77337751.

(3)

X. Huang, Z. P. Aguilar, H. Li, W. Lai, H. Wei, H. Xu, Y. Xiong.

Fluorescent Ru(phen)32+doped silica nanoparticlesbased ICTS
sensorforquantitativedetectionofenrofloxacinresiduesinchicken
meat.Anal.Chem.(2013)85,51205128.

94

Sntesisdenanopartculasdesliceysuaplicacin

JournalofFluorescence(2010)20:171180

Synthesisandcharacterizationofoxazinedopedsilica
nanoparticlesfortheirpotentialuseasstablefluorescent
reagents
J.GodoyNavajas,M.P.AguilarCaballos,A.GmezHens
DepartmentofAnalyticalChemistry.UniversityofCrdoba.CampusofRabanales.
MarieCurieAnnexbuilding.14071Crdoba
Abstract
The synthesis process to obtain silica nanoparticles (NPs) dopedwith
two oxazine dyes, nile blue and cresyl violet, has been investigated using a
modification of the reverse micelle microemulsion method and a procedure
based on the Stber method. A micellar medium provided by the nonionic
surfactant Triton X100 in a hexanol:water mixture and an ethanol:water
mixture, have been used to provide the synthesis medium in each case.
Tetraethoxysilane has been used as the initiator of the polymerization and
condensationreactionsafteritshydrolysisinbasicmediumusingammonium
hydroxide. Dyesilane precursor NPs have been also synthesized in order to
compare their potential advantages against the NPs obtained by the direct
encapsulationoftheoxazinedyes.
SizedistributionandfluorescenceofthesynthesizedNPs,whichwere
monitored using Transmision Electron Microscopy (TEM) and a microplate
reader,respectively,dependonthemolarratioandtotalconcentrationofthe
reagents involved in the synthesis. NPs obtained using the developed
synthesis procedures had sizes below 400 nm in most instances and the best
luminescentpropertieswereobservedforNPswithsizesrangingfrom100to
300nm.Lowersizesresultinadecreaseinthefluorescenceintensitiesofthese
nanomaterials.ParametersrelatedwiththeluminescencefeaturesoftheseNPs
were calculated in order to compare the feasibility of both synthesis
approaches.Therepeatabilityofthereversemicellemicroemulsionprocedure
performedindifferentdaysgavearelativestandarddeviationof10%forthe
fluorescenceintensityvalues.

Keywords: Dyedoped silica nanoparticles; cresyl violet; nile blue; longwavelength

fluorescence

95

Captulo2

1. Introduction
Theuseofnanomaterialsishavingahugeimpactinthebioanalysis
field [19], being their composition and physicochemical properties the
mainfactorstochoosethedetectionsystembestsuitedforeachassay.The
useofsilicaNPsaslabelsisinterestinginbioassayswithopticaldetection
because of the transparency of silica to visible light. Other desirable
properties are: 1) silica polymerization chemistry is well known, 2) silica
material is not microbiologically degraded, and 3) porosity or swelling
changesofsilicaNPsdonothappenatmoderatepHvariations.However,
the porosity of these materials can originate drawbacks in their
performanceaslabelsduetolossesoftheencapsulatedsubstancesasitwill
bediscussedbelow.
Differentsilicaprecursors,suchastetramethoxysilane(TMOS)and
tetraethoxysilane (TEOS) are commonly used to synthesize silica NPs.
These precursors undergo hydrolysis and polycondensation reactions,
which result in the formation of monodisperse spherical silica particles
[10].TherearetwogeneralroutestosynthesizesilicaNPs,namelyreverse
micelle microemulsion and Stber methods. The reversemicelle
microemulsion method relies on the formation of a waterinoil
microemulsionformedbyasmallamountofwater,anorganicsolventand
asurfactant.Waternanodropletsinsidethereversemicellesactasreactors
inwhichthegrowthofsilicaNPstakesplace.TheStbermethodinvolves
the hydrolysis of the silica precursor in an alcohol/water mixture and the
silicic acid formed nucleates and condenses to give rise to spherical
monodisperseNPs.Thechoiceofeverysynthesisapproachdependsonthe
physicochemical properties of the species to be encapsulated [2, 4, 6]. In

96

Sntesisdenanopartculasdesliceysuaplicacin

general, hydrophobic and hydrophilic dyes can be encapsulated by using

theStbermethodwhereasthereversemicellemicroemulsionmethodhas
beenmainlyusedforthesynthesisofmetalchelatedopedsilicaNPs[2,4].
Anadvantageofthereversemicellemicroemulsionmethodisthatusually
providesNPswithnarrowersizedistributionsthanthoseobtainedbythe
Stbermethod2.
Acommonissuetobothsynthesisapproachesistheprobabilityof
dye leakage, especially for organic dyes. This phenomenon can be
minimized or avoided by coupling the organic fluorophor to hydrophilic
specieswithhighmolecularweight,suchasdextrans[11]orproteins[12],
for two reasons: 1) the resulting derivative is hydrophilic as the silica
matrix, and 2) the large size of the macromolecule can prevent the
diffusion through silica matrix pores. Another option is the synthesis of
precursors by covalent linking of the dye or a derivative, usually
containing carboxyl [13], sulfonyl chloride [14] and isothiocyanate [15]
groups,toasilaneprecursor,previouslytothesynthesisofsilicaNPs.
Metal chelates and organic dyes have been traditionally used as
labels in bioassays with luminescent detection [7]. Conventional fluoro
phores,suchasfluoresceinisothiocyanate,fluorescamineorumbelliferone
derivatives,providetheachievementofrelativelylowdetectionlimits,but
these compounds suffer from photobleaching processes due to the
relatively high energy of their incident excitation wavelengths. However,
this drawback can be minimized by embedding these dyes into a silica
matrix,whichprotectsthemfromenvironmentalfactorsthatwouldaffect
theirfluorescence[2,6].Anotherlimitationofconventionalfluorophoresis
that their emission can be overlapped by static background fluorescence

97

Captulo2

signalsfromsamplematrix,whichusuallyhappensatshortwavelengths.
Analternativeistheuseoflongwavelengthfluorophores,suchasorganic
dyes (cyanines, oxazines, alexa dyes) and lanthanide and ruthenium
chelates[1618].
The work presented here encompasses different approaches to
synthesize longwavelength emitting silica NPs doped with cresyl violet
acetate and nile blue chloride for the first time. Two different solgel
methods, based on the modification of the reversemicelle microemulsion
and Stber approaches and on the use of the silane precursor TEOS and
ammonium hydroxide, have been developed for the direct encapsulation
of the fluorophore. The fluorescence of the NPs obtained by the first
methodhasbeenimprovedintheabsenceofcyclohexane,whichisusedin
the conventional procedure. The reversemicelle microemulsion method
has been also applied to the encapsulation of a newly synthesized silane
precursorfromnilebluechlorideusingglutaraldehydeand3aminopropyl
triethoxysilane (APS). The influence of the different reagents on the
featuresoftheNPsformedinbothsynthesisprocedureshasbeenstudied
andoptimizedbymeasuringthefluorescenceintensityusingamicroplate
reader and the NP size by Transmission Electron Microscopy (TEM). The
luminescent features of the NPs synthesized by both methods have been
comparedinordertoselectthesynthesisproceduremoresuitabletoobtain
nanomaterialsusefulasstablefluorescentreagentsforanalyticalpurposes,
such as labels in fluoroimmunoassays. Some properties of these long
wavelength emitting NPs, such as photodegradability, chemical stability
anddyeleakagearealsodiscussed.

98

Sntesisdenanopartculasdesliceysuaplicacin

2.Experimental
2.1.Instrumentation
A 1420 Multilabel counter Victor 3V microplate reader (Perkin
Elmer and Analytical Sciences, Wallac Oy, Turku, Finland) was used to
perform

fluorescence

measurements.

Different

filters

(nominal

wavelength/passband) were used to select the excitation (531/25 nm and

590/7nmforcresylviolet,620/8nmfornileblue)andemission(620/8nm
for cresyl violet, 680/10 nm for nile blue) wavelengths of the doped NPs.
An SLMAminco (Urbana, IL, USA), Model 8100 photoncounting
spectrofluorimeter, equipped with a 450 W xenon arc source and a R928
photomultiplier tube, was used to perform fluorescence excitation and
emission scans of pure dyes and synthesized NPs, as well as
photobleaching experiments. Size characterization was performed by
Transmision Electron Microscopy (TEM), using a CM10 Philips
Microscope.Coppergrids(200CFC)coatedwithaFormvarcarbonfilm
200 mesh supplied by Aname (Madrid, Spain) were used as support in
TEMexperiments.

2.2.Reagents
All

reagents

were

of

analytical

grade.

3Aminopropyl

triethoxysilane (APS) was obtained from Aldrich (Aldrich, Milwaukee,

USA), and tetraethoxysilane (TEOS) and Triton X100 from Fluka (Buch,
Switzerland). Cresyl violet acetate, nile blue chloride and 25%
glutaraldehyde aqueous solution (Grade II) were supplied by Sigma (St
Louis,MO,USA)andabsoluteethanol,acetoneandammoniumhydroxide
(25% as NH3) by Panreac (Castellar del Valls, Spain). Cyclohexane was

99

Captulo2

supplied by Probus (Badalona, Espaa) and 1hexanol by Merck

(Schuchardt, Germany). Cresyl violet and nile blue solutions (1 x 103 M)
were prepared by dissolving the appropriate amount of each dye in the
minimum amount of methanol and then, raised up with distilled water
untilafinalconcentrationofmethanolof10%,andstirringthemixturefor
24h.

2.3.Procedures
2.3.1. Synthesis of cresyl violet and nile bluedoped silica NPs by a modified
reversemicellemicroemulsionmethod
Thesynthesiswasperformedaccordingtothefollowingprocedure:
an amount of Triton X100 (510 530 mg or 0.79 0.82 mmol) was
dissolvedin9.6ml(0.53mol)ofdistilledwaterbystirringvigorouslythis
mixture for 5 min. Then, a volume of 100 l (0.44 mmol) of TEOS was
addedandthesolutionwasstirredfor5min.Avolume(1.8ml)of103M
(1.8 mol) cresyl violet or nile blue was added and the mixture stirred
againfor5min.Afterwards,3ml(0.024mol)ofhexanolwereaddedand
the microemulsion formed was stirred for 15 min. Concentrated
ammoniumhydroxide(70l,0.9mmol)wasthenaddedandthemixture
stirredfor5mintostarttheTEOShydrolysisandcondensationreactions.
Themixturewasthenplacedinathermostatedtankat35Cfor7.5hinthe
dark.
The reaction mixture of each fluorophoredoped NPs, which was
composedbytwophasesclearlydifferenciated,wascentrifugedfor5min
at 2.000 rpm to complete the separation of two phases. The upper phase,
which was strongly colored, was extracted and 5 7 ml of acetone were

100

Sntesisdenanopartculasdesliceysuaplicacin

added to break the microemulsion formed, and the precipitate was

separated by centrifugation for 20 min at 3000 rpm. Then, NPs were
washed with ethanol using ultrasound sonication for 30 s to desorb the
fluorophore from the NP surface and then centrifuged for 5 min at 10000
rpm to get rid of unreacted precursors and the excess of surfactant and
fluorophore.Thewashingprocesswasrepeatedseveraltimeswithethanol
anddistilledwateruntilthefluorescencesignalofthesupernatantwasthe
sameastheblanksignal.NPswerefinallyredispersedin1mlofdistilled
water.

2.3.2.SynthesisofcresylvioletdopedNPsusingaStbermethodbasedprocedure

Toavolumeof25mlofethanol(0.43mol)wereadded500lof103

M(0.5mol)cresylviolet.Then,1ml(4.4mmol)ofTEOSand1.5ml(19.5
mmol) of concentrated ammonium hydroxide solution were added. The
mixturewasstirredfor1houratroomtemperature.Afterthistime,itwas
centrifuged at 3.000 rpm for 10 minutes. The NPs synthesized were
purified by washing them with ethanol and water for several times as
mentionedaboveforthereversemicellemicroemulsionmethod.

2.3.3.SynthesisofNPsusingnileblueAPSprecursorasfluorophore

A volume (12 ml) of 8.9x 104 M (10 mol) nile blue solutionwas

mixedwith100lof1%glutaraldehyde(10mol)aqueoussolutionand,
immediately after, 20 l (85 mol) of APS were added and the mixture
stirred for 5 min before its use in the reversemicelle microemulsion
method above described. A volume (1.2 ml) of the reaction mixture,

101

Captulo2

withoutanyadditionalpurificationstep,wasaddedtothemixtureinstead
oftheunchangedfluorophore.

2.3.4.CharacterizationofcresylvioletandnilebluedopedsilicaNPs

Synthesized NPs were characterized by TEM and fluorescence

intensitymeasurements.TEMexperimentswerecarriedoutbyspotting10
ldropsofNPsuspensionsinethanolontothecoppergrids,whichwere
placedaboveafilterpaperandlettodryatroomtemperatureforseveral
minutes.Fluorescencemeasurementswereobtainedbydispensingaliquots
of 200 l of NP suspensions onto microwells in triplicate and using the
filters above described to choose the adequate excitation and emission
wavelengths.

2.3.5.FluorescencestabilityofoxazinedopedsilicaNPs
SilicaNPsweredividedinfive1mlaliquotsinEppendorftubes.
All of them were washed several times with ethanol and water until the
supernatantspresentedafluorescenceintensitycorrespondingtotheblank
signaland,then,NPswerestoredat4C.Atthetimeoftheperformanceof
the fluorescence measurements, each aliquot was sonicated for 30 s and
thencentrifugedat10.000rpmfor5mintoremovethesupernatant.Then,
theywereagainreconstitutedandredispersedinwater.Avolume(200l)
oftheNPsuspensionwasaddedtoamicrowellplateandthefluorescence
intensitywasmeasuredintriplicate.

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Sntesisdenanopartculasdesliceysuaplicacin

2.3.6.PhotobleachingstudyofoxazinedopedsilicaNPs
FreeorganicdyesolutionordyedopedNPdispersionwasplaced
in a quartz cuvette and the fluorescence intensity was measured at the
corresponding maximum excitation and emission wavelengths by
continuosly irradiating the cuvette with light from the 450 W xenon arc
lamp of the photon counting spectrofluorimeter. Fluorescence intensity
measurements were monitored at room temperature for 1 h, with an
integrationtimeof1.0min.

3.ResultsandDiscussion
3.1.SynthesisofcresylvioletandnilebluedopedsilicaNPs

Both synthesis approaches were optimized by using mainly the

univariate method although, in some instances, the influence of variable

ratiosofthereagentswasalsostudied.

3.1.1Optimizationofreversemicellemicroemulsionmethod
As indicated above, the formation ofmicroemulsions involves the
useofasurfactant,anaqueoussolutionandanorganicsolvent.Depending
on the relative amounts of the components, waterinoil microemulsions
(richinorganicsolvent)oroilinwatermicroemulsions(richinwater)can
be formed. In the first case, a cosurfactant, generally a medium or long
hydrocarbon chain alcohol, is often incorporated in the micelles.
Cyclohexane, nhexanol, Triton X100 and water were chosen to develop
the reversemicelle microemulsion method, according to the procedures
previously reported [2, 4, 6, 1214]. These approaches involve continuous
mechanical stirring to originate stable waterinoil microemulsions.

103

Captulo2

However,theuseoftheexperimentalconditionspreviouslydescribeddid
not give rise to satisfactory results for the synthesis of the oxazinedoped
silica NPs. The study of the stirring conditions showed that, after mixing
the reagents in the addition order described in the procedure and then
leavingthemixturetostand,twophasesappeared:anupperphase(richin
organicsolvent)andalowerphase(richinwater).Theupperphase,which
wasstronglycoloured,wasastablewaterinoilmicroemulsioncontaining
thebrightestNPs.
Studiescarriedoutaboutthemicroemulsioncompositionrevealed
that NPs can be synthesized in the absence of cyclohexane, yielding NPs
with similar features (number, size) but with slightly higher fluorescence
intensity than that obtained using cyclohexane. Figure 1 shows TEM
images of the NPs obtained in the presence of different volumes of
cyclohexane. The water volume was also modified to keep constant the
totalvolume.Ascanbeseen,theamountandsizesofNPssynthesizedin
theabsenceofcyclohexane(Figure1a)werecomparabletothoseachieved
using7.5mlofcyclohexane(Figure1c).However,theuseofintermediate
volumes of cyclohexane (Figure 1b) provided larger NPs with wider size
distributions. According to these results, the use of cyclohexane was
precluded and only water, Triton X100 and 1hexanol were the main
componentsofthemicroemulsion.

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Sntesisdenanopartculasdesliceysuaplicacin

Figure 1. TEM images of nile bluedoped NPs synthesized using different

cyclohexanevolumes(a,bandc),differentTritonX100concentrations(d,e,f)
andthosefornileblueAPSdopedsilicaNPs(g).Experimentalconditions:100
l(0.44mmol)TEOS,0.2mlof1x103M(0.2mol)nileblue,3ml(0.024mol)
hexanol, 70 l (0.9 mmol) NH4OH. In (Figure 1ac): 510520 mg (0.790.82
mmol) Triton X100. In Fig. 1a11.3 ml(0.63 mol) water, 0 ml cyclohexane,in
Figure1b,7.5ml(0.42mol)water,3.75ml(0.034mol)cyclohexane,inFigure
1c3.8ml(0.21mol)water,7.5ml(0.068mol)cyclohexane.InFigure1df11.3
ml(0.63mol)ofwater,inFigure1d0mg,inFigure1e310.9mg(0.48mmol),in
Figure 1f 510.9 (0.79 mmol) of Triton X100. Scale bar: 1 m. In Figure 1g
Experimentalconditions:510520mg(0.790.81mmol)ofTritonX100,10.3ml
(0.57 mol) of water, 100 l (0.44 mmol) TEOS, 1.2 ml precursor reaction
mixture,3ml(0.024mol)hexanol,70l(0.9mmol)ofNH4OH.Scalebar:2m.

105

Captulo2

The phases obtained were separated by centrifugation of the

mixtureat2.000rpmfor5min.Aprecipitateappearedinthebottonofthe
lower phase at reagent concentrations higher than the optimum values,
whichindicatedthesaturationoftheupperphase.TheanalysisbyTEMof
theresuspendedsolidrevealedthatitwasmainlyconstitutedbylargesize
particles.
The distribution of the reagents in both phases is related with the
total volume of solution, the volume of hexanol and the amount of
surfactantused. The reductionof thewater volume from 11.5ml to 5 ml,
keeping constant the amount of the other components, resulted in the no
appearance of NPs after breaking the microemulsion, which would be
ascribedtothechangeoftheeffectivereagentconcentrations.
Theinfluenceofwateramountwasstudiedbykeepingconstantthe
total mixture volume, and hence, the final concentrations of the synthesis
components. Water volume was decreased from 12.5 ml to 7.3 ml,
increasing hexanol volumes from 1.8 ml to 7 ml. The number of NPs
decreased and their sizes were higher than 200 nm when the hexanol
volumewashigherthan3ml,choosingthisvalueasoptimum.
TheinfluenceoftheamountofTritonX100(Figure2)showedthat
the number of nile bluedoped NPs increases and their size decreases as
Triton X100 amount increases. The best results were obtained using 510
520 mg of Triton X100 (Figure 1f), because lower amounts gave NPs of
sizes higher than 200 nm (Figure 1d and e). Very small and partially
aggregated nanoparticles were obtained when the Triton X100 amount
wascloseto1.0g.ItwasalsocheckedthattheTritonX100/hexanolratiois
a critical variable, as Figure 2 shows. As it can be seen, a ratio of 170 mg

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Sntesisdenanopartculasdesliceysuaplicacin

Triton X100/ml hexanol gave the maximum fluorescence intensity of the

synthesizedmaterial.Theamountofthesurfactantusedisrelativelyhigh,
but is in agreement with the fact that the critical microemulsion
concentration of a nonionic surfactant in waterinoil microemulsions is
higher[10]thanthecriticalmicelleconcentrationofthesamesurfactantin
water.

RELATIVE FLUORESCENCE INTENSITY

500000

400000

300000

200000

100000

0
0

100

200

300

400

mg Triton X-100/ml hexanol ratio

Figure2.InfluenceofTritonX100/hexanolratioonthefluorescenceintensity
ofnilebluedopedNPs.Experimentalconditions:11.3ml(0.63mol)ofwater,
100l(0.44mmol)TEOS,0.2mlof1x103M(0.2mol)nileblue,3ml(0.024
mol)hexanol,70l(0.9mmol)NH4OH.

The fluorescence intensity and the size of the NPs were evaluated
modifying the temperature and the reaction time. Figure 3 shows the
influenceofthesevariablesinthefluorescenceintensityofnilebluedoped
NPs, finding that the fluorescence increased from 25 to 35 C, but it
decreasedat45C.Althoughthefluorescenceintensitywashigherafter9h

107

Captulo2

ofreaction,thesizeoftheNPsobtainedalsoincreasedso,atimeof7.5h
was chosen as optimum. The NP sizes slightly decreased with increasing
temperaturesintherangeof2545C.Thisstudywasalsoperformedfor
cresylvioletdopedNPsfindingsimilarresults.

RELATIVE FLUORESCENCE INTENSITY

700000
35 C

600000
500000

45 C

400000
25 C
300000
200000
100000
0
2

10

Time, h

Figure 3. Influence of reaction time and temperature on the fluorescence

intensity of nile bluedoped NPs. Experimental conditions: 510520 mg (0.79
0.81 mmol) of Triton X100, 11.3 ml (0.63 mol) of water, 100 l (0.44 mmol)
TEOS,0.2mlof1x103M(0.2mol)nileblue,3ml(0.024mol)hexanol,70l
(0.9mmol)NH4OH.

ThefeaturesofnilebluedopedNPsobtainedafterareactiontime
of7.5handat35CarecomparedinTable1withthefeaturesofsimilar
NPs obtained after 24 h at 25 C. Some parameters were calculated in a
similar way as described elsewhere [19] using 200 l of 0.1 mg/ml NP
dispersionsineachwell.ThenumberofNPswasobtainedbydividingthe
amountofNPsintotheweightofoneparticle,whichwascalculatedfrom

108

Sntesisdenanopartculasdesliceysuaplicacin

the volume of one particle (assuming that it is a perfect sphere and

expressingthevolumeinmm3)andmultiplyingthisvolumebythespecific
gravityofsilica(2.3).
The intensity/particle ratio is defined as the fluorescence intensity
divided by particle amount, and the specific intensity is the
intensity/particleratiodividedbythevolumeofoneparticle.Anincrease
inthesynthesistimeyieldslargerNPs,whicharemoreluminescent,since
thespecificintensity,whichisindependentontheparticlevolume,isabout
1.6timeshigherthanthatofNPsobtainedat7.5hand35C.

Table 1. Comparison of the properties of nile bluedoped silica nanoparticles

obtainedattworeactiontimeandtemperaturevaluesbythemodifiedreverse
micellemicroemulsionmethod

Conditions

24h,25C

7.5h,35C

Concentration

0.1mg/ml

0.1mg/ml

Amount(mg)

0.02

0.02

Meandiameter,nm

323

170

Particlevolume,mm3

1.76x1011

2.57x1012

Particlenumber

4.92x108

3.38x109

Intensitya

180927

111708

Intensity/particleratio

3.67x104

3.3x105

Specificintensity

2.09x107

1.29x107

Measurements performed in microplates (200 l) using excitation and

emissionfiltersof620/8and680/10nm,respectively

109

Captulo2

Theinfluenceofammoniumhydroxidewasstudiedintherangeof
35 140 l of a commercial concentrated solution (0.45 1.8 mmol). The
number of nile blueNPs formed was scarce at the limits of the assayed
interval, being also the fluorescence intensity obtained quite low (Figure
4a).ThesizeoftheNPsobtainedincreasedintherangeof70610nmof
diameter as NH4OH was increased. The decrease in the fluorescence
intensity at high NH4OH values would be ascribed to the partial
degradation of the fluorofore in the alkaline medium, as it has been
reportedelsewhereforacyaninedye[20].Thevolumechosenasoptimum
was70l(0.9mmol).
TheinfluenceofTEOSwasevaluatedfornileblueNPsintherange
of50800l(0.223.52mmol).IncreasingTEOSvolumes,thesizeofNPs
increasedfrom140to300nm,obtainingsizeslargerthan200nmabove100
l.Theseresultswereobtainedundertheoptimalexperimentalconditions,
exceptingforthefluorophoreamount,whichwastentimeslowerthanthe
optimum value. As it was found that the fluorescence intensity also
increased with the TEOS amount, a compromised solution was adopted
and 100 l (0.44 mmol) of TEOS was chosen as the optimum value to
obtainintermediatesizesandintensities.
Theamountoffluorophoreusedisacriticalvariable.Thevolume
of1x103Maqueousnilebluesolutionwasvariedfrom100lto7.2mlby
adjusting the total volume of the aqueous phase to 11.5 ml with distilled
water, in order to keep constant the initial concentrations of the other
reagents. The influence of this variable on the fluorescence intensity is
shown in Figure 4b, in which can be seen that the maximum value was
obtained using 1.8 mol of nile blue. The decrease in the fluorescence

110

Sntesisdenanopartculasdesliceysuaplicacin

intensity at higher values is ascribed to the appearance of a coloured

precipitate in the tube bottom after the centrifugation process, which was
mainly formed by large size particles. The formation of fluorophore
aggregatescanalsocontributetothisbehaviourastheyarelessfluorescent
thantheirmonomers[6].

500000

RELATIVE FLUORESCENCE INTENSITY

RELATIVE FLUORESCENCE INTENSITY

A)
400000

300000

200000

100000

0.0

0.4

0.8

1.2

1.6

2.0

mmol NH4OH

2400000

B)
2000000

1600000

1200000

800000

400000

0
0

mol nile blue

Figure4.Influenceoftheamountofammoniumhydroxide(A)andnileblue
(B)addedonthefluorescenceintensityofnilebluedopedNPs.Experimental
conditions:510520mg(0.790.81mmol)ofTritonX100,11.3ml(0.63mol)of
water,100l(0.44mmol)TEOS,3ml(0.024mol)hexanol.InA)0.2mlof1x
103M(0.2mol)nileblue,inB)70l(0.9mmol)ofNH4OH.

The fluorophore concentration also affects the size of the NPs,

which decreases until the amount of nile blue is 0.43 mol, from which
their diameter remains constant, with a mean value of about 170 nm. A
similarbehaviourwasobservedforcresylvioletdopedNPs.
The synthesis of precursors involving the covalent linking of the
primary amino groups of the fluorophore and the silane derivative APS
was assayed to obtain NPs less susceptible to dye leakage. It was found

111

Captulo2

thattheuseofequimolarratiosofnileblueandglutaraldehyde,andAPS
in an 8.5fold molar excess, gave the best results in terms of fluorescence
intensity of the NPs obtained. The time of reaction of the mixture was
studiedintheintervalof010min,and5minwerefoundtobeenough,
sincelargerirregularparticleswereobtainedatlongerreactiontimes.This
mixture was used for the NP synthesis without any further purification
step. The influence in the fluorescence intensity of the precursor volume
addedtothesynthesisprocedurewasstudiedintherangeof0.62.4ml,
providing1.2mlofprecursorthehighestvalue.ThemeansizeoftheNPs
synthesizedbyusing1.2mlofprecursormixturewas150nm,decreasing
thesizeathighervolumes.Figure1gshowstheaspectoftheNPsobtained
using 1.2 ml of precursor, which are not completely spherical, although
theyhaveauniformsizewitharelativestandarddeviationaround10%.

3.1.2.OptimizationofthesynthesisbytheStbermethod

The approach used was similar to previously described methods

for the encapsulation of a fluoresceinsilane precursor and a ruthenium

chelate[19,21],whichencompassedtheuseofanethanol:watermixtureas
the medium for the hydrolysis and condensation of silane reagent. The
application of this method has been carried out using cresyl violet as
fluorophore, with the aim of comparing the features of the NPs obtained
withthoseoftheNPsobtainedwiththeabovedescribedmethod.
Ethanol volume was assayed in the range of 1025 ml (0.17 0.43
mol), obtaining the best fluorescence intensity values with the highest
ethanol amount. This would be ascribed to the increase in the effective

112

Sntesisdenanopartculasdesliceysuaplicacin

fluorophore concentration at low volumes of ethanol, which gives rise to

theformationofthenonfluorescentfluorophoredimersabovementioned.
The influence of water was studied in the range of 0.525 ml,
yieldingsizesbelow180nm,whichincreased(300350nm)whenthewater
volume was 510 ml. This behaviour could be explained bearing in mind
that the TEOS hydrolysis, which would be the limiting step at low water
volumes, should be favoured by an increase in water volumes leading to
fasterNPsynthesisandlargerNPsizes.Theuseof25mlofwaterprovided
aloweramountofNPs,owingtothedilutionofthereagentsinvolvedin
thesynthesis.
Theoptimizationofthefluorophoreamount,performedfrom0.5to
10mlof1x103M(0.510mol)cresylvioletatafixedwatervolumeof
0.5 ml, showed that the best results were obtained for 0.5 mol of
fluorophore.TheinfluenceofTEOSwasassayedintherangeof0.110ml
finding that NPs obtained at low TEOS volumes (0.1 2 ml) were
practically monodisperse whereas those synthesized at higher TEOS
volumes(510ml)werequiteirregularinshapeandinsizedistribution.
Table2showsthecomparisonofthecresylvioletdopedsilicaNPs
obtained by the modified reversemicelle microemulsion and the Stber
methods.Theexcitationfilterusedforcresylvioletwas531/25nm,which
is an excitation wavelength shorter than the maximum, in order to
minimizescatteringphenomena.ThespecificintensityoftheNPsobtained
by the modified microemulsion method was about 12 times higher than
that obtained by the Stber procedure. This would be ascribed to the fact
that cresyl violet is less degraded by the alkaline conditions owing to the
protectionthatmicellescanconfer.

113

Captulo2

Table2.ComparisonofthepropertiesofcresylvioletdopedNPsobtainedby
theproposedreversemicellemicroemulsionandStbermethods

Reversemicelle

microemulsionmethod

Stbermethod

Concentration,mg/ml

0.1

0.1

Particleamount,mg

0.02

0.02

Diameter,nm

178

133

2.94x109

7.06x109

125563

10138

Intensity/particleratio

4.27x105

1.44x106

Specificintensity

1.45x107

1.17x106

Particlenumber
Intensitya

Measurementswereperformedusingmicroplates(200l)andexcitationand
emissionfiltersof531/25and620/8nm,respectively

3.2.FluorescencespectraofcresylvioletandnilebluesilicaNPs
The spectral features of the synthesized NPs were compared to
those showed by pure dyes and by bare silica NPs. Figure 5 shows the
emission spectra obtained at the maximum excitation wavelength of each
fluorophore,inwhichcanbeseenthatthereisnotappreciablefluorescent
signalfrombaresilicaNPs.Theemissionbandsoftheoxazinedopedsilica
NPs show a slight shift of approximately 9 nm towards shorter
wavelengths.Thisbehaviourisinaccordancetotheresultsobtainedafter
encapsulating other fluorophores, such as fluorescein isothiocyanate or
ruthenium dyes [6], which experienced shifts towards shorter and longer
wavelengths,respectively.Thisfactcouldbeascribedtotheinteractionof
the dyes, which are cationic, with the negatively charged silanol groups
fromthesilicamatrix.Figure5alsoshowsthattheemissionspectrumfor

114

Sntesisdenanopartculasdesliceysuaplicacin

nile blueAPSdoped NPs is practically the same as that obtained for nile
bluedopedsilicaNPs.

800

A)

RELATIVE FLUORESCENCE INTENSITY

RELATIVE FLUORESCENCE INTENSITY

600
33

400

200
1
1

0
580

600

620

640

660

680

700

720

(nm)

2000
B)

22

1500

33
1000

500

11
0
640

660

680

700

720

(nm)

Figure 5. Emission spectra in distilled water of: bare silica NPs (A.1, B.1), 0.07 M
pure cresyl violet (A.2) and 0.5 M nile blue (B.2) dye solutions, and cresyl violet(A.3) and nile blue-doped (B.3) NPs, respectively.

3.3.FluorescencestabilityofNPs
ThesuspensionsofcresylvioletandnileblueNPsweredividedin
fivealiquotsof1mleach,whichwerestoredat4C.Attheassaytime,NPs
were washed, centrifuged and redispersed in water. The fluorescence
intensitywasmeasuredthesamedaythatNPsweresynthesizedand1,2,5
and9dayslater,usinginallinstancesthesamenumberofwashestoeach
aliquot (three times with 1 ml of ethanol and four times with distilled
water), until supernatant fluorescence close to blank signal. Under these
conditions,thefluorescenceintensityremainedalmostconstantforatleast
9dayswithoutappreciablechangesinthefluorescenceintensitycausedby
dyeleakage.ThefluorescenceintensityfromnileblueandnileblueAPS
dopedNPsdecreasedafterthesevenwashes,beingthedecreaseofthefirst

115

Captulo2

NPs two fold that of the second ones, which would be ascribed to the
covalentbindingofthefluorophoretothesilanereagent,whichminimizes
dyeleakage.
OncetheNPsweretotallypurified(supernatantfluorescenceclose
toblanksignal),theyweresubjectedtoadditionalsubsequentwasheswith
1mlofdistilledwatertostudythepotentialdyeleakagefromthecoreof
NPs, finding that the fluorescence of both nile blue and nile blueAPS
dopedNPsremainedalmostconstant.ThestabilityofnilebluedopedNPs
couldbeascribedtotheabovementionedfactthatnileblueisacationicdye
andwouldinteractwiththenegativesilanolgroupsfromsilicamatrix.In
thisway,dyeleakagewouldbelessfavouredthanforanionicdyes,suchas
Cy5,whicharemorepronetorepulsionforceswiththesilanolgroupsfrom
silicamatrix[12].

3.4.Photostabilityexperiments
ToinvestigatethepotentialphotobleachingofthesynthesizedNPs
in aqueous solution, they were irradiated for 1 h and the results were
comparedtothoseobtainedforpuredyesolutions(Figure6).Theintensity
ofpurecresylviolet(Figure6a,curve1)andnileblue(Figure6b,curve1)
dropped until the 72% and the 65%, respectively, of the initial intensity.
However, the fluorescence intensity of both cresyl violet NPs (Figure 6a,
curve 2) and nile bluedoped NPs (Figure 6b curve 2) remained almost
constant and the NPs prepared by using the nile blueAPS precursor
(Figure6b,curve3)experiencedadecreasebya10%oftheinitialintensity.
SimilarresultshavebeenreportedinrecentlysynthesizedNPscontaining

116

Sntesisdenanopartculasdesliceysuaplicacin

Cy5[12]andfluorescein[19],whichconfirmtheincreasedphotostabilityof
encapsulateddyesduetotheprotectionthatsilicamatrixconfersthem.

A)

1.0

0.8

0.6

0.4

1000

2000

NORMALIZED FLUORESCENC INTENSITY

NORMALIZED FLUORESCENCE INTENSITY

B)

0.8
1
0.6

0.4

3000

TIME (s)

1.0

1000

2000

3000

TIME (s)

Figure6.Photobleachingexperimentsforpurecresylviolet(A.1)andnileblue
(B.1)solutions,respectively;cresylviolet(A.2)andnilebluedoped(B.2)NPs,
respectively;andnileblueAPSdopedNPs(B.3).

4.Conclusions

Theworkpresentedherereportsthesynthesisofcresylvioletand

nile bluedoped silica NPs for the first time. The emission at long
wavelengths of these NPs is a useful option to avoid the potential
interferences of static background signals from sample matrix. The
systematic study of the experimental variables involved in the synthesis
process has given rise to the achievement of NPs with homogeneous size
and high and stable fluorescence intensity. The modified reverse
microemulsion method proposed precludes the use of cyclohexane as
organicsolvent,whichisreplacedby1hexanol.Theuseoftheoxazinedye
and the APS precursor to obtain the NPs has shown that the second one

117

Captulo2

reduces dye leakage in the purification process. The comparison of the

luminescent properties of cresyl violetdoped NPs synthesized by
microemulsion and Stber methods has shown the usefulness of the first
onetoobtainmoreluminescentNPs.
TheincreasedphotostabilityofthesynthesizedNPswithrespectto
the pure dye proves the succesful dye encapsulation and the protection
thatsilicamatrixconfersthem.ThefeaturesofthesynthesizedNPsseemto
make them suitable as analytical reagents in fluoroimmunoassays after
theirfunctionalization,whichiscurrentlyunderresearch.

Acknowledgments

Authors gratefully acknowledge the financial support from the

Spanish Ministerio de Educacin y Ciencia (Grant No. CTQ200603263)

and from the Autonomous Andalusian Government (Grant No. P06
FQM1356).TheyalsoacknowledgethetechnicalassistanceoftheElectron
Microscopy Section of the Central Service of Support to Research (SCAI)
fromtheUniversityofCordoba.

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M. Seydack. Nanoparticles labels in immunosensing using optical

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(12) X.He,J.Chen,K.Wang,D.Qin,W.Tan.Preparationofluminescent
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Heterogeneousimmunoassayforsoyproteindetermination
usingnilebluedopedsilicananoparticlesaslabelsandfront
surfacelongwavelengthfluorimetry
J.GodoyNavajas,M.P.AguilarCaballos,A.GmezHens
DepartmentofAnalyticalChemistry,InstituteofFineChemistryandNanochemistry
(IAQFN).CampusofRabanales.MarieCurieBuilding(Annex).Universityof
Cordoba.14071Cordoba.Spain.

Abstract
A longwavelength fluoroimmunoassay for the determination of soy
proteinisreportedforthefirsttimeusingaconjugatecomposedofantisoyprotein
antibodies bound to nileblue doped silica nanoparticles (NPs). These NPs have
been synthesized by a reversemicelle microemulsion method and functionalized
by using 3(aminopropyl)triethoxysilane (APS) and 3(trihydroxysilyl)propyl
methylphosphonate (THPMP) to avoid NP aggregation. The tracer has been
obtained by linking the functionalized NPs with antisoy protein antibodies
previouslyoxidisedwithsodiumperiodate.Theimmunoassayhasbeendeveloped
in 96well microplates using a heterogeneous competitive format with antibody
capture.Soyproteinsareimmobilisedontothewellsandbovineserumalbuminis
added to block the surface, thus minimising nonspecific binding. After washing,
the microplates can be stored ready to use. At the analysis time, soy protein
standards or sample and tracer are added and incubated and, after the
corresponding washing and drying steps, the fluorescence is measured onto the
solidsurfaceatex620andem680nm.Themethodfeaturesadynamicrangeof0.1
10 mg L1 and a detection limit of 0.05 mg L1. The precision of the method has
been assayed at 0.5 and 5 mg L1 protein concentrations, obtaining the values of
relativestandarddeviationof9.6%and6.1%,respectively.Thisnewimmunoassay
has been applied to the analysis of food containing soy protein and the results
obtainedhavebeencomparedtothoseprovidedbyacommercialELISAkitwith
no statistically differing results. Also, a recovery study has been performed,
providingpercentagesintherangeof81.5111.0%.

Keywords: soy protein, food samples, longwavelength fluorimetry, heterogeneous

immunoassay,nilebluedopedsilicananoparticles

123

Captulo2

1.Introduction
In recent years, the global consumption of soy foodstuffs has
increased because of their reported beneficial effects on nutrition and
health,suchasadecreaseoftheplasmacholesterol,preventionofcancer,
diabetesandobesity,andprotectionagainstbowelandkidneydisease[1].
Soybean proteins are used as additives in human foodstuffs, thus being
marketedasdifferentcommercialformulations,suchassoyflour,protein
concentrates and isolates. These formulations are used to obtain better
mechanical properties in soy milks, drinks, tofu and vegetarian meat
substitutes and a number of new food varieties is being developed.
However,soybeanscontainproteinsthatcanproduceallergicresponsesin
some people. The safe choice of processed foods free from soy proteins
may become more critical for these patients because these proteins and
their derivatives have been increasingly incorporated in a number of
processedfoods.Theallergenicityoftheseproteinslargelydependsonthe
foodprocessing,e.g.hightemperaturetreatmentsandconjugationtosome
organiccompounds,suchaspolysaccharides[2],amongothers.
Nativesoyproteinsareglobularproteins,mainlyconstitutedby7S
and 11S (glycinin and conglycinin) fractions [3], which have been
characterized

and

determined

by

liquid

chromatography

[4],

electrophoresis [5] and immunoblotting [6]. Also, a number of enzyme

linked immunosorbent assays (ELISA) methods have been described for
soy protein determination in different food samples [711], thus existing
somecommercialkitsdevelopedforthispurpose[11].Onlyfewalternative
approaches to the use of enzymes as labels have been reported for soy
proteindetermination.Acommercialfourchannelopticalbiosensorbased

124

Sntesisdenanopartculasdesliceysuaplicacin

on surface plasmon resonance (Biacore 3000) has been applied to the

determinationofseveralnonmilkproteins,includingsoyproteins,inmilk
products [12]. An advantage of this method is that proteins are directly
detected, avoiding the use of a label, but the relatively high cost of the
instrument hinders its generalized use. Also, a more complex
immunoassay has been described using an expensive flow analyzer
(Luminex),fluorescentmicrospheresandafluorophore(Alexa532)aslabel
oftheproteins[13].Theuseofnanoparticles(NPs)aslabelsforsoyprotein
determination has been previously described in a homogeneous
immunoassay in which the tracer, formed by gold NPssoy protein
antibodies,reactswiththeanalyteincreasingthelightscatteringintensity
ofthesolution[14].
The method presented here is a longwavelength fluorimetric
heterogeneous immunoassay using nile blue (NB)doped silica NPs as
label, which have been obtained by a relatively simple reversemicelle
microemulsion method [15]. This procedure has been slightly changed to
achieveNPsurfacemodificationwithaminogroupstobefurtherlinkedto
soy protein antibodies in order to develop the longwavelength
luminescent tracer. The use of silica NPs as labels in immunoassays with
optical detection has very interesting features: 1) transparency of silica to
visible light, 2) almost negligible photobleaching phenomena of
encapsulated organic dyes, 3) stability of silica matrix at mediumterm
sinceitisnotsusceptibletomicrobiologicaldegradation,and4)porosityor
swellingchangesdonothappenatmoderatepHvariations.Anadditional
advantage of the use of NPs as labels is the decreased number of steps
needed to perform this heterogeneous immunoassay compared to those

125

Captulo2

requiredforthedevelopmentofELISAassays,sincetheuseoftheenzyme
conjugateandsubstratesolutionsisavoided.Todate,alimitednumberof
immunoassays have been described which make use of biofunctionalized
dyedoped silica nanoparticles [16]. These assays mainly involve the
encapsulation of fluorescein and rhodamine derivatives, and ruthenium
and lanthanide chelates to obtain luminescent silica NPs. The results
obtained in these methods have shown that the sensitivity is greatly
increased when compared with the corresponding immunoassays
performedwithdirectfluorophorelabeling.
Tothebestofourknowledge,themethodreportedhereisthefirst
heterogeneous immunoassay described for soy protein determination in
food samples using NPs as labels. Two competitive formats involving
antibodyandantigencapturewereassayed,butonlythefirstone,inwhich
soy proteins were immobilised on the wells of a microplate, gave rise to
satisfactoryresults.
Themethodhasbeenappliedtotheanalysisoffruitandsoyjuice,
soy yoghourt and milk samples and the results obtained have been
compared to those provided by a commercial ELISA with no statistically
relevantdifferences.Also,thedetectionlimitusingthereportedmethodis
about10timeslowerthantheaffordedbythecommercialELISA.

2.Experimental
2.1.Instrumentation
A 1420 Multilabel counter Victor 3V microplate reader (Perkin
Elmer and Analytical Sciences, Wallac Oy, Turku, Finland) was used to
perform

fluorescence

measurements.

126

Two

filters

(nominal

Sntesisdenanopartculasdesliceysuaplicacin

wavelength/passband) were used to select the excitation (620/8 nm) and

emission (680/10 nm) wavelengths of the doped NPs. Fluorescence
mesurements were performed using the stabilized energy mode of the
instrument with an integration time of 1 s. NP size characterization was
performed by transmission electron microscopy (TEM), using a CM10
Philips Microscope. Copper grids (200CFC) coated with a Formvar
carbon film 200 mesh supplied by Aname (Madrid, Spain) were used as
support for TEM experiments. Both 300L well Optiplate and 100L
well black and shallow Proxyplate 96well microplates (Perkin Elmer)
were assayed as supports for the development of the heterogeneous
immunoassaymethod.

2.2.Reagents
All the reagents were of analytical grade and used as supplied by
the manufacturer. Tetraethyl orthosilicate (TEOS) and Triton X100 were
obtained

from

Fluka

(Germany).

Nile

Blue

chloride,

(trihydroxysilyl)propyl methylphosphonate monosodium salt, bovine

serum albumin (BSA), polyclonal antisoy protein antibodies (raised in
rabbits),sodiumborohydride,sodiumacetateandsodiumcarbonatewere
supplied by SigmaAldrich (USA), and (3aminopropyl)triethoxysilane
(APS) by Aldrich (Germany). 1Hexanol and dipotassium hydrogen
phosphate were acquired from Merck (Germany). Ammonium hydroxide
(25% in NH3), acetone and absolute ethanol were obtained from Panreac
(Spain), sodium metaperiodate from Scharlau (Spain) and soy protein
isolate,PROTEINASUPRO500E(90%proteincontent),waskindlygifted
by DOSCADESA (Spain). Phosphate (0.02 M, pH 7.5), acetate (0.1 M, pH

127

Captulo2

5.5 and pH 4.8), and carbonate (0.05 M, pH 9.5) buffer solutions were
preparedbydissolvingtheappropriateamountofthesesaltsandadjusting
the pH with either hydrochloric acid or sodium hydroxide when
appropriate. A commercial ELISA kit (ELISA SYSTEMS, Windsor,
Australia)forthedeterminationofsoyprotein11wasusedforcomparative
purposes.

2.3.Procedures
2.3.1.SynthesisofaminofunctionalizedNBdopedsilicaNPs
TheprocedureusedtosynthesizeNBdopedsilicaNPsissimilarto
a reversemicelled microemulsion method previously reported [15], but it
has been modified in the last step to provide the doped silica NP surface
withaminogroupstobelinkedtoantisoyproteinantibodies,inorderto
giverisetotheconjugateusedastracer.
Briefly,anamountofTritonX100(510530mgor0.790.82mmol)
wasdissolvedin9.6mL(0.53mol)ofdistilledwaterbystirringvigorously
thismixturefor5min.Then,avolumeof100L(0.44mmol)ofTEOSwas
addedandthesolutionwasstirredfor5min.Avolume(1.8mL)of103M
(1.8mol)NBsolutionwasaddedandthemixturestirredagainfor5min.
Afterwards, 3 mL (0.024 mol) of hexanol were added and the
microemulsion formed was stirred for 15 min. Concentrated ammonium
hydroxide(70L,0.9mmol)wasthenaddedandthemixturestirredfor5
mintostarttheTEOShydrolysisandcondensationreactions.Themixture
wasthenplacedinathermostatedtankat25Cfor24hinthedarkand,
afterwards,itwascentrifugedfor5minat2000rpmtoseparatethephases
involved.Theupperphase(about3mL)wastransferredtoa10mLbeaker

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Sntesisdenanopartculasdesliceysuaplicacin

and 40 L of APS and 80 L of THPMP were added. This mixture was

magneticallystirredduring1.5hinawaterbathtokeepthetemperatureat
25 C. Afterwards, the microemulsion was broken by adding 10 mL of
acetone and stirring for 5 min. The supernatant was discarded and NPs
were recovered from the bottom with 8 10 mL of distilled water. The
mixturewascentrifugedfor5minat10000rpmandtheNPprecipitatewas
washed with ethanol and water until the fluorescence intensity of
supernatants was similar to the blank. Finally, the NPs were redispersed
in1mLofphosphatebuffersolution.

2.3.2. Preparation of the NBdoped silica nanoparticles antisoy protein antibody

conjugate
A volume (50 L) of antisoy protein were diluted with 0.1 M
sodiumacetatesolution(pH5.5)untilavolumeof2mL,and200Lof0.1
Msodiummetaperiodatesolutionwereaddedtothisvolume.Themixture
was incubated for 20 min in the freezer at 0 C and the reaction volume
was layered on a HiTrap Desalting Column (GE Healthcare) equilibrated
with25mL0.1Msodiumacetate0.5Msodiumchloridesolution(pH4.8),
at a 5 mL min1 flow rate, and 300L fractions were taken. Their
absorbance was monitored at 280 nm and the antisoy protein was
recovered from the first seven fractions, accounting for about 2 mL of
solution. This volume was added to the aminomodified NBdoped silica
NPs, which had been previously centrifuged to remove phosphate buffer
solution. The mixture was incubated for 20 h at 4 C, in a similar way as
previouslydescribedelsewhere[17].Then,50Lof0.66MNaBH4solution
were added to the dispersion and allowed to react for 20 min at 4 C.

129

Captulo2

Afterwards, the reaction mixture was centrifuged and the NPantisoy

protein conjugate obtained was washed with phosphate buffer solution.
Finally, NPs were redispersed in 1 mL of the same phosphate buffer
solutionandstoredat4Cforfurtheruse.

2.3.3.Preparationofsoystandard
The procedure used involves a denaturationrenaturation process,
according to the procedure indicated by the antisoy protein antibody
supplier,asfollows:anamountofsoyproteinisolate(50mg)wasplacedin
a glass test tube and mixed with 3 g of urea, 1 mL of 0.001 M TRISHCl
buffersolutionofpH8.6,0.1mLof2mercaptoethanolandwater.Thetotal
volume of the reaction mixture did not exceed 5 mL. The test tube was
sealedwithacapandaluminumfoilandthetubewasplacedinaboiling
water bath for 2 h. After this time, the tube was cooled for 5 min in cold
waterandthereactionmixturewasdilutedto10mLwithdistilledwater.
This solution can be stored at 4 C in the fridge for a month. Working
standard solutions were daily prepared by diluting this stock solution in
phosphatebuffersolution.

2.3.4.Determinationofsoyprotein
A volume (80 L) of a 5 mg L1 soy protein solution prepared in
carbonatebuffersolutionwasaddedtoeachwell,andthemicroplatewas
incubated overnight at 4 C. Afterwards, wells were washed three times
withdistilledwaterandthen,80Lofa0.01%BSAsolutionpreparedin
phosphatebufferwereaddedtoeachwell.Themicroplatewasstirredfor1

130

Sntesisdenanopartculasdesliceysuaplicacin

h at room temperature, it was washed for three times with phosphate

buffersolutionandthen,itwasstoredat4Cuntiluse.
Toperformtheimmunoassay,avolume(60L)ofapreincubated
mixture, prepared by mixing 75 L of NBdoped silica NPslabeled anti
soyproteinantibody(20nM)inphosphatebuffersolution(0.02M,pH7.5),
and 225 L of soy protein standard or sample solution (0.110 g mL1)
wereaddedtoeachwell.Themicroplatewasincubatedfor1.5hat37C,
the wells were washed three times with the same phosphate buffer
solution,andtheplatewasdriedfor10minatroomtemperature.Then,the
measurementsweredirectlyperformedontothesolidsurfaceofthewell.

2.3.5.Determinationofsoyproteininfoodsamples

An amount (0.85 mL or g of soy milk and yoghourt, respectively,

and1.7goffruitandsoyjuice)offoodsamplewastreatedinthesameway
asdescribedaboveforsoyproteinstandards.
The sample extracts were diluted (1:2000) in phosphate buffer
solution and 225 L of this solution were analysed following the above
mentionedprocedureforsoyproteindetermination.

3.Resultsanddiscussion
3.1.

Choiceandoptimizationoftheimmunoassaysystem
The synthesis and optical properties of NBdoped silica NPs used

to obtain the tracer have been recently described [15]. These NPs feature
lowphotobleachingphenomenabecauseoftheprotectionthatsilicamatrix
confersthemandgoodstabilityowingtotheinteractionofthefluorophor
NBwithsilicamatrix.However,thesurfacefunctionalizationtointroduce

131

Captulo2

reactive groups to couple these NPs to either analytes or antibodies, is

described here for the first time. The NP surface was functionalized with
aminogroupsusingAPSasreagent,whichhasbeenpreviouslydescribed
for this purpose [18]. This process can lead to aggregation phenomena
because of the accumulation of positive charges at physiological pH onto
their surface. Thus, the aminemodified silica NPs can back bond to
residualsilanolgroups,asithasbeenreportedelsewhere[19],leadingtoa
verylowchargeinthesurface,whichoriginatesNPaggregation.Theuse
of a silane reagent containing phosphonate groups, such as THPMP, can
partially or totallyavoidthis effect since more electrostatic repulsionsare
createdamongNPs,whichcontributetotheirdispersion.Thus,attheend
of the synthesis of NPs once the phase separation was performed, the
upper layer, which is rich in NB and hexanol, was taken and different
mixturesofAPSandTHPMPwereassayed.Volumesrangingfrom10to40
LofAPSandfrom20to160LofTHPMP,involumeratiosintherange
of 1:1 to 1:16, were used. It was found by TEM that the addition of these
reagents in an 1:2 APS:THPMP volume ratio gave rise to nonaggregated
silica NPs. Several volumes at the 1:2 APS:THPMP ratio were assayed to
check out which volumes would give rise to a suitable amount of amino
groups, and the confirmation of their presence was followed by a
fluorescamine method [20]. The highest fluorescence intensity was
obtained using 40 L of APS and 80 L of THPMP, being these volumes
chosentoperformthefunctionalization.
Thetemperatureofthefunctionalizationwasassayedintherange
of037C.Temperaturesabove25CincreasethehydrolysisrateofAPS
and can lead to aggregation phenomena, thus selecting 25 C for the

132

Sntesisdenanopartculasdesliceysuaplicacin

development of the reaction. The reaction time was also studied from 30
minto2h,findingthat1.5hprovidedasilicashellincorporatingenough
amino groups to perform the further conjugation chemistry. Under these
conditions,theNBdopedsilicaNPsobtainedfeaturedameandiameterof
257nm,measuredbyTEM(Figure1).

Figure1.TEMimageoffunctionalizedNBdopedsilicaNPs.

These NPs were coupled to soy protein and to antisoy protein

antibodies to study the potential use of the corresponding tracers for the
development of two different heterogeneous competitive formats: with
antigen capture and with antibody capture. The first involved the
immobilisation of antisoy protein antibodies onto the wells, after the
previous immobilisation of antirabbit IgG antibodies, and the use of the
tracer composed of soy protein linked to NBdoped silica NPs. Although
the well surface was coated with BSA at different concentrations, there
were still nonspecific interactions of the tracer with antirabbit IgG

133

Captulo2

antibodies. To overcome this problem, the use of the antibody capture

format was assayed. Thus, soy proteins were immobilised first onto the
wells,andtheremainingsitesofthewellwereblockedwithBSA.Then,a
preincubated mixture of soy protein standards or sample together with a
conjugate antisoy proteinNBdoped silica NPs (tracer) was added,
competing both immobilised and in solution soy proteins for the active
sites of the conjugate. After washing, the fluorescence from bound tracer
fraction was measured. The performance of fluorescence measurements
onto the well surface was investigated assaying 100L proxyplate and
conventional 300L Optiplate microplates. The signal obtained using
proxyplates was slightly better, which could be ascribed to that well
bottomcanbemoreeasilyapproachedbythedetectionsystemusingthese
plates. Also, an additional advantage is the use of lower volumes, which
considerablyreducesimmunoassaycosts.
Thesynthesisofthetracerencompassedthepreviousoxidationof
carbohydrate moieties of antibody molecule to give rise to aldehyde
moieties, which react to NP surface amino groups giving rise to Schiff
basesthatcanbefurtherreducedusingsodiumborohydride[17].
After the oxidation of the antibody with sodium metaperiodate, it is
necessary to perform a purification step using commercial desalting
columnstoremovetheexcessofoxidantinordertoproceedwiththenext
step, taking 300L fractions. These fractions were selected by measuring
theirabsorbanceat280nm.OncetheantibodyandNPswerecoupled,the
reaction mixture was purified by subsequent washing and centrifugation
stepswithphosphatebuffersolution,beingfinallystoredinthisbuffer.

134

Sntesisdenanopartculasdesliceysuaplicacin

The optimisation of the system was carried out by modifying the

experimental conditions at different soy protein concentrations. The
concentration of immobilized soy protein onto the wells is an important
variable on the system, which influences the assay sensitivity, since
immobilisedandinsolutionsoyproteincompetefortheactivesitesofthe
tracer.Theinfluenceofthisvariablewasassayedintherangeof1to50mg
L1, finding that 5 mg L1 provided the best results as the dynamic range
obtained for the calibration curve was wider and the sensitivity obtained
wasalsobetterthanthoseobtainedathigherorlowerconcentrations.BSA
has been used to block the remaining free sites of plate surface after soy
protein immobilization. The influence of BSA concentration was checked
outintherangeof0.0010.1%,findingthat0.01%BSAwasenoughatthe
incubationconditionsassayed(1h,roomtemperature)(Figure2A).
BSA concentrations above 0.01% led to a sensitivity decrease,
whereas some nonspecific interactions with the well surface were
observed at lower BSA concentrations, since a higher background signal
wasobtainedforhighanalyteconcentrations.
The sensitivity of the assay also depends on the tracer
concentration,asFigure2Bshows,beingthebestresultsobtainedat20nM.
Althoughthesignalsobtainedatconcentrationsabove20nMwerehigher,
theassayswerelesssensitive,sincethedynamicrangewasshiftedtosoy
proteinconcentrationshigherthan0.1mgL1.

135

Captulo2

RELATIVE FLUORESCENCE INTENSITY

RELATIVE FLUORESCENCE INTENSITY

30000
16000
B)
A)
1
14000
25000
1
2
12000
2
20000
10000
3

15000
8000
3

4
6000

10000
5
4000
5000
2000
4
5

0
0
-0.02 0.00
0.02
0.04
0.06
0.08
0.10
0.12
5
10
15
20
25
30
35
40
45

[BSA], (w/v%)
[tracer], nM

Figure2.InfluenceofBSA(A)andtracer(B)concentrationsontheassayinthe
presenceofdifferentsoyproteinconcentrations(mgL1):1)0.0;2)0.1;3)1.0;4)
5.0; 5) 10.0. [tracer] = 20 nM in (A). [BSA] = 0.01% in (B). Other experimental
conditionsinboth(A)and(B)are:[immobilizedsoyprotein]=5mgL1,assay
incubationtime=1.5h,temperature=37C.

The study of incubation temperature influence showed that the

assaysensitivitywasbetterat37C.Theincubationtimewasinvestigated
from15minto3h,findingthat1.5hwasenoughtoachieveanadequate
signal. Although the signal increased slightly at longer times, the results
werelessreproducible,whichcouldbeascribedtoahigherprobabilityof
nonspecificinteractionswiththeplate.

3.2Analyticalfeatures
Fluorescenceintensitymeasurementswereperformedbyselecting
the most appropriate filters to fix the maximum excitation and emission
wavelengthsforthetracer,ex620andem680nm.Themethodpresenteda
dynamicrangeof0.110mgL1(Figure3).

136

Sntesisdenanopartculasdesliceysuaplicacin

16000

RELATIVE FLUORESCENCE INTENSITY

14000
12000
10000
8000
6000
4000
2000
0
0.001

0.01

0.1

10
-1

[soy protein], mg L

Fig.3.Calibrationcurveobtainedunderoptimumconditions.

The calibration curve was adjusted to a 4parameter sigmoidal

curveusingthesoftwareSigmaPlot2001,whichequationis:

y y0 (
1

a
( x x 0 )
e b

) ,

whereyisthefluorescenceintensityandxisthedecimallogarithmofsoy
protein concentration expressed in mg L1. The values of the regression
parametersa,b,x0andy0were(1.120.04)x104,0.170.02,0.130.02,
and (1.9 0.2) x 103, respectively. The value of the regression coefficient
was 0.9989. The detection limit, calculated according to IUPAC
recommendations [21], was 0.05 mg L1, which is about ten times lower
than that provided by the ELISA kit used as reference method [11]. The
precision, expressed as the percentage of relative standard deviation and
assayedattwodifferentsoyproteinconcentrations,0.5and5mgL1,gave
valuesof9.6%and6.1%,respectively.

137

Captulo2

The selectivity of the method mainly depends on antisoy protein

antibodiesselectivity.Accordingtotheantibodyspecificationsheet,these
antibodiespresentsomecrossreactivitytowardshotureaextractsofwheat
and ovalbumin, but no crossreactivity is observed in extracts from meat,
cornorcasein,andriceorpotatoflour.
Additionally, in a previous work that involved the use of the same
antisoyproteinantibodies[14],itwasfoundthatbovineserumalbumin,
globulins, myoglobin and hemoglobin were tolerated in excess higher
than80foldtheanalyteconcentration.

3.3 Applications
Themethodwasappliedtotheanalysisofmilk,yoghourtandfruit
juice samples containing soy proteins. The sample treatment includes the
same procedure used for soy protein standard preparation to obtain soy
protein with a suitable conformation to be recognized by the antibody.
Similarprocedureshavebeenusedforthetreatmentofsomefoodsamples
in immunoassays intended for soy protein determination [22]. These
sampleswerealsoanalysedusingacommercialELISAkit[11],thusbeing
extracted using a commercial extraction solution for samples containing
polyphenols,accordingtotheprotocolgivenbythemanufacturer.
Table 1 shows the soy protein content found by the reported
immunoassayandtheELISAmethod,whichwerecomparedbyapairedt
test carried out at a 95% significance level, finding that there were not
statisticallyrelevantdifferencesbetweenthem.

138

Sntesisdenanopartculasdesliceysuaplicacin

Table1.Determinationofsoyproteincontentinfoodsamples

Sample

Reportedmethoda,b

CommercialELISAa,b

746

843

8.40.7

10.50.3

644

603

Soymilk
Fruitandsoyjuice
Soyyoghourt

MeanSD(n=3)
Units:fruitandsoyjuice,soyyoghourtareexpressedingkg1,soymilkunits
aregL1

A recovery study was also carried out to validate the method

(Table 2). It was performed by adding three different amounts of soy
proteintoeachsampleandsubtractingtheresultsobtainedfromsimilarly
treated non spiked samples. The values obtained ranged from 81.5 and
111.0%.

Table2.Recoveryofsoyproteinaddedtofoodsamples

Addeda,b

Founda,b

Recovery(%)

Soymilk

24
72
123

272
694
11010

111.0
95.7
89.4

Fruitandsoyjuice

2.7
8.1
13.5

2.20.2
7.70.6
121

81.5
95.1
88.9

Sample

Soyyoghourt
23
212
91.3

65
614
93.2

113
957
84.1
aMeanSD(n=3)
bUnits:fruitandsoyjuice,soyyoghourtareexpressedingkg1,soymilkunits
aregL1

139

Captulo2

4. Conclusions
The reported method can be regarded as a useful alternative to
enzymelinkedimmunosorbentassays,sincetheuseofenzymeconjugate
and substrate solutions are avoided, thus decreasing the number of steps
required. In addition, the detection limit achieved with the reported
method is lower than that provided by some ELISA assays described for
thispurpose[11].Theuseofthe96wellformatensurestheautomationof
the assay together with a relativelyhigh sample throughputcompared to
othermethods,whichincludesequentialoperations.Proxyplatesallowthe
use of lower sample and tracer volumes than conventional 300L wells,
whichcontributetominimizethecostsassociatedtotheimmunoassay.
The results obtained for the analysis of real food samples using
the reported method are in accordance with those provided by an ELISA
methodusedasareference,whichindicatesthepracticalusefulnessofthe
developedmethod.

Acknowledgements
Authors gratefully acknowledge financial support from the
MICINN(GrantNo.CTQ200908621),fromtheJuntaofAndalucia(Grant
No. P09FQM4933) and from the FEDERFSE Program (Grant No. P09
FQM4933). J. Godoy Navajas thanks the Junta of Andalucia (Grant No.
P09FQM4933)forthefinancialsupportofhispredoctoralfellowship.

140

Sntesisdenanopartculasdesliceysuaplicacin

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Determinationofmonensininmilksamplesbyfrontsurface
longwavelengthfluoroimmunoassayusingnilebluedoped
silicananoparticlesaslabels
JuanGodoyNavajas,MariaPazAguilarCaballos,AgustinaGmezHens
DepartmentofAnalyticalChemistry.InstituteofFineChemistryandNanochemistry
(IQFN).CampusofRabanales.AnnextoMarieCurie(C3)Building.Universityof
Cordoba.14071Cordoba.Spain.
Abstract
A heterogeneous immunoassay for monensin determination in milk
samplesusingatracerformedbyantimonensinantibodiesboundtonileblue
(NB)doped silica nanoparticles (NPs), 96well microplates as solid supports
and longwavelength fluorescence measurements is described for the first
time. The assay relies on the competition of the monensin present in the
samples with a monensinbovine serum albumin conjugate, which was
immobilized onto the well surface, for the active sites of antimonensin
antibodies.Aftersubsequentincubationandwashingsteps,thefluorescenceof
the bound tracer fraction is measured onto the dry surface of the well. An
antigen capture format was also assayed by immobilizing antisheep IgG
previously to the incubation of sheep antimonensin antibodies and using a
tracer formed by monensin bound to nile bluedoped silica NPs, which
competeswiththeanalyteforbindingtheimmobilizedantibody.Althoughthe
fluorescence signal obtained in both formats can be correlated to the analyte
concentration,betterresultswereobtainedusingtheantibodycaptureformat.
Aftertheoptimizationofthesystemusingthisformat,themethodfeaturesa
detectionlimitof0.015gL1andadynamicrangefrom0.05to5gL1.The
precision,assayedattwodifferentanalyteconcentrations,0.2and1gL1,and
expressed as relative standard deviation, gave values of 5.9% and 4.0%,
respectively. The method was satisfactorily applied to the analysis of milk
samples,whichonlyrequiredasimpleextractionstepinordertoremovethe
proteinsfromsamples,givingrecoveriesintherange83.3107.5%.

Keywords: monensin; milk samples; longwavelength frontsurface fluorescence;

immunoassay;nilebluedopedsilicananoparticles

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1.Introduction

Monensin is a veterinary drug polyether ionophore produced by

Streptomycescinnamonensisthatexhibitsbothantibacterialandanticoccidial
activitiesandhasbeentraditionallyusedtopreventcoccidiosisinpoultry
[1]. This antibiotic has been considered as a growthpromoter, since this
disease prevents the growth of poultry owing tothe bloody diarrheaand
weightlosesassociated.Althoughtheuseofmanyveterinaryantibioticsas
growth promoters has been banned since 2006, they can be still
administeredtosomespeciesaccordingtotheEURegulation1831/2003/EC
[2]. Also, the treatment with monensin has been extended to calves for
whichtheEUhassetmaximumresiduelimits(MRL)of2gkg1inbovine
muscle, kidney and milk, 10 g kg1 in fat and 30 g kg1 in liver, the
residues being determined in all the samples as unchanged monensin, as
statedinthe37/2010/ECregulation[3].
The concern on food safety has led to the search for reliable
analytical methods to screen and confirm the presence of antibiotic
residues in foodstuffs to reduce the increasing bacterial resistance to
antibiotics.Thedeterminationofpolyetherionophoreantibioticshasbeen
traditionally performed using liquid chromatographic methods with UV
and fluorometric detection after their derivatization [4, 5]. This step is
required owing to the lack of suitable chromophore and/or fluorophore
groupsintheirstructure,exceptforlasalocid,whichhasbeendetermined
bymeasuringitsintrinsicfluorescence[4].
Nowadays, several LCMS/MS methods have been developed for
the determination of cocciodiostats [6 10] and for multiclass residue
analysis including the ionophore class [11 13]. These confirmatory

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Sntesisdenanopartculasdesliceysuaplicacin

methodsrequiretheavailabilityofasophisticatedandexpensivetechnique
and, sometimes, are timeconsuming owing to the extraction and further
cleanup procedures involved. For this reason, the use of screening
methods plays two essential roles: in one hand, the number of samples
subjectedtoconfirmatoryanalysisislowerand,ontheother,thecostofthe
analysis is reduced. Immunoassays, whenever used in a quantitative or
semiquantitativeway,haveshowntheirusefulnessasscreeningmethods
for antimicrobial residues in foodstuffs [14 21]. Several commercial and
noncommercial enzymelinked immunosorbent assays (ELISAs) have
been described for the determination of monensin in biological [14 18]
andenvironmental[19]samples.Mostofthesemethodsinvolvetheuseof
photometric measurements, reaching detection limits close to 1 ng mL1,
but a lower detection limit (0.06 ng mL1) has been reported using
chemiluminescence

detection

[18].

Also,

timeresolved

fluoroimmunoassaysinvolvingtheuseofaneuropium(III)chelateaslabel
havebeendescribedformonensindetermination[20,21],butthedetection
limit reached is very similar to those obtained using ELISA with
photometricdetection.
The use of functionalized inorganic matrix nanoparticles (NPs) as
alternative labels in immunoassay is a relatively new trend justified by
theirversatilephysicochemicalpropertieswhichallowtheimprovementof
thefeaturesoftheseassays[2225].AmongtheseNPs,dopedsilicaNPs
are a useful option owing to their chemical and thermal stability, fine
dispersion in aqueous solution, transparency to visible light, capability to
encapsulate a wide variety of compounds and relatively inert
environmental behavior, in addition to their large surface area and easy

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surface functionalization [25]. These properties have given rise to a great

varietyofimmunoassaymethods,whichhavebeenmainlydevotedtothe
determinationofmacromolecules,suchastumormarkers[23,24].
Theimmunoassayreportedhereshowstheusefulnessofnileblue
(NB)dopedsilicaNPsaslabelsforthedeterminationofmonensininmilk
samples using longwavelength fluorescence measurements, which
provide the spectral selectivity required to avoid interferences from the
samplematrix.ThesynthesisandopticalpropertiesoftheseNPshavebeen
recentlyreported[26].AlthoughalimitationofdyedopedsilicaNPsisthe
potential dye leakage, owing to the porosity of silica material, NBdoped
silicaNPsshowanexcellentstability,whichisascribedtotheinteractionof
thecationicdyewiththenegativesilanolgroupsfromsilicamatrix.These
NPs have been used as labels in a heterogeneous immunoassay for soy
protein determination in several food samples [27], reaching a detection
limit about 10 times lower than the afforded by a commercial ELISA kit
availableforthedeterminationoftheseproteins.
Tothebestofourknowledge,themethodproposedhereisthefirst
immunoassay for monensin determination using NPs as labels, which
shows their capability to improve the analytical features, such as the
detection limit, of the immunoassays previously described for the
determinationofthisdrug[1418,20,21].Althoughmostoftheseassays
areappliedtotheanalysisofplasmaandliversamples,milksampleshave
been chosen in this instance becausethey are easily obtainedandallow a
noninvasive detection of monensin for its safe use in milk producing
animals. The sample treatment is quite simple as it only requires a
deproteinizationstepandasuitabledilution.

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Sntesisdenanopartculasdesliceysuaplicacin

2.Experimental
2.1.Instrumentation
A 1420 Multilabel counter Victor 3V microplate reader (Perkin
Elmer and Analytical Sciences, WallacOy, Turku, Finland) was used to
perform

fluorescence

measurements.

Two

filters

(nominal

wavelength/passband) were used to select the excitation (620/8 nm) and

emission (680/10 nm) wavelengths of the doped NPs. An additional filter
(531/25 nm) was used to perform absorbance measurements using the
Komarowsky reaction in order to calculate monensin concentrations.
Fluorescence measurements were performed using the constant voltage
mode of the instrument with an integration time of 1 s. NP size
characterization was performed by TEM as described elsewhere [26, 27].
Black and shallow 100L well proxyplate 96well microplates (Perkin
Elmer)wereassayedassupportforthedevelopmentoftheheterogeneous
immunoassaymethod.

2.2.Reagentsandsolutions
Allthereagentswereofanalyticalgradeandwereusedassupplied
by the manufacturer. Triton X100, NB chloride, sodium acetate, sodium
chloride, sodium borohydride, monensin sodium salt, anhydrous
Ndimethylformamide (DMF), Nhydroxysulfosuccinimide sodium salt
(sulfoNHS), N(3dimethylaminopropyl)Nethylcarbodiimide hydro
chloride(EDAC),bovineserumalbumin(BSA)andsodiumcarbonatewere
obtained from SigmaAldrich (USA). Tetraethyl orthosilicate (TEOS), (3
aminopropyl)triethoxysilane

(APS),

3(trihydroxysilyl)propylmethyl

phosphonate monosodium salt (THPMP) and vanillin were supplied by

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Aldrich (Germany). 1Hexanol and dipotassium hydrogen phosphate

wereacquiredfromMerck(Germany).Acetone,absoluteethanol,sodium
tetraborate, sulphuric acid and methanol were obtained from Panreac
(Spain),sodiummetaperiodatefromScharlau(Spain)andsheeppolyclonal
antibodytomonensinwassuppliedbyAbcam(UnitedKingdom).
NB solution was prepared in distilled water according to the
aforementionedprocedures[26,27].A1.0gL1stocksolutionofmonensin
was prepared by dissolving the appropriate amount of monensin sodium
salt in ethanol. Intermediate and working monensin solutions were
prepared by further dilution in phosphate buffer solution (0.015 M, pH
8.0).Phosphate(0.015M,pH8.0),acetate(0.1M,pH5.5andpH4.5),and
carbonate (0.05 M, pH 9.0) buffer solutions were prepared by dissolving
the appropriate amount of these salts and adjusting the pH with either
hydrochloric acid or sodium hydroxide when appropriate. The
Komarowsky reagent solution [5,28]was prepared by dissolving vanillin
(4g)inmethanol,adding2mLofconcentratedsulphuricacidtoadjustthe
pH and raising the mixture to 100 mL with methanol. This solution was
freshlypreparedwheneverused.

2.3.Procedures
2.3.1.SynthesisandfunctionalizationofNPs
TheprocedureusedtosynthesizeNBdopedsilicaNPsissimilarto
a reversemicelle microemulsion method previously reported [26, 27].
Briefly:anamountofTritonX100(510530mgor0.790.82mmol)was
dissolvedin9.6mL(0.53mol)ofdistilledwaterbystirringvigorouslythis
mixture for 5 min. Then, a volume of 100 L (0.44 mmol) of TEOS was

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Sntesisdenanopartculasdesliceysuaplicacin

addedandthesolutionwasstirredfor5min.Avolume(1.8mL)of103M
(1.8 mol) NB was added and the mixture stirred again for 5 min.
Afterwards, 3 mL (0.024 mol) of hexanol were added and the
microemulsion formed was stirred for 15 min. Concentrated ammonium
hydroxide(70L,0.9mmol)wasthenaddedandthemixturestirredfor5
mintostarttheTEOShydrolysisandcondensationreactions.Themixture
wasthenplacedinathermostatedtankat25Cfor24hinthedarkand,
afterwards, it was centrifuged for 5min at 537 x g to separate the phases
involved.Theupperphase(about3mL)wastransferredtoa10mLbeaker
and 40 L of APS and 80 L of THPMP were added to introduce amino
groupsontoNPsurface.Thismixturewasmagneticallystirredduring1.5
hinawaterbathat25C.Afterwards,themicroemulsionwasbrokenby
adding 10 mL of acetone and stirring for 5 min. The supernatant was
discarded and NPs were recovered from the bottom with 8 10 mL of
distilled water. The mixture was centrifuged for 5 min at 9300 x g to
separate the NPs from unreacted reagents. Then, the NP precipitate was
washed with ethanol and water until the fluorescence intensity of
supernatants was similar to the blank and, finally, the NPs were re
dispersedin1mLofphosphatebuffersolution.

2.3.2.PreparationoftracersusingnilebluedopedsilicaNPs

NBdoped silica NPs were coupled to either antimonensin

antibodiesormonensintoobtainthetracersrequiredtostudythepotential
determination of this drug using antibody or antigen capture format,
respectively. The synthesis of the NPsantimonensin antibodies tracer
involves an oxidation step of carbohydrate moieties of the antibody

151

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molecule to give rise to aldehyde groups capable of reacting to amino

groupsoftheNPsurface.Firstly,200Lof1.2Mantimonensinantibody
solution prepared in 0.1 M phosphate buffer solution at pH 7.5, were
dilutedwith0.1Msodiumacetatebuffersolution(pH5.5)untilavolume
of2mL,and200Lof0.1Msodiummetaperiodatesolutionwereadded
tothisvolume.Themixturewaslettostandfor20minat0C,layeredona
HiTrapDesaltingColumn(GEHealthcare)equilibratedwith25mLacetate
buffer solution (pH 4.5), at a 5 mL/min flowrate, and 400L fractions
were taken. Their absorbance was monitored at 280 nm and the anti
monensin protein was recovered from fractions 7 to 11, accounting for
about2mLofsolution.ThisvolumewasaddedtotheaminomodifiedNP
doped silica NPs, which had been previously centrifuged to remove
phosphate buffer solution. The mixture was incubated for 5 min at room
temperatureandthen,50Lof0.66MNaBH4solutionwereaddedtothe
dispersion and allowed to react for 20 h at 4 C. Afterwards, the reaction
mixture was centrifuged and the NPantimonensin protein conjugate
obtained was washed with phosphate buffer solution (0.015 M, pH 8.0).
Finally,thetracerwasredispersedin1mLofthesamephosphatemolar
concentrationsolutionandstoredat4Cforfurtheruse.

The synthesis of the NPsmonensin tracer was performed using a

carbodiimide reaction in which 200 l of a 2 g L1 monensin solution in

DMF were mixed with the same volume of a 0.2 M EDAC solution
prepared in 0.05 M 2(Nmorpholino)ethanesulphonic acid (MES) at pH
6.2and21.6mgofsulfoNHSwerethenadded,beingthemixtureraisedto
2 mL with the same MES buffer solution and incubated at room
temperaturefor15min.Then,40Lof1M2mercaptoethanolwereadded

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Sntesisdenanopartculasdesliceysuaplicacin

and the mixture was left to stand for 10 min. An appropriate volume (50
L)ofthismixturewasmixedtogetherwiththeaminofunctionalizedNB
doped silica NPs and 950 L of phosphate saline buffer were added and
theresultingmixturewasincubatedfor2hatroomtemperature.Afterthis
time, the synthesized tracer was washed using absolute ethanol and
phosphate buffer solution, reconstituted in 1 mL of the same phosphate
molarconcentrationsolutionandstoredat4Cuntiluse.

2.3.3.SynthesisofBSAmonensinconjugate

TheuseofaBSAmonensinconjugatewasrequiredtoimmobilize

monensinontothewells.ThecouplingofmonensintoBSAmoleculewas
performedviaacarbodiimidereaction[29]usingthefollowingprocedure:
an amount of monensin (0.2 mmol) was dissolved in 1 mL of dry DMF,
thensulfoNHS(23.0mg,0.1mmol)andEDAC(41.2mg,0.2mmol)were
added in order to activate the carboxylic acid group of monensin. The
mixturewasstirredatroomtemperaturefor4h,andthenwascentrifuged
to remove the precipitate from the acylisourea derivative formed. An
aliquot(250L)oftheactivatedhaptensolutionwasaddeddropwisetoa
stirredBSAsolutionpreparedbydissolvingBSA(50mg)in5mLof0.05M
boratebuffer(pH7.8)andDMF(1.05mL).Thesynthesizedconjugatewas
purified using a HiTrap Desalting column (GE Healthcare) by a similar
procedure to that described above for the synthesis of NPanti monensin
conjugate.

The concentration of monensin in the conjugate was determined

using the Komarowsky reaction [5, 28] by reacting 500 L aliquots of

monensin standard or BSAmonensin conjugate dilutions prepared in

153

Captulo2

methanolto1mLofKomarowskyreagentinglasstesttubes.Thesetubes
weresealedandplacedinawaterbathat80Cfor5min.Theredpurple
color indicative of monensin presence was measured at 520 nm
immediately after cooling the tubes to room temperature. The BSA
concentration in the synthesized conjugate was calculated from the
absorbance of the protein measured at 280 nm. As a convention, the
concentration of the conjugate will be given in terms of the monensin
concentrationfoundbyusingtheabovementionedKomarowskyreaction.

2.3.4.Determinationofmonensin

The competitive heterogeneous immunoassay with antibody

capture involves the previous immobilization of the BSAmonensin

conjugateinthewell,whichwasperformedbyadding60Lofa10mgL1
BSAmonensin conjugate prepared in carbonate buffer solution (0.05 M,
pH 9.0) to each well, and incubating the microplates overnight at 4 C.
Afterwards,wellswerewashedthreetimeswithphosphatesolution(0.015
M,pH8.0)andtheplateswerestoredat4Cuntiluse.

Toperformtheimmunoassay,avolume(60L)ofapreincubated

mixture, prepared by mixing 75 L of NBdoped silica NPslabeled anti

monensin antibody (10 nM) in phosphate molar concentration solution
(0.015 M, pH 8.0), and 225 L of monensin antibiotic standard or sample
solution (0.05 5 g L1) were added to each well. The microplate was
incubatedfor1.5hat37C,thewellswerewashedforthreetimeswiththe
samephosphatebuffersolution,anddriedfor10minatroomtemperature.
Then, fluorescence measurements were directly performed at ex 620 and
em680nmontothesolidsurfaceofthewell.

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Sntesisdenanopartculasdesliceysuaplicacin

2.3.5.Analysisofmilksamples

The sample preparation involved the deproteinization of food

samples using a slight modification of a previously described procedure

[21]:0.4gofmilksamplewasacidifiedwith0.1mLof0.1Mhydrochloric
acidandmixedwith1.6mLofpureacetonitrileinordertoquantitatively
precipitateallproteins.Then,themixturewasvigorouslymixedduring30
min and centrifuged (2722 x g, 10 min, 10 C). The supernatant was
collected in an Eppendorf tube and was evaporated to dryness under a
gentlestreamofnitrogenat65Cinordertoachieveamediumcompatible
with the immunoassay. Then, the samples were reconstituted in 3 mL of
phosphatebuffersolution0.015MpH8.0.

Spiked samples were prepared by adding suitable amounts of

monensin standard to 10 g of skimmed milk, semi skimmed milk and

whole milk and allowing sample matrix and standards to equilibrate for
1.5 h. Aliquots of 0.4 g were treated as described above for nonspiked
samples.

3.Resultsanddiscussion
3.1.Choiceandoptimizationoftheimmunoassaysystem

Two

heterogeneous

competitive

immunoassays,

involving

antibodyorantigencaptureformat,wereassayedtochoosethebestoption
formonensindeterminationusingNBdopedsilicaNPsaslabels.Thefirst
formatwasbasedontheimmobilizationofaBSAmonensinconjugateand
theuseofanantimonensinantibodyNPtracer,whichwassynthesizedby
a similar procedure to that described in an immunoassay for soy protein
determination [27]. The second assay needed a monensinNP tracer,

155

Captulo2

synthesized via a carbodiimide reaction using EDAC, and the

immobilizationofantimonensinantibodiesbycoatingthewellswithanti
sheepIgG.Preliminaryassaysperformedusingbothformatsshowedthat
the difference in the fluorescence signals obtained for 0.0 and 0.2 g L1
monensin standard using the antibodycapture format was about 3fold
higher than that obtained using the analytecapture assay, so the first
format was chosen to develop the new immunoassay for monensin
determination.
Sincemonensinisahapten,itsimmobilizationrequiredtheuseofa
proteinhapten conjugate to avoid potential steric hindrance that would
prevent its reaction with the tracer. BSA was chosen to produce the
conjugate via a carbodiimide reaction with the carboxylic acid of
monensin, which also allows the simple immobilization of monensin by
passive adsorption onto the well surface. It was found that under the
reaction conditions, one molecule of BSA was coupled to approximately
one molecule of monensin, by calculating the BSA concentration by
measuring the absorbance at 280 nm and the monensin concentration by
the Komarowsky reaction [5, 28]. A study of the potential nonspecific
adsorptionof the tracerusing this format was performed by assaying the
system in the absence and presence of BSAmonensin at different
monensinconcentrations(Figure1).

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Sntesisdenanopartculasdesliceysuaplicacin

25000

IF

10 mg L-1 BSA-monensin
0 mg L-1 BSA monensin

20000

15000

10000

5000

0
0.0001

0.001

0.01

0.1

10

[monensin], g L

Figure 1. Fluorescence intensity signals obtained using 0 and 10 mg L1 BSA

monensin conjugate at different monensin concentrations. [Tracer] = 10 nM;
[phosphate]=0.015M;pH=7.5.

-1

In the presence of BSAmonensin conjugate, the signal can be

correlated to monensin concentration, as a result of the competition
between monensin and BSAmonensin conjugate. The results obtained in
the absence of BSAmonensin conjugate showed that the signal obtained
wasalmostconstantatallthemonensinconcentrationsassayedandlower
than the signal obtained at the highest analyte concentration assayed.
Theseresultsshowthatthetracerconcentrationbeingattachedtothewell
in the absence of BSAmonensin conjugate is almost negligible and, thus,
nonspecificadsorptiondoesnotoccur.
The optimization of the system was carried out by modifying the
experimental conditions at different monensin concentrations. The

157

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concentration of the immobilized BSAmonensin conjugate on the well

surface is an important variable on the system, which influences the
sensitivity of the assay, since immobilized and in solution monensin
competefortheactivesitesofthetracer.Theinfluenceofthisvariablewas
assayedintherangeof060mgL1(expressedasmonensinconcentration)
finding that 10 mg L1 provided the best results as the dynamic range
obtained for the calibration curve was wider and the sensitivity obtained
was also better than those obtained at concentrations below and above
(Figure2A).
Another important variable that influences the sensitivity and
working range of the assay is the tracer concentration (Figure 2B), which
was assayed in the range from 5 to 12.5 nM, finding that a 10 nM
concentrationprovidedthebestsensitivityfortheassay.

24000

IF

24000

A)

B)

IF

20000

20000

16000

16000

12000

12000

8000

8000

4000

4000

20

40

60

10

12

14

[BSA-monensin], mg L

Figure 2. Influence of BSAmonensin (A) and tracer (B) concentration on the

assay in the presence of different monensin concentrations (g L1): 1) 0.0; 2)
0.1;3)0.5;4)1.0;5)5.0.[tracer]=10nMinfigureA.[BSAmonensin]=10mgL
1infigureB.Otherexperimentalconditionsinbothfiguresare:[phosphate]=
0.015M,pH7.5,assayincubationtime=1.5h,temperature=37C.
-1

[tracer], nM

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Sntesisdenanopartculasdesliceysuaplicacin

The influence of pH and phosphate molar concentration on the

system was also studied to optimize the reaction conditions. The pH was
investigatedintherangeof6.08.5,findingthattherewasanincreasein
thesignalatpHvaluesabove7.0,beingthebestresultsobtainedatpH8.0,
fromwhichaslightdecreaseinthefluorescencesignalwasobserved.The
study of buffer concentration showed that the assay sensitivity remained
independentonthisvariableintherangeof0.010.03M,beinga0.015M
concentration chosen as the optimum value. The incubation temperature
and time were studied in the ranges 25 40 C and 30 min 2 h,
respectively,choosing37Cand1.5hasoptimumvalues.

3.2.Analyticalfeatures

Figure3showsthecalibrationcurveobtainedformonensinunder

optimalexperimentalconditionsandmeasuringthefluorescencesignalsat
ex620andem680nm.

22000

IF
20000

18000

16000

14000

12000
0.001

0.01

0.1

10

[monensin], g L

-1

Figure3.Calibrationcurveobtainedunderoptimumconditions.

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Captulo2

The method presents a dynamic range of 0.05 5 g L1. The

calibration curve was adjusted to a 4parameter logistic curve using the
SigmaPlot2001software,whichequationis:

where y is the fluorescence intensity and x is the concentration of

monensinexpressedingL1.Thevaluesoftheregressionparametersa,b,
x0andy0were(8.90.4)x103,0.830.08,0.210.03,and(1.210.03)x104
respectively, and the regression coefficient was 0.9992. The detection and
quantification limits, calculated according to IUPAC recommendations
[30], were 0.015 and 0.05 g L1, respectively, which would correspond to
0.12and0.40gkg1inthemilksamples.Thelattervalueisabout5times
lowerthantheMRLsetbythe37/2010/ECCommissionRegulation[3]for
monensin in milk samples. Also, the detection limit is four times lower
than that reported using ELISA with chemiluminescence detection [18].
The precision, expressed as the percentage of relative standard deviation
andassayedattwodifferentmonensinantibioticconcentrations,0.2and1
gL1,gavevaluesof5.8and4.0%respectively.
The selectivity of the system was studied by assaying different
antibiotics of veterinary use belonging to the ionophore group, such as
lasalocid, or to other antibiotic groups such as aminoglycosides,
tetracyclines and fluoroquinolones, which can be used for therapeutic
purposes in animals. Neomycin, oxytetracycline and enrofloxacin were

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Sntesisdenanopartculasdesliceysuaplicacin

chosen as representative components of these groups. A compound was

considered not to interfere at a given concentration when the analytical
signalobtainedinthepresenceofthissubstancewaswithinonestandard
deviationofthevalueobtainedinitsabsence.Themaximumconcentration
testedforallthesecompoundswas200gL1 usinga0.2gL1 monensin
concentration, finding that all of them were tolerated at this 1000:1
concentrationratio.Thisresultagreeswiththeresultsalreadyreportedfor
other monensin immunoassays [15 17, 20, 21], which shows the good
selectivityofantimonensinantibodies.

3.3.Applications

The method was applied to the analysis of skimmed, semi

skimmedandwholemilksamples.Thesampletreatmentwasquitesimple
and consisted in a deproteinization step after which the solvent was
evaporated owing to the lack of compatibility of the organic solvent with
the immunoassay performance. This compatibility was studied using
methanolandethanolandtheyprovedtodecreasethefluorescencesignal
atpercentagesabove3%ofeachsolvent.Theseextractionandevaporation
steps are common to those required by immunoassays for monensin
determination in sample extracts of feedstuff [15, 16] and foodstuff [21]
samples.Themonensinconcentrationinthemilksampleswasdetermined
according to the procedure above described. A recovery study was also
carriedouttovalidatethemethod(Table1),obtainingvaluesintherange
of83.3107.5%.

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Table1.Recoveriesobtainedformonensinaddedtomilksamples
Monensin
Added/

Founda/

Recovery

Sample

gkg1

gkg1

(%)

Skimmedmilk

1.5

1.60.1

106.7

1.960.09

98.0

4.10.2

102.5

1.5

1.50.1

100.0

2.00.1

100.0

3.40.3

85.0

1.250.07

83.3

1.70.1

85.0

4.30.3

107.5

Semiskimmedmilk

Wholemilk

1.5

MeanSD(n=3)

4.Conclusions

TheresultsobtainedhaveshowntheusefulnessofNBdopedsilica

NPs as labels for the determination of monensin using immunoassay,

reporting a lower detection limit than those previously reported using
otherlabels[1418,20,21].Also,thenumberofstepsoftheassayislower
than those required using an enzyme as label. The development of the
assay in 96well microplates, using 100L proxyplate, allows the
automation of the method together with a relatively high sample
throughput, and reduces the sample and tracer volumes required in
comparison with the conventional 300L wells, which contribute to
minimizethecostsassociatedtotheimmunoassay.

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Sntesisdenanopartculasdesliceysuaplicacin

The low quantification limit of this method allows the

determinationofmonensinatlevelsbelowitsMRLformilksamples.The
use of these samples to control the potential presence of monensin in
foodstuffsisausefulalternativetotheanalysisofotherbiologicalsamples,
sincetheycanbeeasilyobtained.

Acknowledgments
Authors gratefully acknowledge financial support from the
MICINN (grant no. CTQ200908621), from the Junta of Andalucia (grant
no. P09FQM4933) and from the FEDERFSE Program (grant no.P09
FQM4933).J.GodoyNavajasthankstheJuntaofAndalucia(grantno.P09
FQM4933)forthefinancialsupportofhispredoctoralfellowship.

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nutrition.Off.J.Eur.Union(2003)L268,2943.

(3)

Commission Regulation (EU) No 37/2010 on pharmacologically

active substances and their classification regarding maximum
residuelimitsinfoodstuffsofanimalorigin.Off.J.Eur.Union(2010)
L15,172.

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R. Galarini, L. Fioroni, S. Moretti, L. Pettinacci, G. Dusi.

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167

CAPTULO 3
CHAPTER 3

Nuevas aportaciones para la

determinacin de antioxidantes en
alimentos
New approaches for the
determination of food antioxidants

Determinacindeantioxidantesenalimentos

Este captulo recoge las investigaciones realizadas para la

propuesta de nuevas metodologas analticas para la determinacin de
antioxidantesenalimentos.Enconcreto,sehatrabajadoeneldesarrollode
un mtodo para la determinacin de la capacidad antioxidante de
alimentos y, en segundo lugar, se ha abordado la puesta a punto de un
mtodo para la determinacin de polifenoles en muestras de vinos. Estos
estudioshanoriginadodosartculoscientficos:

J. GodoyNavajas, M.P. AguilarCaballos, A. GmezHens.

Longwavelength

fluorimetric

determination

of

food

antioxidant capacity using nile blue as reagent. Journal of

AgricultureandFoodChemistry,59(2011)22352240.

J. GodoyNavajas, M.P. AguilarCaballos, A. GmezHens.

Automaticdeterminationofpolyphenolsinwinesusinglaccase
andterbiumoxidenanoparticles.FoodChemistry,enviado.

El inters de estas determinaciones emana de las numerosas

investigaciones realizadas en las ltimas dcadas sobre el impacto del
estrsoxidativoenlasaludhumanaylascausasqueloproducen.Elestrs
oxidativo se define como el desequilibrio entre la produccin de especies
reactivasdeoxgeno(ROS)odenitrgeno(RNS)yladefensaantioxidante,
encontrndose que desempea un papel fundamental en distintas
condiciones patofisiolgicas [1]. La existencia de este estrs oxidativo se
atribuye a la incapacidad de los antioxidantes endgenos para evitar el
daooxidativodebiomolculasclaves.
Los antioxidantes se han definido como cualquier sustancia que,
cuando se encuentra presente a bajas concentraciones con respecto a un

171

Captulo3

sustratooxidable,retrasasignificativamenteoinhibelaoxidacindedicho
sustrato[2].Estadefinicinsemodificmstarde,en2007,parapasara
definirlos como cualquier sustancia que retrasa, evita o elimina el dao
oxidativoaunamolculadiana[3].Surelacinconlasespeciesreactivas
de oxgeno (ROS) se estableci ese mismo ao mediante la definicin de
Khlebnikov y colaboradores [4], en la que se definen los antioxidantes
como cualquier especie que atrapa directamente ROS o indirectamente
actapararegularlasdefensasantioxidantesoparainhibirlaproduccin
de ROS. Despus de atrapar las ROS, las sustancias antioxidantes deben
ser capaces de formar un nuevo radical estable mediante la formacin de
puentesdehidrgenointramoleculares[5].Losantioxidantesrealizanesta
actividad de diversas formas: 1) como inhibidores de las reacciones de
oxidacin que transcurren mediante radicales libres, en las que actan
inhibiendolaformacinderadicaleslibresdelpidos;2)interrumpiendola
propagacin de las reacciones de autoxidacin; 3) como inhibidores del
oxgeno singlete; 4) mediante accin sinrgica con otros antioxidantes; 5)
como agentes reductores que convierten los hidroperxidos en otras
sustancias ms estables; 6) como agentes quelatantes de metales que
transforman metales prooxidantes (derivados de hierro y cobre) en
productosestablesy,porltimo,7)actuandocomoinhibidoresdeenzimas
prooxidantes (p.ej. lipooxigenasas). Actualmente se est trabajando en la
determinacin de la actividad de los antioxidantes a nivel celular para
expandirladefinicindesustanciasantioxidantesaaquellassustanciasque
activan factores de transcripcin capaces de inducir la expresin de
enzimasconactividadantioxidante[1].

172

Determinacindeantioxidantesenalimentos

Las sustancias antioxidantes pueden clasificarse en tres grupos:

naturales, sintticas y sustancias con carcter prooxidante [6]. Dentro de
losantioxidantesnaturales,sehaconsideradolosqueactansobrelasalud
humana (Figura 1), que se dividen en un primer nivel en antioxidantes
enzimticosynoenzimticos.

o Enzimasprimarias
o Enzimassecundarias

ENZIMTICOS

ANTIOXIDANTES
NATURALES

NOENZIMTICOS
o Cofactores
(coenzimaQ10)

o Vitaminas (A:retinol,C:cidoascrbico,Etocoferol)

o Especiesinorgnicas
(zinc,selenio)
o Carotenoides (caroteno,licopeno,lutena,zeaxantina)

o Compuestosnitrogenados (noprotecos,cidorico)
o Compuestosorganosulfurados (indoles,glutatin)

o Flavonoides (flavonoles,flavanoles,flavonas,flavanonas,isoflavonoides,antocianinas)
o cidosfenlicos (cidoshidroxicinmicos,cidoshidroxibenzoicos)

Figura1.Clasificacindelassustanciasantioxidantesnaturales.

Las enzimas antioxidantes se clasifican en defensas primarias y

secundarias,segnneutralicenlapresenciaderadicaleslibresoparticipen
en

reacciones

de

regeneracin

de

sustancias

antioxidantes,

respectivamente. Dentro de los antioxidantes no enzimticos, se

173

Captulo3

encuentran las vitaminas, coenzimas, especies inorgnicas, carotenoides,

compuestosorgnicosdeazufreydenitrgenonoproteico,flavonoidesy,
por ltimo, cidos fenlicos. A pesar de que muchas de estas sustancias
antioxidantes se encuentran de forma endgena en nuestro sistema, se
necesita un aporte externo mediante la dieta para mantener una baja
concentracinderadicaleslibres.Algunosantioxidantesseencuentranen
relativamente alta concentracin como metabolitos secundarios en las
plantas. De esta forma, los zumos naturales, extractos de plantas y vinos,
entre otros, presentan contenidos elevados de antioxidantes como
vitaminas,especiesinorgnicas,flavonoidesycidosfenlicos[7].
Los antioxidantes sintticos son compuestos qumicos que se
adicionan a los alimentos para mantener sus propiedades despus de los
tratamientos incluidos en el procesado de alimentos y, tambin, para
prolongarsuduracin.LaFigura2muestraalgunoscomponentesdeesta
familia, tales como butilhidroxianisol (BHA), butilhidroxitolueno (BHT),
terbutilhidroquinona, steres del cido glico (ms conocidos como
galatos) y derivados de otros fenoles como, por ejemplo, el resorcinol. La
Autoridad Europea de Seguridad Alimentaria (EFSA) revis en 2012 las
concentracionesmximaspermitidasdeestosaditivos,quesiguenestando
enusoapesardealgunosestudiosqueindicanefectosnocivosenlasalud
[8].

174

Determinacindeantioxidantesenalimentos

OH

OH

OH

OH

butilhidroxianisol
(BHA)

terbutilhidroquinona
(TBHQ)

butilhidroxitolueno
(BHT)

OH

OH

HO

HO

HO
O

O
galatodeoctilo
(GO)

galatodepropilo
(GP)

HO

OH

HO

4hexilresorcinol

Figura2.Estructurasqumicasdealgunosantioxidantessintticos.

Las sustancias prooxidantes se definen como especies qumicas

que inducen estrs oxidativo, normalmente mediante la formacin de
especies reactivas o mediante la inhibicin de sistemas antioxidantes. Los
radicales libres son conocidos prooxidantes, pero las sustancias
antioxidantes pueden tener tambin un comportamiento prooxidante [6].
Porejemplo,lavitaminaCesunconocidoantioxidantepero,enpresencia

175

Captulo3

de hierro, puede descomponer perxido de hidrgeno en radicales

hidroxilo, lo que le confiere naturaleza prooxidante. El comportamiento
antioxidanteoprooxidantedependedelantioxidanteconsideradoydesu
concentracin, siendo el estudio de la relacin antioxidante:prooxidante
unreadeinvestigacinmuyactivaenlaactualidad.
Lagranvariedaddesustanciasantioxidantesquepuedenexistiren
los alimentos dificulta la determinacin individual de cada una de las
especiesdeestegrupoy,porotraparte,elpoderantioxidantetotaldeun
alimentoesunaherramientamuytilparaevaluarlosefectosbeneficiosos
de la ingesta de antioxidantes en la salud humana. Cabe distinguir entre
dos conceptos que se utilizan a menudo de forma indistinta pero que
tienen connotaciones ligeramente diferentes: actividad y capacidad
antioxidante.Laactividadantioxidanteserelacionaconlacinticadeuna
reaccinentreunantioxidanteyunprooxidanteounradicalalquereduce
o atrapa, mientras que la capacidad antioxidante mide la eficacia de la
transformacintermodinmicadelareaccindeunprooxidanteconuna
sustanciaantioxidante.Sehandesarrolladodiversosensayosparaevaluar
elpoderantioxidantetotaldeunalimento,peronoproporcionanmedidas
que puedan correlacionarse entre s, debido fundamentalmente a que no
existenmtodosnormalizadosparaestasdeterminaciones[9].Engeneral,
losensayosqumicosdeactividadantioxidantesepuedenclasificarendos
grandesgrupos,atendiendoaltipodereaccininvolucrada:

Ensayos basados en transferencia de tomos de hidrgeno

(HAT)

Ensayosbasadosentransferenciadeelectrones(ET)

176

Determinacindeantioxidantesenalimentos

LosensayosbasadosenreaccionesHATmidenlacapacidaddeun
antioxidante para inhibir los radicales libres (generalmente radicales
perxido)mediantelacesindetomosdehidrgeno.Elmecanismoporel
que se interpreta la accin antioxidante de un compuesto fenlico que
transfiereunprotnaunradicaleselsiguiente:

donde el radical oxiarilo (ArO) formado por la reaccin del antioxidante

fenlico con el radical perxido se estabiliza mediante resonancia y las
especies AH y ArOH se corresponden con las biomolculas protegidas y
las de antioxidante fenlico, respectivamente. En estos ensayos, se utiliza
un fluorforo que compite con la sustancia antioxidante por los radicales
perxido.Laactividadantioxidantesedeterminamediantemedidasdela
diferenciaenlainhibicindelafluorescenciadelfluorforoenausenciay
presencia de las sustancias antioxidantes. Algunos ensayos basados en
HAT son el que mide la capacidad de absorcin de radicales de oxgeno
(ORAC), el de poder antioxidante total (TRAP) y los ensayos de
decoloracindecrocinaycarotenos.

En los ensayos mediante reacciones ET, el antioxidante reacciona

con un cromforo o un fluorforo con naturaleza oxidante en lugar de

reaccionar con los radicales perxido. As, la presencia de sustancias
antioxidantesoriginaunavariacinenlaabsorbanciaoenlafluorescencia
del cromforo y fluorforo, respectivamente, de forma que estos cambios
se pueden correlacionar con la concentracin de sustancias antioxidantes
presentes en la muestra. Algunos ejemplos de ensayos basados en estas

177

Captulo3

reaccionessonelquemidelacapacidadantioxidantetotalequivalentede
Trolox (TEAC), el ensayo DPPH (que utiliza radicales 2,2
difenilpicrilhidracilo), el mtodo de FolinCiocalteu, el del potencial
antioxidantereductordeionesfrricos(FRAP)ylacapacidadantioxidante
reductoradeionescpricos(CUPRAC).

LasinvestigacionesdeestaMemoriaqueabordaneldesarrollode

un mtodo para la determinacin de la capacidad antioxidante estn

basadas en la aplicacin del mtodo ORAC. Este mtodo utiliza un
iniciador azobis, como el diclorhidrato de 2,2azobis(2amidinopropano)
(AAPH),elcualliberaradicalesperxidocuandosecalientaconayudadel
oxgenodisuelto[10].

Los radicales perxido liberados reaccionan con un fluorforo e

inhibensufluorescencia.Losfluorforosutilizadosdeformaconvencional
han sido ficoeritrina [11], que se sustituy posteriormente por
fluorescena y diclorofluorescena [12], debido a que stas presentaban
mayor fotoestabilidad y una reactividad prcticamente nula con los
polifenoles.

178

Determinacindeantioxidantesenalimentos

El estudio realizado ha estado orientado a investigar la utilidad

analticadelosfluorforosdelargalongituddeondaconobjetodemejorar
algunas propiedades analticas del ensayo ORAC, tales como la
selectividad espectral, como se discutir ms adelante. Esta mejora es
posibleyaquelasmedidasserealizanalongitudesdeondamslargasque
las que presentan los fluorforos tradicionalmente utilizados con este fin.
Estasinnovacionesestnenconsonanciaconlasdirectricesdeuninforme
tcnicodelaIUPAC[9]publicadorecientemente,enelqueseproponenlas
siguientesaccionesdemejora:

Desplazarelfocodeatencindesdeelscreeningalestudiodelos
mecanismosdereaccindelassustanciasantioxidantes.

Sustituir la fluorescena por otros fluorforos que no presenten

inhibicin de la fluorescencia por irradiacin, reacciones o
interaccionescolaterales.

Analizarpotencialesinterferenciasmedianteelanlisisdemuestras
blanco en las que se analice la contribucin de las muestras sin
antioxidantes junto con el fluorforo, en presencia y ausencia del
reactivogeneradorderadicales.

Ensayar intervalos de concentraciones de antioxidantes para

proponermecanismosdereaccindelassustanciasantioxidantesy
conocer a partir de qu concentraciones se comportan como pro
oxidantes.

Obtener informacin para la normalizacin de los procedimientos

ORAC: relacin fluorforo:generador de radicales para eliminar
efectos de autoinhibicin del fluorforo que oculten la accin del
antioxidante,estudiosistemticodelainfluenciadelosnivelesde
179

Captulo3

oxgenodisueltoenlavelocidaddereaccin,determinacindelos
niveles de oxgeno disuelto mnimos para que la reaccin tenga
lugar y desarrollo de mtodos para asegurar niveles de oxgeno
adecuados,deacuerdoaloexpuestoanteriormente.

Disearmtodosparacontrolaradecuadamentelatemperatura.

Desarrollarnormasparaeldesarrollodemtodosenelformatode
microplacasenensayosdeactividadantioxidanteyconseguirque
losfabricantesdiseenlainstrumentacinapropiadaparatalfin.

Ponerapuntomtodosnormalizadosparaelclculodereasbajo
lacurva.

Proporcionardatosconjuntosdecapacidadantioxidanteydeotros
parmetroscomolaconcentracintotaldepolifenolesparaconocer
losperfilesdeactividad.

Conocer la capacidad antioxidante en extractos naturales despus

de su tratamiento con polivinilpirrolidona para la eliminacin de
compuestosfenlicosydeducirlacontribucindeloscompuestos
nofenlicosadichoensayo.
Delosdosltimospuntosdemejoradelinformetcnicoanteriorse

deducelaimportanciadeproporcionardatoscomplementarios,talescomo
la concentracin total de antioxidantes fenlicos y la de antioxidantes no
fenlicos, ntimamente relacionada con la capacidad antioxidante para
conocer los perfiles de actividad antioxidante de cada alimento. En la
segunda seccin de este captulo se aborda la determinacin de
antioxidantesfenlicosmedianteelusodelaenzimalaccasaenpresencia
denanopartculasdexidodeterbiocomoactivador.

180

Determinacindeantioxidantesenalimentos

Las enzimas de la familia de las laccasas son fenoloxidasas que

oxidan las sustancias antioxidantes con ayuda del oxgeno disuelto en el
medio.Lasenzimaslaccasassonactivasfrenteacompuestosantioxidantes
que incorporan grupos orto y paradifenol, aunque presentan mayor
afinidad por los compuestos fenlicos sustituidos en para. Estas enzimas
presentan una relativa selectividad por los sustratos, que pueden tener
mayor o menor potencial de reduccin dependiendo del tipo de laccasa.
Las enzimas laccasas catalizan la oxidacin de una amplia variedad de
sustratos,entrelosqueseincluyenmono,diypolifenoles,aminofenoles,
metoxifenoles,aminasaromticasyascorbato,conlareduccindeoxgeno
aaguamedianteelintercambiodecuatroelectrones.
Estas enzimas pueden obtenerse de diversas fuentes, ya sean
plantas,comoeselcasodelaplantajaponesaRhusvernicifera,delacualse
aisllaprimeravariedaddelaccasa,insectosybacterias[13].Noobstante,
la mayora de las laccasas actualmente utilizadas se obtienen de hongos
superiores,comoTrametesversicoloryTrameteshirsuta,entreotros.
Estas enzimas presentan numerosas aplicaciones industriales y
analticascuandoseencuentraninmovilizadassobreunsoporteslido.Se
han revisado recientemente [13] las diversas opciones de inmovilizacin
tales como atrapamiento, adsorcin, encapsulamiento, entre otras, para
finalmenteconcluirquenosiempreserecuperalaactividadenzimticade
forma satisfactoria tras el proceso de inmovilizacin. Las aplicaciones
analticas ms destacables de la enzima laccasa han estado orientadas al
desarrollo de biosensores con deteccin electroqumica [14]. El modo de
operacindeestosbiosensoresesgeneralmentesecuencial,loquelimitasu
velocidad de muestreo, a diferencia de otros ensayos que utilizan
181

Captulo3

microplacas de pocillos donde las medidas se realizan de forma

prcticamente simultnea. ste es un aspecto que se ha estudiado y
optimizadoenlasinvestigacionesdescritasenestaMemoria.Enconcreto,
elsegundoapartadodeestecaptulohatenidocomoobjetoeldesarrollode
un mtodo de alta velocidad de muestreo para la determinacin de
polifenolesenvinosconelquesepretendereducirelconsumodeenzima
conlasnanopartculasdexidodeterbiocomoactivador,almismotiempo
que se acorta el tiempo requerido para las determinaciones analticas
basadasenelusodeestaenzima.

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European Food Safety Authority (EFSA). Statement on the safety

assessmentoftheexposuretobutylatedhydroxyanisoleE320(BHA)
byapplyinganewexposureassessmentmethodology.EFSAJ.(2012)
10:2759,116.

(9)

R. Apak, S. Gorinstein, V. Bhm, K.M. Schaich, M. Ozyrek, K.

Gl. Methods of measurement and evaluation of natural
antioxidant capacity/activity (IUPAC Technical Report). Pure Appl.
Chem.(2013)85,957998.

(10)

R.L. Prior, X. Wu, K. Schaich. Standardized methods for the

determination of antioxidant capacity and phenolics in foods and
dietarysupplements.J.Agric.FoodChem.(2005)53,42904294.

(11)

G.H. Cao, H.M. Alessio, R.G. Cutler. Oxygenradical absorbance

capacityassayforantioxidants.FreeRadicalBiol.Med.(1993)14,303
465.

(12)

B.Ou,M.HampschWoodill,R.L.Prior.Developmentandvalidation
of an improved oxygen radical absorbance capacity assay using
fluorescein as the fluorescent probe. J. Agric. Food Chem. (2001) 49,
46194621.

(13)

M. FernndezFernndez, M.A. Sanromn, D. Moldes. Recent

developments and applications of immobilized laccase. Biotechnol.
Adv.(2013)31,18081825.

(14)

A.SnchezArribas,M.MartnezFernndez,M.Chicharro.Therole
of electroanalytical techniques in analysis of polyphenols in wine.
TrendsAnal.Chem.(2012)34,7896.

183

Chapter3

Thischaptercontainstheinvestigationsperformedtodevelopnew
analytical methodologies for the determination of antioxidants in food
samples. More specifically, these investigations have been focused to the
development of a method to determine the antioxidant capacity in food
samples, and secondly, on the proposal of a method to determine
polyphenols in wines. These studies have given rise to two scientific
articles:

J. GodoyNavajas, M.P. AguilarCaballos, A. GmezHens.

Longwavelength

fluorimetric

determination

of

food

antioxidant capacity using nile blue as reagent. Journal of

AgriculturalandFoodChemistry,59(2011)22352240.

J. GodoyNavajas, M.P. AguilarCaballos, A. GmezHens.

Automaticdeterminationofpolyphenolsinwinesusinglaccase
andterbiumoxidenanoparticles.FoodChemistry,submittedfor
publication.

The interest on these determinations comes from the numerous

investigations performed in the latest decades on the impact of the
oxidativestressonhumanhealthanditscauses.Theoxidativestresscanbe
definedastheunbalancebetweentheproductionofreactiveoxygen(ROS)
ornitrogen(RNS)speciesandtheantioxidantdefence,findingthatitplays
asignificantroleindifferentpathophysiologicalconditions[1].Thereason
forthisoxidativestressistheinabilityofendogenantioxidantstoavoidthe
oxidativedamageofkeybiomolecules.
Antioxidantsubstanceshavebeendefinedasanysubstancethatat
verylowconcentrationcansignificantlydelayorinhibittheoxidationofa

184

Determinationoffoodantioxidants

substrate [2]. This definition was later modified in 2007, to define an

antioxidant as any substance that delays, prevents or removes the
oxidative damage to a target molecule [3]. The relationship of oxidative
stresswithROSwasestablishedinthesameyearbyKhlebnikovetal.[4],
who defined antioxidants as any species that scavenges ROS directly or
indirectly acts to upregulate antioxidant defences or to inhibit ROS
production.AfterscavengingROS,properantioxidantsubstancesmustbe
able to give rise to new stable radicals by means of intramolecular
hydrogen bonds [5]. Antioxidants can act in many different ways: 1) as
inhibitors of redox reactions happening via free radicals, by mainly
inhibiting lipid free radicals; 2) breaking the chain of autooxidation
reactions; 3) as inhibitors of singlet oxygen; 4) through synergism with
otherantioxidants;5)asreducingagentsthatchangehydroperoxidesinto
more stable compounds; 6) as metal chelating agents that convert pro
oxidativemetalspecies(mainlyderivedfromironandcopper)intostable
products, and, 7) as inhibitors of prooxidant enzymes (e.g.
lipooxygenases).Nowadays,anactiveareaofresearchisthedetermination
of cellular antioxidant activity to expand the definition of antioxidant
compounds to those substances that activate transcription factors able to
inducetheexpressionofantioxidantenzymes[1].
Antioxidant compounds can be categorized into three different
groups: natural, synthetic and prooxidant substances [6]. Figure 1 shows
the most important natural antioxidants involved in the human system,
which can be divided in first instance in enzymatic and nonenzymatic
antioxidants.

185

Chapter3

o Primary enzymes
o Secondary enzymes

ENZYMATIC

NATURAL
ANTIOXIDANTS

NONENZYMATIC
o Cofactors
(coenzyme Q10)

o Vitamins (A:retinol,C:ascorbic acid,E:tocopherol)

o Inorganic
species (zinc,selenium)
o Carotenoids (carotene,lycopene,luteine,zeaxanthine)

compounds (nonprotein,uric acid)

o Nitrogen
o Organosulfur compounds (indoles,glutathione)

o Flavonoids (flavonols,flavanols,flavones,flavanones,isoflavonoids,anthocyanins)
o Phenolic acids (hydroxycinnamic acids,hydroxybenzoic
acids)

Figure1.Classificationofnaturalantioxidantsubstances.

Antioxidant enzymes either classify as primary or secondary

defences depending on whether they neutralise the presence of free
radicals or they participate in regeneration reactions for antioxidant
compounds. Among nonenzymatic compounds, we can find vitamins,
coenzymes,inorganicspecies,carotenoids,organosulfurcompounds,non
protein nitrogen compounds, flavonoids and phenolic acids. Although
some of them are endogenous substances, an external intake from diet is
needed to preserve the free radical concentration at low levels. Some

186

Determinationoffoodantioxidants

antioxidant compounds can be found in relatively high concentrations as

secondary plant metabolites. Thus, fruit and extract juices and wines,
among others, exhibit a high content of some antioxidants as vitamins,
inorganicspecies,flavonoidsandphenolicacids[7].
Synthetic antioxidants are chemical compounds that are added to
foods to keep them unaltered after food processing and to elongate shelf
life. Figure 2 shows some components of this group, such as butylated
hydroxyanisole

(BHA),

butylated

hydroxytoluene

(BHT),

tertbutylhydroquinone,estersofgallicacid(betterknownasgallates)and
somephenolderivatives,suchase.g.resorcinolderivatives.

OH

OH

OH

butylated hydroxyanisole
(BHA)

tertbutilhydroquinone
(TBHQ)

butylated hydroxytoluene
(BHT)

OH

OH

HO

HO

HO

HO

octyl gallate
octylgallate
GO)
(OG)

propylgallate
propyl
gallate
(GP)
(PG)
OH

HO

4hexylresorcinol

Figure2.Chemicalstructuresofsomesyntheticantioxidants.

187

OH

Chapter3

The European Food Safety Authority (EFSA) revised in 2012 the

maximumallowedconcentrationsfortheseadditives,whicharestillinuse,
though the results obtained in some studies indicated they can pose a
hazardtohumanhealth[8].
Prooxidantsubstancesaredefinedaschemicalspeciesthatinduce
oxidativestress,usuallybygivingrisetotheformationofreactivespecies
or by inhibiting antioxidant systems. Free radicals are well known pro
oxidant substances, but antioxidant compounds can also bear a pro
oxidantbehavior[6].For instance,vitaminCisa wellknownantioxidant
that in presence of iron can decompose hydrogen peroxide into hydroxyl
radicals, as a prooxidative substance would do. The antioxidant or pro
oxidant behavior depends on the antioxidant substance and on the
concentration, the study of antioxidant:prooxidant ratio for each
compoundbeinganactivecurrentresearcharea.
Thegreatvarietyofantioxidantcompoundsthatcanexistinfood
makes it difficult the individual determination of every single species
belonging to this family, and on the other hand, the total antioxidant
powerofafoodsampleisaveryusefultooltoassessthebeneficialeffects
of antioxidant intake on human health. There are two terms, which are
often indistinctly used, but have slightly different meanings: antioxidant
activity and capacity. The antioxidant activity is related to the kinetics of
reactionbetweenanantioxidantcompoundandaprooxidantoraradical
towhichreducesorscavenges,whereastheantioxidantcapacitymeasures
theefficiencyofthethermodynamictransformationthroughthereactionof

188

Determinationoffoodantioxidants

aprooxidantwithanantioxidant.Severalassayshavebeendevelopedfor
the assessment of the total antioxidant power of foods, but they do not
provide measurements that correlate mainly owing to the lack of
standardized methods [9]. In general, the chemical assays of antioxidant
activity can be divided into two main groups according to the type of
reactioninvolved:

Assaysbasedonhydrogenatomtransfer(HAT)

Assaysbasedonelectrontransfer(ET)

The assays based on HAT reactions measure the ability of an

antioxidant compound to inhibit free radicals (generally peroxyl radicals)
by the donation of hydrogen atoms. The mechanism interpreting the
antioxidant action of a phenol compound that transfers a proton to a
radicalisthefollowing:

wherethearyloxyradical(ArO)formedafterthereactionofthephenolic
antioxidant with the peroxyl radical is stabilized by resonance, and AH
and ArOH species are the protected biomolecules and the antioxidant
molecules, respectively. In these assays, a fluorophore competes with the
antioxidantsubstancefortheperoxylradicalsandtheantioxidantactivity
is determined by measuring the difference in the inhibition of the
fluorophore signals obtained in the presence and in the absence of
antioxidant compounds. Some HATbased assays are the oxygen radical

189

Chapter3

absorbance capacity (ORAC), the total radical antioxidant power (TRAP)

andthecrocinandcarotenebleachingassays.

For the ETbased assays, the antioxidant reacts to an oxidant

chromophore or fluorophore instead of reacting with peroxyl radicals.

Thus,thepresenceofantioxidantcompoundsgivesrisetoachangeinthe
absorbance or the fluorescence of the chromophore or fluorophore,
respectively,sothatthesechangescanbecorrelatedtotheconcentrationof
antioxidant compounds in the sample. Some assays based on these
reactions are the Trolox Equivalent Antioxidant Capacity (TEAC), the
DPPH assay (that uses 2,2diphenylpicrylhydrazyl radical), the Folin
Ciocalteu method, the ferric reducing antioxidant power (FRAP) and
cupricreducingantioxidantcapacity(CUPRAC).

The investigations of this Dissertation dealing with the

developmentofamethodforthedeterminationofantioxidantcapacityare
basedontheapplicationoftheORACapproach.Thismethodinvolvesthe
use of a bisazo initiator, such as 2,2azobis(2methylpropionamidine)
dihydrochloride(AAPH),whichgeneratesperoxylradicalswhenheatedin
thepresenceofdissolvedoxygen[10].

190

Determinationoffoodantioxidants

The peroxyl radicals generated react to a fluorophore thus

inhibiting its fluorescence. The fluorophores traditionally used have been
phycoerythrin [11], which was further replaced by fluorescein and
dichlorofluorescein[12]owingtothefactthattheselaterpresentedhigher
photostabilityandapracticallynegligiblereactivitytowardpolyphenols.
Theinvestigationsperformedhavebeenfocusedtothestudyofthe
analytical usefulness of longwavelength fluorophores with the aim of
improvingthespectralselectivityofORACassays,asitwillbediscussed
below in this Dissertation. This improvement is possible because the
measurements are performed at longer wavelengths than those exhibited
by the fluorophores conventionally used for this purpose. These
innovations are in agreement with the guidelines of a recently published
IUPACtechnicalreport[9],wherethefollowingimprovementactionsare
suggested:
To shift the focus of these assays from screening to elucidation of
theantioxidantmechanism.

To replace fluorescein by other fluorophores insensitive to

photobleaching,tosidereactionsorinteractions.

Toanalyzepotentialinterferencesbytheanalysisofblanksamples
where the contribution of samples and fluorophore can be
analyzed in the absence and in the presence of the radical
generator.

To test antioxidant concentration ranges to suggest reaction

mechanisms of antioxidants and to identify prooxidant
concentrationranges.

191

Chapter3

To obtain information in order to standardize ORAC procedures:

fluorophore:radical generator ratio to avoid quenching effects of
the fluorophore that can hide the antioxidant action, systematic
study of the influence of dissolved oxygen levels on the reaction
rate,determinationofminimumdissolvedoxygenlevelstoobserve
the reaction and development of methods to ensure suitable
oxygenlevelsaccordingtotheabovementioned.

To devise methods for suitable temperature monitoring and

control.

To establish standards for the development of microplatebased

methods for the determination of antioxidant capacity and to
encouragemanufacturerstodesignappropriateinstrumentation.

Todevelopstandardizedmethodsforthecalculationofareaunder
thecurve.

Toprovidedataofantioxidantcapacityandotherparameters,such
as for instance, the total polyphenol concentration to know the
activityprofile.

To know the capacity profile in natural extracts after their

treatment with polyvinylpyrrolidone for the removal of phenolic
compounds and to obtain the contribution of nonphenolic
compoundstothisassay.
From the last two actions described above, it can be seen the

significanceofcomplementarydata,suchastotalconcentrationofphenolic
antioxidantsandthatofnonphenolicantioxidants,intimatelyrelatedwith
the antioxidant activity to know the activity profile of each food. The
second section of this chapter tackles the determination of phenolic

192

Determinationoffoodantioxidants

antioxidant concentration by using laccase enzyme in the presence of

terbiumoxidenanoparticlesasactivators.
Laccase enzymes are phenoloxidases that can oxidise antioxidant
compounds using dissolved oxygen. These enzymes have activity for
antioxidant compounds with ortho and paradiphenol groups, although
their affinity is higher toward parasubstituted phenolic compounds.
Laccases have a relative selectivity toward their substrates, which have
higher or lower reduction potential depending on the laccase type. These
enzymes catalyze the oxidation of a wide variety of substrates, such as
mono, di and polyphenols, aminophenols, methoxyphenols, aromatic
amines and ascorbate, with the reduction of oxygen to water by
exchangingfourelectrons.
These enzymes can be obtained from different sources, such as
fromplants,liketheJapaneselacquertreeRhusvernicifera,whichthefirst
laccasevarietywasisolatedfrom,insectsandbacteria[13].However,most
laccase currently in use are obtained from higher fungi, such as Trametes
versicolorandTrameteshirsuta,amongothers.
These enzymes have found a great number of industrial and
analytical applications when immobilised onto a solid support. Different
immobilisation approaches, such as entrapment, adsorption and
encapsulation, among others, have been recently reviewed [13], to finally
concludethattheinitialenzymeactivitycannotbesatisfactorilyrecovered
in all instances after these procedures. The most salient analytical
applications of laccase enzyme have been focused to the development of
electrochemical biosensors [14]. These biosensors often operate
sequentially, which limits their sample throughput, unlike other assays

193

Chapter3

basedontheuseofwellmicroplates,wheremeasurementsareperformed
almost simultaneously. This is an aspect that has been investigated and
optimizedinthisDissertation.Morespecifically,thesecondsectionofthis
chapter hasdealt with the development of a highthroughput method for
the determination of polyphenols in wines, where the enzyme
consumption has been lowered by using terbium oxide nanoparticles as
activators.Themethodalsodecreasestheanalysistimesrequiredwhenthe
enzymeisusedintheabsenceofnanoparticles.

LITERATURE
(1)

B.D. Craft, A.L. Kerrihard, R. Amarowicz, R.B.Pegg. Phenolbased

antioxidants and the in vitro methods used for their assessment.
Compr.Rev.FoodSci.FoodSafety.(2012)11,148173.

(2)

B. Halliwell, J.M. Gutteridge. The definition and measurement of

antioxidantsinbiologicalsystems.FreeRadic.Biol.Med.(1995)18,125
126.

(3)

B. Halliwell. Biochemistry of oxidative stress. Biochem. Soc. Trans.

(2007)35,11471150.

(4)

A.J.Khlebnikov.I.A.Schepetkin,N.G.Domina,L.N.Kirpotina,M.T.
Quinn.Improvedquantitativestructureactivityrelationshipmodels
to predict antioxidant activity of flavonoids in chemical, enzymatic,
andcellularsystems.Bioorg.Med.Chem.(2007)15,17491770.

(5)

B.Halliwell.Howtocharacterizeabiologicalantioxidant.FreeRadic.
Res.(1990)9,132.

(6)

M.Carocho,I.C.F.R.Ferreira.Areviewonantioxidants,prooxidants
and relative controversy: Natural and synthetic compounds,
screening and analysis methodologies and futureperspectives. Food
Chem.Tox.(2013),51,1525.

194

Determinationoffoodantioxidants

(7)

B.Lorrain,I.Kay,L.Pechamat,P.L.Teissedre.Evolutionofanalysis
ofpolyphenolsfromgrapes,winesandextracts.Molecules.(2013)18,
10761100.

(8)

European Food Safety Authority (EFSA). Statement on the safety

assessmentoftheexposuretobutylatedhydroxyanisoleE320(BHA)
byapplyinganewexposureassessmentmethodology.EFSAJ.(2012)
10:2759,116.

(9)

R. Apak, S. Gorinstein, V. Bhm, K.M. Schaich, M. Ozyrek, K.

Gl. Methods of measurement and evaluation of natural
antioxidant capacity/activity (IUPAC Technical Report). Pure Appl.
Chem.(2013),85,957998.

(10)

R.L. Prior, X. Wu, K. Schaich. Standardized methods for the

determination of antioxidant capacity and phenolics in foods and
dietarysupplements.J.Agric.FoodChem.(2005),53,42904294.

(11)

G.H. Cao, H.M. Alessio, R.G. Cutler. Oxygenradical absorbance

capacityassayforantioxidants.FreeRadicalBiol.Med.(1993),14,303
465.

(12)

B.Ou,M.HampschWoodill,R.L.Prior.Developmentandvalidation
of an improved oxygen radical absorbance capacity assay using
fluorescein as the fluorescent probe. J. Agric. Food Chem. (2001) 49,
46194621.

(13)

M. FernndezFernndez, M.A. Sanromn, D. Moldes. Recent

developments and applications of immobilized laccase. Biotechnol.
Adv.(2013)31,18081825.

(14)

A.SnchezArribas,M.MartnezFernndez,M.Chicharro.Therole
of electroanalytical techniques in analysis of polyphenols in wine.
TrendsAnal.Chem.(2012)34,7896.

195

Determinacindeantioxidantesenalimentos

J.Agric.FoodChem.2011,59,22352240

LongWavelengthFluorimetricDeterminationofFood
AntioxidantCapacitybyUsingNileBlueasReagent
J.GodoyNavajas,M.P.AguilarCaballos,A.GmezHens
DepartmentofAnalyticalChemistry,UniversityofCordoba,CampusofRabanales.
AnnextoMarieCurieBuilding.14071Cordoba.Spain

ABSTRACT

A method for the determination of the antioxidant capacity by using

longwavelength fluorescence measurements is described for the first time.
This method is a modification of the conventional oxygen radical absorbance
capacity (ORAC) method that uses fluorescein or phycoerythrin and the
generator of peroxyl radicals 2,2 azobis(2methylpropionamidine)
dihydrochloride (AAPH). The longwavelength fluorophor nile blue is
proposed as an analytical reagent alternative to these conventional
fluorophores.
Kinetic curves have been obtained by monitoring the fluorescence
variation(ex620,em680nm)withtime,usingthe96wellmicroplateformat.
The vitamin E analogue 6hydroxy2,5,7,8tetramethylchroman2carboxylic
acid(Trolox)hasbeenchosenasmodelanalyte,andthenormalizedareaunder
thedecaycurvehasbeenusedastheanalyticalparameter.Thedynamicrange
ofthecalibrationcurveis0.88.0Mandthedetectionlimitis0.45M.The
precisionofthemethod,expressedasrelativestandarddeviationandassayed
using 1 and 5 M Trolox concentrations, was 5.6 and 2.9%, respectively. The
method has been applied to the analysis of fruit juices and wines, obtaining
results that did not differ significantly from those provided using the ORAC
methodwithfluoresceinasreagent.

Keywords:nileblue,antioxidantcapacity,longwavelengthfluorimetry,96wellplate

197

Captulo3

1.Introduction
Oxidation processes involving free radicals contribute to the
development of many types of illnesses, such as, e.g., cardiovascular
disease, Alzheimers disorder, and cancer. This redox phenomenon is
produced by reactive oxygen species (ROS), which damage cell
membranes, with this effect being reduced by the ingest of antioxidant
compounds. The antioxidant capacity of foods is mainly given by
polyphenols,suchasflavonoids,andvitaminsCandE,amongothers[1].
From an analytical point of view, there are two types of methods for
assessingthisparameter.Thefirstgroupinvolvesasingleelectrontransfer
reaction, which can be followed by a change in the colour as the oxidant
speciesisreduced.Thesecondinvolvestheuseofhydrogenatomtransfer
reactions,inwhichtheantioxidantandthesubstratecompeteforthefree
radicalsgenerated.Theoxygenradicalabsorbancecapacity(ORAC)assay
isanexampleofthelattermethods.Thisassayinvolvestheuseof2,2azo
bis(2methylpropionamidine)dihydrochloride (AAPH) to give rise to
peroxyl radicals that directly attack absorbing or fluorescent probes,
leadingtothequenchingoftheirabsorbanceorfluorescence,respectively.
Thedyesphycoerythrin[24]andfluorescein(FL)[1,514]havebeenby
far the most used fluorescent probes, although Pyrogallol Red has been
alsodescribedtodevelopphotometricapproachesforthedeterminationof
the antioxidant capacity in berry extracts [15] and human blood plasma
and urine [16]. This assay relies on the performance of photometric
measurementsofPyrogallolRedbleachingbyactionoftheperoxylradical
generator,AAPH.Ithasbeenreportedthatthisdyeallowsfortheseparate
estimationofthecontributionofascorbicacidandsomepolyphenolstothe

198

Determinacindeantioxidantesenalimentos

total antioxidant capacity of fruits with high ascorbic acid concentration,

thus allowing for the determination of ascorbic acid in complex samples.
TheORACassayhasbeenalsousedtoassess,e.g.,theantioxidantcapacity
ofhumanmilk[14].
Despitethehighsensitivitythattheabovementionedfluorophores
offer, these compounds feature a relatively short Stokes shift (about 27
nm), which favors scattering phenomena. Also, phycoerythrin is
relativelyexpensiveandsuffersfromphotobleaching,withthisfactbeing
themainoneresponsibleforthealmostwidespreaduseofFLasprobefor
theORACassay.However,thisfluorophorshowsalimitationcommonto
organic conventional fluorophores that emit in the 400 550 nm range,
which is an increased influence of static background signals from the
sample matrix. The usefulness of longwavelength dyes as fluorescent
probes to overcome this limitation has been previously demonstrated in
several analytical methods [17], but their potential application to the
determinationoftheantioxidantcapacityhasnotbeenstudieduptonow.
Nile blue (NB) is a dye from the oxazine group (Figure 1), which
emitslightintheredregionofthespectrumandfeaturesaStokesshiftof
about 60 nm. This reagent has been previously used for the fluorimetric
determinationofthesyntheticantioxidantbutylhydroxyanisole(BHA)[18]
and the photometric determination of cerium(IV) [19]. Other recent
applications of this fluorophor have been its use in energy and electron
transferreactionsincovalentlyfunctionalizedcarbonnanotubes[20]andto
describe a hydrogen peroxide biosensor by its immobilization, together
withhorseradishperoxidase[21].

199

Captulo3

N
Cl-

H2N

N
CH3

CH3

Figure1.ChemicalstructureofNBchloride

The work described here involves a modification of the ORAC

assay by using NB as the fluorescent probe and 6hydroxy2,5,7,8
tetramethylchroman2carboxylic acid (Trolox) as the reference standard
forantioxidantcapacity.Themethodhasachieveditsautomationbyusing
a microplate reader, which monitored the variation of the fluorescence
intensity with time using 620 and 680 nm as excitation and emission
wavelengths, respectively. The proposed method has been applied to the
analysisoffruitjuiceandwinesamples,andtheresultsobtainedhavebeen
compared to those provided by the ORAC approach using FL as the
reference method, with no statistically relevant differences between the
results given by both methods. Also, a recovery study was carried out to
validate the usefulness of the proposed method to the analysis of real
samples, which has been scarcely reported in the ORAC methods
described up to date. These results confirm the usefulness of NB as an
analytical reagent for antioxidant capacity determination with better
spectral selectivity than the ORACFL assay, owing to the use of long
wavelengthfluorescencemeasurements.

200

Determinacindeantioxidantesenalimentos

2.Materialsandmethods
2.1.Instrumentation
A 1420 Multilabel counter Victor 3V microplate reader (Perkin
Elmer and Analytical Sciences, Wallac Oy, Turku, Finland) was used to
perform

fluorescence

measurements.

Different

filters

(nominal

wavelength/passband)wereusedtoselecttheexcitation(485/15nmforFL
and620/8nmfor NB)andemission (535/25nmforFLand680/10nmfor
NB) wavelengths used to monitor ORACFL and ORACNB systems,
respectively.Eachmeasurementwasobtainedin0.5s,andthesubsequent
measurementsforeachwellwithtimeweremadein1minintervals.

2.2.Reagents
All reagents used were of analytical grade. NB chloride was
supplied by Sigma (St Louis, MO), and Trolox and AAPH were obtained
from Aldrich (Milwaukee, WI, and Steinheim, Germany). Dipotassium
hydrogen phosphate was purchased from Merck (Darmstadt, Germany).
Phosphate buffer solutions (0.085 M pH 6.9 and 0.075 M pH 7.5) were
preparedbydissolvingappropriateamountsofdipotassiumhydrogensalt
andadjustingthepHvalueswithhydrochloricacidtodevelopORACNB
and ORACFL methods, respectively. A NB stock solution (29.4 M) was
prepared by dissolving the appropriate amount of the dye in distilled
water by magnetic stirring for 24 h and stored at room temperature.
Workingstandardsolutionsof89nMNBwereprepareddailybydiluting
theappropriatevolumeofthestocksolutioninphosphatebuffersolution
(0.085 M at pH 6.9). A stock 4.5 x 104 M FL solution was prepared by
dissolvingtheappropriateamount

of sodium fluorescein in distilled

201

Captulo3

water, and subsequent dilutions were made daily in phosphate buffer

(0.075 M at pH 7.5) to prepare working 70 nM FL solutions. A 0.012 M
AAPHsolutionwasalsoprepareddailyinphosphatebuffersolution(0.085
MatpH6.9)andkeptatroomtemperatureprotectedfromlighttoprevent
itsdegradation.

3.Procedures
3.1.DeterminationofAntioxidantCapacitybytheORACNBMethod
A volume (20 L) of standard (0.8 8 M Trolox), wine or juice
diluted sample, or blank (0.085 M phosphate buffer at pH 6.9) solutions
was added to each well together with 120 L of 89 nM NB. This mixture
waspreincubatedinsealedplatesat37Cfor15minutes,andthen,60L
of AAPH 0.012 M was added to each well by using an eightchannel
electronicmicropipettoachievethesimultaneousadditionofthisreagent.
Immediately, the plate was inserted into the microplate reader, and the
variationofthefluorescenceintensitywithtimewasmonitoredat37Cfor
60 min, using the instrumental conditions indicated above. Each
measurement was performed in triplicate, and a blank triplicate was
recorded at the beginning of each series to control potential changes that
may occur, owing to the lack of stability of AAPH, which was kept
protected from light at room temperature to ensure identical thermal
conditions at time 0 of the reaction. The decay curves were integrated by
usingOriginsoftware,andthen,thenormalizednetareaunderthecurve
(AUC) was calculated by subtracting the blank signal from the signal
obtainedinthepresenceofthestandardorsampleanddividingtheresult
bytheblanksignal.

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Determinacindeantioxidantesenalimentos

3.2.DeterminationoftheAntioxidantCapacityusingtheORACFLMethod

The ORACFL measurements were done according to the

instructionsdescribedelsewhere[5],whichinvolvetheuseofamicroplate
reader and FL as reagent. Briefly, 20 L of standard or diluted sample
solutionsweremixedwith120Lof70nMFLinphosphatebuffer(0.075
M, pH 7.4) for 15 min at 37 C, and then, 60 L of 0.012 M AAPH were
added.Thevariationofthefluorescenceintensitywithtimewasmonitored
for 60 min using 485 and 535 nm filters to select excitation and emission
wavelengths,respectively.Thecurvesobtainedwereprocessedinthesame
wayasthatdescribedfortheORACNBmethod.

3.3.DeterminationofAntioxidantCapacityinCommercialWineandFruitJuice
Samples
Several wines (white, semidry and red) and fruit juices (peach,
pineapple and apple) were bought in a local market and analysed
immediatelyaftertheywereopenedaccordingtothefollowingprocedure:
A volume (4.5 mL) of sample was treated with 300 L of 1 M NaOH to
increase the pH to neutral values, and it was raised up to 5 mL with
phosphate buffer. Then, an adequate dilution (1:500 1:10000 dilutions)
with the same phosphate buffer was performed to match the dynamic
rangesofthecalibrationcurvesofeitherORACFLorORACNBmethod.
A volume (20 L) of the diluted sample was treated according to the
proceduresindicatedaboveforbothmethods.

203

Captulo3

4.Resultsanddiscussion
4.1.SelectionoftheLongWavelengthFluorophor
ROS, such as peroxyl radicals (ROO), hydroxyl radicals (OH),
superoxide ion (O2), and singlet oxygen (1O2) are involved in the
physiology of some diseases. Radical chainbreaking antioxidants convert
reactive free radicals into stable and nonaggressive molecules by
mechanisms in which AAPH radicals formed in airsaturated solutions
reactrapidlywithmolecularoxygentogiverisetoperoxylradicals,ROO,
as described elsewhere [6]. The presence of antioxidant compounds gives
rise to the formation of a hydroperoxide and a stable antioxidant radical
that breaks the action of peroxide radicals. Although a wide variety of
antioxidant compounds can be used as reference standards in ORAC
assays [13], such as gallic, caffeic and ascorbic acids, the vitamin E
analogue Trolox has been chosen to develop the ORACNB method
presentedhere.
The reactions involving the formation of free radicals can be
followed by the decrease in the inhibition of the fluorescence from some
organic molecules, such as phycoerythrin and FL, in the presence of
sampleswithantioxidantcapacity[114].However,theshortStokesshiftof
these compounds can give rise to lightscattering phenomena that could
affecttheperformanceoffluorescencemeasurements.
Also, phycoerythrin shows a low photostability, and its price is
relatively high. With the aim of studying the potential use of fluorescent
dyesthatemitintheredregionofthespectrumasalternativereagentsfor
thispurpose,severaloxazineandthiazinedyes,namelyNB,azureAand
azure B, were assessed. Figure 2 shows the curves obtained for each

204

Determinacindeantioxidantesenalimentos

fluorophor, in the presence and absence of Trolox, which was used as

standard.

18000

18000

22

16000

a)

Relative fluorescence intensity

Relative fluorescence intensity

16000
14000
12000
10000

11

8000
6000
4000

b)

14000
12000
10000

8000
6000

4000

2000

2000

0
0

20

40

60

80

100

20

40

Time (min)

60

80

100

Time (min)

18000

Relative fluorescence intensity

16000

c)

14000
12000

10000
8000

6000

4000
2000
0
0

20

40

60

Time (min)

80

10

Figure6:CurvesobtainedfortheORACassayusing(a)nileblue,(b)azureA,
and(c)azureBfluorophores:(a)blankand(2)2MTrolox.

Although it was possible to measure the difference between the

blank and standard with all the fluorophors assayed, NB gave the best
results. Also, NB was chosen because it has a wider Stokes shift that the

205

Captulo3

other two reagents, which avoids potential lightscattering signals. The

decaycurvesobtainedinthepresenceofNB,AAPH,anddifferentTrolox
concentrationsareshowninFigure3,inwhichtheAUCincreasedwiththe
Troloxconcentration.

16000

Relative fluorescence intensity

14000

14

12000
10000
8000
6000
4000
2000
0
0

10

20

30

Time (min)

40

50

Figure 3. Antioxidant capacity curves obtained at different Trolox

concentrations: (1) 0 M, (2) 1 M, (3) 2 M, and (4) 4 M. Experimental
conditions: [NB], 89 nM; [AAPH], 0.012 M; pH, 6.9; [phosphate], 0.085; and
temperature,37C.

4.2.OptimisationoftheORACNBMethod

Thevariablesaffectingthesystemwereoptimisedbytheunivariate
method.Eachresultwastheaverageofthreemeasurements.Theanalytical

206

Determinacindeantioxidantesenalimentos

parameter used to optimise the system was the net AUC, calculated as
indicatedaboveintheprocedureoftheORACNBmethod.
The influence of the pH was investigated in the range of 4 11,
using0.075Macetate,phosphate,borateand,carbonatebuffersolutionsto
keepthepHconstantinthebufferingregionofeachsolution.AsFigure4A
shows, there was not appreciable net AUC at pH values below 5.8 and
above8.ThebehaviourofthesystematlowpHvaluescouldbeascribedto
thefactthatthepKavalueofTroloxisabout3.89,showingalowsolubility
at this pH [22]. The solubility increases with the pH, which improves the
valueofthenetAUC,obtainingthebestresultsinthepHrangeof6.07.0.
A pH of 6.9 was chosen, which is slightly lower than that required to
develop the ORACFL method (pH 7.4). The influence of buffer
concentration was studied by assaying phosphate concentrations in the
range of 0.02 0.15 M, finding that a 0.085 M phosphate buffer solution
gavethebestsignal.
NB and AAPH concentrations are two critical variables that are
interrelated.Theinfluenceofthefluorophorconcentrationwasstudiedby
adding a fixed volume (120 L) of solutions with NB concentrations
ranging from 60 to 370 nM (Figure 4B). The system was practically
independentonthisvariableintherange80130nM,choosing89nMNB
for the development of the method. The influence of the peroxyl radical
generator, AAPH, was evaluated in the range of 0.006 0.024 M, finding
thattheAUCsignalsofbothanalyteandblanksolutionsdecrease,withthe
difference also decreasing between both signals, when the AAPH
concentration increases. However, the curves obtained at low AAPH
concentrations were less defined

and

207

showed

low

Captulo3

reproducibility.Thus,a0.012MAAPHconcentrationwaschosen.

0.16

0.10

A)

B)

0.08

0.12

AUC

AUC

0.06

0.08

0.04
0.04
0.02
0.00

0.00

5.5

6.0

6.5

7.0

7.5

8.0

8.5

pH

100

200

300

400

[NB], nM

Figure 4. Influence of pH (A) and (B) NB concentration on the ORACNB

method. In panel A, [NB], 120 nM; [phosphate], 0.075 M; [AAPH], 0.024 M;
[Trolox],2M;andtemperature,27C.InpanelB,[AAPH],0.012M;[Trolox],
2M;pH,6.9;[phosphate],0.085M;andtemperature,37C.

The temperature is an important variable in kinetic studies, and

also,ithasaremarkableeffectonthedecompositionofAAPH[7],mainly
at values close to 37 C. The influence of this variable on the system was
studied by mixing NB with Trolox for 15 min at 37 C and, and after the
addition of 60 L of 0.012 M AAPH, the mixture was kept at different
temperatures,intherange25C40C.Thekineticcurvesobtainedat25
C were relatively slow, providing a net AUC value 1.5 times lower than
thatobtainedat37C.Thistemperaturewaschosentodevelopthemethod
becausethecurvesobtainedwerealsomoredefined,whichcouldprobably
be ascribed to the fact that the decomposition reaction of AAPH was not

208

Determinacindeantioxidantesenalimentos

thelimitingstep.ThemethodusingFLasreagent[8]isalsodevelopedat
37C.

4.3.AnalyticalFeatures
Thekineticcurveswereobtainedunderoptimumconditions,using
ex of 620 nm and em of 680 nm to monitor the variation of the
fluorescence intensity with time for 60 min and the net AUC as the
analyticalparameter.Thedynamicrangeofthecalibrationgraphwas0.8
8 M Trolox. The regression equation was AUC = (0.02 0.01) + (0.074
0.006) X, where X was the Trolox concentration expressed in micromolar.
The regression coefficient (R) is 0.993, which is indicative of a good
linearityofthecalibrationcurve.Thedetectionlimit,calculatedfollowing
International Union of Pure and Applied Chemistry (IUPAC)
recommendations[23],was0.45M,whichislower[6,7]orsimilar[8]to
thoseobtainedinotherORACmethodsinvolvingFL.Theprecisionofthe
method was assessed attwo different Trolox concentrations,1 and5 M,
andexpressedasthepercentageofrelativestandarddeviation,giving5.6
and2.9%,respectively.

4.4.Applications
The proposed method was applied to the analysis of wines,
namely, white, semidry and red, and commercial fruit juices, namely,
pineapple,peachandapple.ThesesampleswereanalyzedbybothORAC
FL and ORACNB methods with a simple sample treatment, which
consisted in the adjustment of the pH to neutral values, because the
samples

featured

acidic

pH

values. Then, the samples were

209

Captulo3

diluted to accommodate the antioxidant content to the dynamic range of

thecalibrationcurve.Figure5showsthecurvesobtainedfortheblankand
those obtained at different sample dilutions for (A) white wine and (B)
pineapple juice samples. It can be seen from these curves that the area
proportionallyincreasedasthesampledilutiondecreased.ThenetAUCof
the samples was measured as (AUCsample AUCblank)/AUCblank, and this
result was interpolated in the calibration curve obtained for Trolox
standards.

20000

20000

16000

A)

B)
Relative Fluorescence Intensity

Relative Fluorescence Intensity

12000

8000

4000

16000

12000

8000

4000

0
0

20

40

60

Time (min)

80

100

20

40

60

80

100

Time (min)

Figure5.Antioxidantcapacitycurvesobtainedinthepresenceof(A)semidry
white wine and (B) pineapple juice samples at different sample dilutions.
Experimentalconditionsareasfollow:[NB],89nM;[AAPH],0.024M,pH,6.9;
[phosphate],0.085M;andtemperature,27C.InpanelA,(1)blankand(2,3
and 4) are 1/4000, 1/2000, and 1/1000 dilutions of the white wine sample,
respectively.InpanelB(1)blank.

After the application of the corresponding dilution factors, the

antioxidant content of each analyzed sample, expressed as millimolar
Trolox Equivalents of sample, was calculated. Table 1 lists the content

210

Determinacindeantioxidantesenalimentos

found in the samples using both ORACNB and ORACFL methods. The
paired t test was applied to the results at a 95% significance level, and it
was found that there were not significant differences in the results
provided by both methods, which confirms the practical utility of the
proposedORACNBmethodtotheanalysisofthesefoodsamples.

Table 1. Antioxidant Capacity (Expressed as millimolar Trolox Equivalents

of Sample) of Food Samples Analyzed by ORACNB and ORACFL
Methods

Sample
ORACNBa
ORACFLa

whitewine(manzanilla)

1.500.09

1.30.2

semidrywine

7.70.9

4.40.4

redwine

15.30.4

10.40.9

2.2220.001

1.930.09

pineapplejuice

2.70.3

2.3990.006

applejuice

2.70.3

3.50.4

peachjuice

Meanstandarddeviation(SD)(n=3)

Also,thevaluesfoundforbothtypesofsamplesagreewithvalues
reportedintheliterature[9,10].

A recovery study was also carried out to validate the method. It

wasperformedbyaddingthreedifferentamountsofTroloxtoeachsample
and by subtracting the results obtained from similarly treated unspiked
samples.Table2showstherecoverypercentages,whichrangedfrom72.7

211

Captulo3

to 113.6%. The mean recovery values obtained were 92.0 and 92.9 % for
wineandjuicesamples,respectively.Thisinternalvalidationalsoconfirms
theusefulnessofthedevelopedORACNBmethodfortheanalysisofreal
samples.

Table2.Recoveryvaluesobtainedforthedifferentsamplesanalyzed
Recoverystudy

Sample
Addeda
Founda,b
Recovery(%)
Whitewine
2.2
2.00.2
92.0
8.9
6.70.9
75.2
11.1
9.20.8
82.9

Semidrywine

4.4
8.8
13.2

3.60.2
8.10.3
12.90.6

81.8
92.1
97.0

Redwine

17.8
35.6
44.4

182
384
444

101.1
106.7
99.1

Peachjuice

2.2
4.4
5.5

2.10.1
3.50.4
4.30.2

95.5
79.5
78.2

Pineapplejuice

2.2
4.4
5.5

2.50.2
4.50.3
5.80.4

113.6
102.3
105.5

Applejuice

4.4
3.20.2
8.8
7.90.5
11.1
111
aUnitsinmillimolarTroloxequivalentsofsample.
bMeanSD(n=3).

72.7
89.6
99.1

The results obtained show the feasibility of long wavelength

fluorimetryforthedeterminationofantioxidantcapacityinfoodsusingthe

212

Determinacindeantioxidantesenalimentos

fluorophor NB for the first time as an analytical reagent. The use of this
reagent instead of other fluorophores previously proposed for this
purpose, such as FL or phycoerythrin, is a useful alternative to avoid
potentialbackgroundsignalsfromthesamplematrix,whichcanappearat
lower wavelengths. Also, the relatively wide Stokes shift of NB allows
analytical measurements to be free of scattering signals, which can be a
limitation when the abovementioned fluorophores are used. Finally, the
probability of photobleaching processes for NB is lower than that for
phycoerythrin,aswellasitscost.

Acknowledgement
The authors gratefully acknowledge financial support from the
MinisteriodeCienciaeInnovacin(MICINN)(GrantCTQ200908621)and
fromtheJuntaofAndalucia(GrantP09FQM4933).

Abbreviationsused
AAPH,2,2azobis(2methylpropionamidine)dihydrochloride;AUC,area
underthecurve;BHA,butylhydroxyanisole;FL,fluorescein;NB,nileblue;
ORAC,oxygenradicalabsorbancecapacity;ROS,reactiveoxygenspecies

213

Captulo3

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A. Dvalos, B. Bartolom, C. GmezCordovs. Antioxidant

properties of commercial grape juices and vinegars. Food Chem.
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J. Tabart, C. Kevers, J. Pincemail, J. O. Defraigne, J. Dommes.

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L. Mller, S. Groyke, A.M. Popken,V. Bhm. Antioxidantcapacity

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217

Determinacindeantioxidantesenalimentos

Automaticdeterminationofpolyphenolsinwines
usinglaccaseandterbiumoxidenanoparticles
J.GodoyNavajas,M.P.AguilarCaballos,A.GmezHens
DepartmentofAnalyticalChemistry,UniversityofCordoba,Campus
ofRabanales.AnnextoMarieCurieBuilding.14071Cordoba.Spain

Abstract
The analytical usefulness of the combined use of laccase, terbium
oxide

nanoparticles

(Tb4O7NPs)

and

8hydroxypyrene3sulfonate

trisodium(HPTS)forthedeterminationofpolyphenolcompoundsinwine
samples is described. The system is based on the temporal inhibition by
polyphenols on the decrease of the HPTS fluorescence in the presence of
laccase and on the activating effect of Tb4O7NPs, which increase the
reactionrateofthesystem,shorteninganalysistimes.

The method has been developed in a microplate format using an

automatic reader, reaching a sample throughput of 35 samples h1. The

dynamicrangeofthecalibrationgraphis0.512Mgallicacid,whichwas
chosenasmodelanalyte,andthedetectionlimitis0.14M.Precisiondata,
expressedasrelativestandarddeviation,rangedbetween2.5and6.3%.The
method was applied to the analysis of several wine samples, obtaining
recoveryvaluesintherangeof80.0108.3%.

Keywords: Polyphenols, terbium oxide nanoparticles, laccase, fluorometric

detection,wineanalysis.

219

Captulo3

1.Introduction

Polyphenols are an important group of natural antioxidant

compounds present in the human diet that, after numerous

epidemiological and clinical studies, have shown their usefulness in the
preventionofvariouschronicdiseases.Theinterestinthedeterminationof
these compounds has given rise to chromatographic (Lorrain, Ky,
Pechamat & Teissedre, 2013; Mediari, Rastija & Boji, 2011) and
capillary electrophoretic (FranquetGriell, Checa, Nez, Saurina,
HernndezCassou & Puignou, 2012; SnchezArribas, Martnez
Fernndez & Chicharro, 2012) methods for their identification and
quantification. However, the high variety of compounds that form this
group justifies the development of methods devoted to determine phenol
content and antioxidant activity, which is another related parameter that
includes nonphenolic antioxidants. Both parameters are widely
determinedinfruitsandbeveragessuchaswines.
The photometric FolinCiocalteu method (Magalhes, Segundo,
Reis,Lima&Rangel,2006;Nenadis,Lazaridou&Tsimidou,2007;Snchez
Rangel, Benavides, Heredia, CisnerosZevallos & JacoboVelzquez, 2013)
isusuallychosenforphenoliccontentdetermination,usingapolyphenol,
such as gallic acid, as calibration standard. However, a limitation of this
method is that usually provides an overestimation of these compounds
duetointerferencesfromsomenonphenolicreducingsubstances.Several
methods involving the measurement of scavenging capacity of
antioxidants against a given radical have been described for antioxidant
activity determination (Sochor et al., 2010; Dawidowicz, Wianowska &
Olszowy, 2012; Nkhili & Prat, 2011; GodoyNavajas, AguilarCaballos &

220

Determinacindeantioxidantesenalimentos

GmezHens,2011).MostofthesemethodsuseTrolox(6hydroxy2,5,7,8
tetramethylchroman2carboxylicacid),whichisawatersolubleanalogue
of vitamin E, as calibration standard. Although several studies have been
carriedoutusingdifferentwinetypestorelatethephenolcontentwiththe
antioxidant activity, the correlation coefficients obtained are usually low
(FernndezPachn,Villao,GarcaParrilla&Troncoso,2004;DiMajo,La
Guardia, Giammanco, La Neve & Giammanco, 2008; Li, Wang, Li, Li &
Wang,2009),whichistheresultofthedifferentantioxidantpotentialofthe
phenolcompoundspresentinwines.
Laccaseisaphenoloxidaseenzymethathasbeendescribedforthe
determination of both phenolic content and antioxidant activity, using
different experimental conditions. This enzyme catalyzes the oxidation of
phenolic compounds to quinones or radicals by reducing the dissolved
oxygen to water. Several laccasebased amperometric sensors have been
described for polyphenol determination in wines (Gamella, Campuzano,
Reviejo & Pingarrn, 2006; Di Fusco, Tortolini, Deriu & Mazzei, 2010;
Montereali,DellaSeta,Vastarella&Pilloton,2010)usinggallicacidasthe
polyphenol standard. Although the detection limits of these methods are
adequatefortheiruseinroutineanalysis,theyshowalimitedlifetimethat
is ascribed to the adsorption of oxidized products on the surface of the
electrode with the consequent negative effect on the enzyme activity. A
fluorometric method has been reported for the determination of
polyphenolcontentinjuiceandteasamplesusingindocyaninegreenand
positively charged gold nanoparticles (AndreuNavarro, Fernndez
Romero & GmezHens, 2012). The method is based on the temporal
inhibitioncausedbypolyphenolsontheoxidationofthefluorophoreand

221

Captulo3

the modification of the enzyme activity by its interaction with the

nanoparticles. Laccase has also been described for the determination of
antioxidant activity using high reactive chromophore substrates, such as
2,2azinobis(3ethylbenzothiazolin6sulfonic) acid (ABTS) (Kulys &
Bratkovskaya, 2007) or syringaldazine (NugrohoPratseyo, Kudanga,
Steiner, Murkovic, Nyanhongo & Guebitz, 2009; NugrohoPratseyo,
Kudanga, Steiner, Murkovic, Nyanhongo & Guebitz, 2010), which are
oxidized to free radicals that react with the phenolic and nonphenolic
antioxidantspresentinthesample.Inadditiontotheuseoflaccaseinfood
analysis,thisenzymehasbeenalsoappliedindetoxificationproceduresof
effluents from chemical industries, in which the degradation of some
compounds,suchasphenolisperformed(Wang,Hu,Guo,Huang&Liu,
2012). Also, it has been used in textile industries for dye decolourization
anddegradation(Benzinaetal.,2013;Ashrafi,Rezaei,Foroontanfar,Mahvi
&Faramarzi,2013;Taha,Shwaish,Mohamed,Haider&Stamatis,2013).
A feature of the methods described for the determination of
polyphenol content and antioxidant capability is that measurements are
usually performed sequentially, so that the sample throughput is lower
thaninsystemsoperatingsimultaneouslysuchasthoseinvolvingtheuse
ofamicroplatereader.Thisapproachhasbeenhereusedforthefirsttime
to develop an automatic method for the determination of polyphenols in
winesusinglaccaseandthefluorophore8hydroxypyrene1,3,6trisulfonic
acid trisodium salt (HPTS or pyranine) in the presence of Tb4O7
nanoparticles (NPs). Other aims of this research were the study of the
potential usefulness of HPTS as a laccase substrate and the capacity of
theseNPstoplayaroleaslaccaseactivators.

222

Determinacindeantioxidantesenalimentos

HPTS has been previously used for the evaluation of the

antioxidantpotentialofpolyphenolsbymeasuringthebleachingofHPTS
by peroxyl radicals using 2,2azobis(2amidinopropane) hydrochloride
(AAPH) as the free radical source (Campos, Sotomayour, Pino & Lissi,
2004).However,thepracticalapplicationofthismethodwasnotdescribed.
A comparative study of the radicalscavenging capacity of red wine has
beencarriedoutusingthebleachingofHPTSandpyrogallolredinduced
by free radicals generated from an azo initiator (Omata, Saito, Yoshida &
Niki, 2008). This study showed that HTPS has much lower reactivity
towardperoxylradicalsthanpyrogallolred,whichallowsantioxidantsto
inhibit HTPS bleaching almost completely to produce a lag phase, from
which the total amounts of free radicals that can be scavenged by
antioxidants, may be estimated. The results obtained in the study carried
out here have shown the usefulness of HPTS as a laccase substrate for
polyphenoldeterminationandthepositiveeffectofTb4O7NPstoimprove
thefeaturesofthedevelopedmethod.

2.Experimental
2.1Instrumentation

A 1420 multilabel counter Victor 3V microplate reader (Perkin

Elmer and Analytical Sciences, Wallac Oy, Turku, Finland) was used to
perform

fluorescence

measurements.

Different

filters

(nominal

wavelength/passband) were used to select excitation and emission

wavelengths of several fluorophores assayed in the study (Table 1) and
eachmeasurementwasmadein0.5s.AnSLMAminco(Urbana,IL,USA),
Model 8100 photoncounting spectrofluorometer, equipped with a 450 W

223

Captulo3

xenon arc source and a R928 photomultiplier tube, was used to perform
fluorescenceexcitationandemissionscansofthedifferentfluorophoresin
solution and photobleaching experiments. A Lambda 35 UV/VIS
spectrometer (PerkinElmer, United Kingdom) was used to perform
photometricmeasurements of the FolinCiocalteumethod. 1/2AreaPlateTM
microplates(PerkinElmer,USA)withatotalvolumeof180 Lwereusedto
recordthekineticcurvesofthesystem.

2.2Reagents
All reagents used were of analytical grade. The fluorophores 8
hydroxypyrene1,3,6trisulfonic acid trisodium salt (HPTS), 2[4
(dimethylamino)styryl]1methylpyridinium iodide (2Di1ASP), 2[4
(dimethylaminophenyl]1,3butadienyl)3ethylbenzothiazolium

toluenesulfonate salt (Styryl 7), nile blue chloride, azure B chloride and
toluidineblueOwereprovidedbySigmaAldrich(St.Louis,Mo,USA),as
wellasTween20,laccaseenzyme(TrametesVersicolor,ECNumber1.10.3.2,
2.05 U/mg), and caffeic acid. The surfactants Triton X100 and hexadecyl
trimethylammoniumbromide(CTAB)werepurchasedfromFluka(Buchs,
Switzerland).Europium(III)oxidenanoparticles(Eu2O3nanopowder,<150
nm), diamond nanoparticles (<10 nm particle size) and gallic acid were
acquired from Sigma. Terbium oxide nanopowder (TEM <150 nm), silver
nanoparticles (10 nm, in aqueous buffer containing citrate as stabilizer),
terbium nitrate pentahydrate and sodium chloride were purchased for
SigmaAldrichandfluoresceinsodiumwaspurchasedforFluka.Ascorbic
acid was acquired from SigmaAldrich while citric acid and sulfuric acid
were purchased from Panreac (Barcelona, Spain). Glucose and sucrose

224

Determinacindeantioxidantesenalimentos

werepurchasedfromSigmaAldrichandFluka,respectively.Dipotassium
hydrogen phosphate, tris(hydroxymethyl)aminomethane (TRIS), sodium
sulfite, malic acid and sodium hydroxide were purchased from Merck
(Darmstadt, Germany). Sodium carbonate, sodium acetate and sodium
dodecyl sulfate (SDS), FolinCiocalteus phenol reagent (2N) and 6
hydroxy2,5,7,8tetramethylchromane2carboxylic acid (Trolox) were
providedfromSigmaAdrich.
Phosphate buffer solution (0.4 M pH 7.0) was prepared by
dissolvinganappropriateamountofdipotassiumhydrogenphosphatesalt
(Merck)indistilledwaterandadjustingthepHwithhydrochloricacid.A
HPTSstocksolution(890M)waspreparedbydissolvingtheappropriate
amount of the fluorophore in distilled water by magnetic stirring for
several minutes and stored at room temperature. Working solutions (10
MHPTS)wereprepareddailybydilutingtheappropriatevolumeofthe
stocksolutioninthephosphatebuffersolution.A10mMstocksolutionof
gallicacidwasprepareddailybydissolvingtheappropriateamountofthe
reagent in distilled water and the different standards solutions were
prepareddilutingtheappropriatevolumeofthestocksolutionindistilled
water. A 4 mg/mL stock Tb4O7 NPs dispersion was prepared daily by
dispersing the appropriate amount of nanopowder in distilled water by
using an ultrasound bath. Working solutions were prepared by diluting
the stock dispersion in the phosphate buffer solution. A laccase stock
solution (4 U/mL) was prepared daily by sonication for 10 s and let
overnighttostandbeforeuse.Workinglaccasesolutionswerepreparedby
diluting the appropriate volume of the stock solution in the phosphate
buffersolution.

225

Captulo3

2.3.Procedures
2.3.1.DeterminationofpolyphenolsusingthelaccaseTb4O7NPmethod
A volume (75 L) of zero standard,gallic acid standards (0.5 12
M) or diluted wine sample was firstly added to each well of the
microplatetogetherwith15Lof10MHPTS.Thismixturewaskeptfor
15minutesat37 oCtoreachconstanttemperatureand,then,avolume(60
L) of a mixture containing 1 mg/mL Tb4O7 NPs and 1 U/mL laccase in
phosphate buffer solution was added to each well by using an electronic
multichannelmicropipette.Immediatelyafter,themicroplatewasinserted
intothemicroplatereader,andthevariationofthefluorescenceintensityof
HPTS with time was monitored at 37 oC for 36 min, using nominal
excitationandemissionwavelengthfiltersof450and535nm,respectively.
Normalized decay curves were obtained by dividing the fluorescence
signals obtained at each time by the fluorescence signal obtained at time
zero for each curve. These curves were later integrated using Origin
software, and then, the net normalized area under the curve (AUC) was
calculatedbysubtractingtheAUCfortheblankfromtheAUCobtainedin
thepresenceofgallicacid.

2.3.2.Analysisofwinesamples

Winesamples(red,whiteandsweetwinesamples)wereboughtat

a local supermarket and analyzed immediately after they were opened.

Samples were diluted according to their content to match the dynamic
rangeofthecalibrationcurve.Thedilutionswereintherangesof1:4000

226

Determinacindeantioxidantesenalimentos

1:8000 for red, 1:100 1:500 for white and 1:500 1:2000 for sweet wine
samples.

2.3.3.FolinCiocalteumethod
Thismethodwasperformedasdescribedelsewhere(Magalheset
al., 2006). Briefly, a volume (2.5 mL) of gallic acid standard (15150 M
preparedindistilledwater)ordilutedwinesamplewasmixedwith500L
ofFolinCiocalteureagent(heteropolyphosphotungstatemolybdate)and5
mL of sodium carbonate (60 g/L). This mixture was kept at room
temperaturefor2hand,afterwards,theabsorbancewasmeasuredat760
nm.

3.ResultsandDiscussion
3.1.Studyofthesystem
This study was carried out to choose the experimental conditions
that allow the development of a fast and automatic fluorometric method
for the determination of polyphenol compounds using laccase. The study
was carried out in an automatic system involving the use of microplates
with a volume of 180 L for each well, so that the sample and reagent
consumptionwaslowerthaninconventionalmicroplates(350400L).
Several potential fluorescent laccase substrates (Styryl 7, 2Di1
ASP, azure B chloride, toluidine blue O, nile blue chloride, sodium
fluorescein and HPTS) (Table 1) were assayed to study their capability to
compete with phenolic antioxidants for the active sites of the enzyme.
Although indocyanine green has been described as a laccase substrate
(AndreuNavarro et al., 2012), it was not assayed because its long

227

Captulo3

excitationandemissionwavelengthswerenotadequateforthemicroplate
readerusedinthisstudy.
Table1.Fluorophoresassayedaspotentialsubstratesoflaccaseenzymein
thisstudy
Fluorophore

O O
HO
S

ONa

NaO
ONa
S
S
O O

O O
8Hydroxypyrene1,3,6trisulfonicacidtrisodiumsalt(HPTS)

I+

N
CH3
CH3

N
CH3

2[4(Dimethylamino)styryl]1methylpyridiniumiodide(2
Di1ASP)

O S O

CH3
H3C
+
N

CH3
S
N

CH3
2[4(Dimethylaminophenyl]1,3butadienyl)3ethyl
benzothiazoliumptoluenesulfonatesalt(Styryl7)

Excitationfilter

Emissionfilter

450/6

535/25

485/15

535/25

531/25

680/10

485/15

535/25

620/8

680/10

620/8

680/10

620/8

680/10

ONa
O

NaO

Sodiumfluorescein
H3C

H2N

Cl+
N CH3
CH3

ToluidineblueO

N
ClH2N

N
CH3

Nilebluechloride

H3C N
S

H
AzureBchloride

CH3
Cl+
N CH3
CH3

228

Determinacindeantioxidantesenalimentos

Figure 1 shows the behavior of some of the fluorophores assayed

onthesystem,inwhichcanbeseenthatonlyHPTSshowsarelativelyfast
decreaseinitsfluorescenceintensity.Theseresultscouldbeascribedtothe
factthatHPTShasaphenolgroup,unliketheotherfluorophoresassayed,
allowingitsenzymaticoxidationbydissolvedoxygen.Anotherfeatureof
HPTS that could be related with its behavior in the presence of laccase is
thatthiscompoundisaweakacidinthegroundstate,withapKof7.4,but
this value decreases up to 0.4 in the excited state (Mondal, Ghosh, Sahu,
Sen & Battacharyya, 2007), behaving as a strong acid when fluorescence
measurementsareobtained.

Normalized fluorescence intensity

1.0

1
2
3

0.8

0.6

0.4

0.2

10

20

30

40

Time (min)

Figure1.Behaviorofdifferentfluorophoresinthepresenceoflaccase.1)azure
Bchloride,2)sodiumfluorescein,3)toluidineblueO,4)2Di1ASP,5)HPTS.
Experimentalconditions:In1),3)and4)[fluorophore]=5M,[laccase]=0.2
U/mL.In2)and5)[fluorophore]=1M,[laccase]=0.1U/mL.Alltheassays
werecarriedoutusingphosphatebuffersolution(0.2M,pH7.0).

229

Captulo3

Thepresenceofgallicorcaffeicacidonthesystemcausedadelay
on the fluorescence decrease of HPTS, ascribed to the sequential catalytic
effectoflaccase,whichactsoverthefluorophorewhenthepolyphenolhas
been oxidized. Figure 2 shows the kinetic curves obtained in the absence
andinthepresenceofgallicacid.

1.2

Normalized fluorescence Intensity

14

1.0

0.8

0.6

0.4

0.2

0.0

10

15

20

Time (min)

Figure 2. Kinetic curves obtained for the HPTSlaccase system in the absence
(curves1and2)andinthepresence(curves3and4)of2Mgallicacid,andin
thepresence(curves1and3)andintheabsence(curves2and4)of1mg/ml
Tb4O7NPs.[HPTS]=10M,[laccase]=1U/mL,pH=7.0,[phosphate]=0.4M,
temperature37C.

230

Determinacindeantioxidantesenalimentos

Another assay carried out to improve the features of the system

was the study of the potential activator effect of different NPs on the
catalyticbehavioroflaccase.ThepositiveeffectofcopperoxideNPsonthe
degrading activity of laccase has been previously described using
syringaldazine, but the activation mechanism of these NPs has not been
elucidated (Mukhopadhyay, Dasgupta & Chakrabarti, 2013). Although
Cu(II) in solution also caused an activation effect, it was lower than that
obtainedinthepresenceoftheNPs.SeveralNPs,suchasdiamond,Eu2O3,
Tb4O7, magneticgold NPs and silver NPs (AgNPs) were assayed in the
range of 0.12 mg/mL, finding that only Tb4O7 NPs notably increased the
reactionrateofthesystem.BothblankandanalyteAUCdecreased,butthe
net AUC difference was better than in the absence of the NPs, as can be
seeninFigure2.Also,theeffectofTb4O7NPsonthesystemwaschecked
in the absence of laccase, finding that the NPs did not modify the
fluorescence of HPTS. Diamond and magneticgold NPs caused the
fluorescencequenchingofHPTSandAgNPsinhibitedthecatalyticeffectof
laccase,whilenochangeonthekineticbehaviorofthesystemwasfound
inthepresenceofEu2O3NPs.Terbium(III)ionsinsolutionwereassayedat
equivalentconcentrationsthanTb4O7NPs,obtaininglowernetAUCvalues
than using the NPs. These results show the usefulness of these NPs as
activators of the enzymatic system. Terbium(III) in solution has been
recently described as an activator of deoxyribozymes, which are DNA
catalysts(JavadiZarnaghi&Hbartner,2013),buttheuseofTb4O7NPsas
acatalystactivatorhasnotbeendescribeduptodate.
The effect of cationic (CTAB), anionic (SDS) and nonionic (Triton
X100 and Tween 20) surfactants on the system was assayed below and

231

Captulo3

abovetheircriticalmicellarconcentration.ThestronginteractionofHPTS
with CTAB, which has been previously described (Pramanik, Banerjee &
Bhattacharya, 2007), gave rise to a decrease of the reaction rate of the
system, which made difficult to develop an assay with a suitable sample
throughput.ThepresenceofSDSandTritonX100causedadecreaseinthe
fluorescenceintensityofHPTS,decreasingalsotheAUCvaluesobtainedin
the absence and the presence of the analyte. Finally, the fluorescence of
HPTSandthereactionrateofthesystemwerenotaffectedinthepresence
ofTween20.Accordingtotheseresults,surfactantswerenotusedforthe
developmentofthesystem.

3.2.Optimizationofthesystem
Thevariablesaffectingthesystemwereoptimizedbytheunivariate
method using the net AUC as the analytical parameter, which was
calculatedasindicatedintheprocedure.However,itwasnecessarytofind
acompromisedsolutionbetweenthisparameterandthetimerequiredto
obtain each measurement, in order to avoid the excessive duration of the
procedure.Eachresultwastheaverageofthreemeasurements.
TheinfluenceofthepHonthesystemwasstudiedintherangeof
4.09.0 using different buffers solutions, such as acetic acid/acetate,
phosphate and carbonate at a 0.2 M concentration. The fluorescence of
HPTS was very low at pH 4.0 in the presence of acetate ions and it
remained constant without undergoing any oxidation reaction with
laccase.HPTSshowedahighfluorescenceatpHvaluesintherangeof8.0
9.0, but it was not modified in the presence of laccase. However,
appropriate net AUC values were obtained at pH 7.0 7.5. Hexamine,

232

Determinacindeantioxidantesenalimentos

imidazole and phosphate buffer solutions were assayed to adjust the pH

withinthisrange,obtainingthebestnetAUCinthepresenceofphosphate
buffersolutionintheconcentrationrangeof0.20.5M.Theinfluenceof
the HPTS concentration was studied adding a fixed volume (15 L) of
solutionswithHPTSconcentrationsrangingfrom2.5to20M,obtaining
that the system was independent of this variablein the range of 812 M
HPTSconcentration.
Laccase activity and Tb4O7 NPs concentration are two critical
variables that have a remarkable effect on both the net AUC and the
durationofthekineticcurve.Bothvariableswerestudiedintherangesof
0.5 2 U/mL and 0.2 2 mg/mL for laccase activity and Tb4O7NP
concentration,respectively.Highvaluesofbothvariablesgaverisetovery
high reaction rates and low net AUC, which has a negative effect on the
sensitivity of the method. Several assays were carried out using different
valuesof these variables, obtainingappropriate net AUC and duration of
theprocessinthepresenceof1U/mLlaccaseand1mg/mLNPs.AsFigure
2shows,theactivationeffectoftheNPsonthecatalyticactionoflaccaseis
demonstrated under these experimental conditions. The temperature was
studiedintherangeof2040C,andthebestresultswereobtainedat37
C.

3.3.Analyticalfeatures

The net AUC values were obtained from the normalized kinetic

curves, monitored at ex 450 nm and em 535 nm under the optimum

experimental conditions for differentgallicacid concentrations (Figure 3).
Thedynamicrangeofthecalibrationgraphwas0.512Mgallicacidand

233

Captulo3

theregressionequationwas:AUC=(0.400.05)+(2.40.6)X,inwhichXis
the gallic acid concentration expressed as micromolar. The regression
coefficient (R) was 0.996, which is indicative of the good linearity of the
calibration curve. The limit of detection (LOD), calculated according to
IUPACrecommendations(Long&Winefordner,1983),was0.14M,while
thevalueobtainedintheabsenceofTb4O7NPswas0.4M,whichshows
thepositiveeffectoftheNPsonthemethod.

Normalized fluorescence intensity

1.0

16

0.8

0.6

0.4

0.2

0.0

10

20

30

40

Time (min)

Figure 3. Kinetic curves obtained for different gallic acid concentrations: 1) 0

M, 2) 0.5 M, 3) 1 M, 4) 2 M, 5) 5 M and 6) 10 M. [HPTS] = 10 M,
[laccase] = 1 U/mL, [Tb4O7 NPs] = 1 mg/mL, pH = 7.0, [phosphate] = 0.4 M,
temperature=37C.

234

Determinacindeantioxidantesenalimentos

The LOD reached in the presence of the NPs is lower than those
describedforgallicacidusingsomeamperometricbiosensors(DiFuscoet
al., 2010; Montereali et al., 2010). Although a LOD of 0.04 M has been
obtained for gallic acid using indocyanine green (AndreuNavarro et al.,
2012), this method involves the sequential analysis of each sample,
requiring about 15 min to carry out each measurement. However, the
automation of the measurements using a microplate reader allows a
samplethroughputof35samplesh1(threemeasurementsforeachsample)
in the new method. The precision of the method, expressed as the
percentage of relative standard deviation, was assessed at 0.7 and 5 M
gallicacidconcentrations,providingvaluesof6.3and2.5%,respectively.
Theselectivityofthemethodwascheckedbyassayingsomenon
phenolic reducing substances, which could be present in wines. A
compoundwasconsiderednottointerfereiftheanalyticalsignalobtained
initspresencewaswithinonestandarddeviationthesignalobtainedinits
absence. Table 2 shows the results obtained after assaying different
potentialinterferingcompounds.Themostseriousinterferencewascaused
byascorbicacid,whichwastoleratedatthesamemolarconcentrationthan
thatoftheanalyte.However,thisinterferencewouldbenegligiblebecause
themaximumlimitallowedforascorbicacidwhenusedasanadditivein
wine is 0.85 mM (Oxford companion to wine, Robinson J. (Ed.), 2006),
which is lower than the usual content of polyphenols in wines. Sulfites
were tolerated in a concentration 20fold of the analyte. Although this
interference could be removed using a pretreatment step (Montereali et
al., 2010), it was not necessary as the presence of sulfites in wines is
controlled according to their total content in reducing substances

235

Captulo3

(OrganisationInternationaledelaVigneetduVin(OIV),2011).Thus,the
maximumlimitofsulfurdioxideis2.5mMforredwine,3.3mMforwhite
wineand6.3mMforsweetwinesamples,whentheycontainupto4g/lof
reducingsubstances.Accordingtotheselimitsandthevaluesforphenolic
antioxidantconcentrationfoundinliterature(Gamellaetal.,2006;DiFusco
et al., 2010), the new method can be directly applied to the analysis of
wines since the concentration of polyphenols is higher than those of
ascorbic acid and sulfites. Trolox, which is usually the standard used for
thedeterminationoftheantioxidantactivity,asindicatedabove,wasalso
assayedintheconcentrationrangeof0.410M,butthecurvesobtained
weresimilartothatobtainedfortheblanksample.Theseresultsshowthat
the new method is suitable for the determination of polyphenols, but it
doesnotallowthedeterminationofantioxidantactivity.

Table2.Influenceofsomereducingagentsontheproposeddeterminationof
phenolicantioxidantsat2Mgallicacid
Compound

Maximumtolerated
interferent/analyteratio*

Malicacid

100

Citricacid

100

Sucrose

100

Glucose

25

Sodiumsulfite

20

Ascorbicacid

*Maximumratioassayedwas100

236

Determinacindeantioxidantesenalimentos

3.4.Applications

Theproposedmethodwasappliedtotheanalysisofred,whiteand

sweet wines. The sample treatment only consisted in the dilution of the
samplestomatchthedynamicrangeofthecalibrationcurve,usinggallic
acidstandardsandthenetAUCasanalyticalparameter.Table3showsthe
content ofantioxidants found in these samples analyzed by the proposed
methodandbytheFolinCiocalteumethod.Thevaluesfoundbyusingthe
new method agree with those reported in the literature (Gamella et al.,
2006; Di Fusco et al., 2010). As it can be seen from the table, the values
providedbyFolinCiocalteumethodwerehigherinallinstancesthanthose
providedbythelaccasemethod,whichisascribedtothecapabilityofthe
later to also detect nonphenolic substances. A recovery study was
performedbyaddingthreedifferentamountsofgallicacidtoeachsample
and subtracting the results obtained from similarly treatment unspiked
samples(Table3).Therecoverypercentagesobtainedrangedfrom81.0to
108.3%andthemeanrecoveriesforred,whiteandsweetwineswere90.9,
92.4and99.5%,respectively.

237

238
1.920.04

2.0280.005

3.640.05

Whitewine(Rueda)

Whitewine(Valdepeas)

Sweetwine(Crdoba)

Units:mMgallicacidequivalentsofsample
MeanSD(n=3)

14.040.18

Redwine(Cuenca)

11.50.1

contenta,b

Redwine(LaRioja)

sample

FolinCiocalteu
method

1.270.09

1.000.03

1.000.06

9.50.5

7.00.4

contenta,b

1
3
5

0.25
0.75
1.25

0.25
0.75
1.25

4
12
20

4
12
20

addeda

1.010.09
3.00.3
4.70.3

0.230.03
0.700.04
1.00.1

0.240.03
0.770.04
1.080.03

3.20.2
11.70.3
18.90.8

4.30.5
10.00.5
161

founda,b

Proposedmethod

Table3.Antioxidantconcentrationfoundandrecoverystudyforthewinesamplesanalyzed

101.5
102.0
95.0

92.0
92.2
83.3

97.2
102.8
86.7

80.0
97.4
94.5

108.3
84.1
81.0

recovery
(%)

Captulo3

Determinacindeantioxidantesenalimentos

4.Conclusions

An automated method has been reported for thedetermination of

polyphenols in wine samples using HPTS as a new fluorescent laccase

substrateandTb4O7NPsasactivatorsoflaccase.ThepresenceoftheseNPs
on the system allows shorter analysis times and lower enzyme
consumption. The microplate reader enables the simultaneous processing
of96tests,reachinganoverallsamplethroughputofabout35samplesh1,
when analysed in triplicate. Also, the use of wells with a total volume of
180 L, in spite of conventional microplates with volumes of 350400 L
perwell,iscosteffective,sincethereagentconsumptionislower.

The analytical usefulness of the proposed method for the

determinationofpolyphenolsinwinesampleshasbeendemonstratedand
theresultshavebeencomparedwiththoseprovidedbytheFolinCiocalteu
method,whichwasusedasreference.Theresultsobtainedforallsamples
analyzed by the new method were lower owing to its better selectivity
compared to the reference method, which is a common aspect for other
laccasebased enzyme methods previously described for wine analysis
(Gamellaetal.,2006;DiFuscoetal.,2010).

Acknowledgements

AuthorsgratefullyacknowledgefinancialsupportfromtheSpanish

MinisteriodeEconomayCompetitividadMINECO(GrantNo.CTQ2012
32941),theJuntadeAndalucaProgram(GrantNo.P09FQM4933)andthe
FEDERFSEprogram.

239

Captulo3

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Nkhili, E. & Brat, P. (2011). Reexamination of the ORAC assay: effect of
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NugrohoPrasetyo,E.,Kudanga,T.,Steiner,W.,Murkovic,M.,Nyanhongo,
G. S. & Guebitz, G. M. (2010). Laccasegenerated tetramethoxy
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NugrohoPrasetyo,E.,Kudanga,T.,Steiner,W.,Murkovic,M.,Nyanhongo,
G. S. & Guebitz, G. M. (2009). Antioxidant activity assay based on
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fluidizedbed.BioresourceTechnology,110,120124.

244

CAPTULO 4
CHAPTER 4

Innovaciones en ensayos de afinidad

mediante el uso de upconverting
phosphors y fenmenos de
transferencia de energa resonante
luminiscente
Innovations in affinity assays by
using upconverting phosphors and
luminescence resonance energy
transfer

Upconvertingphosphors

Este captulo aborda un estudio sistemtico del uso de

upconverting phosphors (UCPs) en ensayos homogneos de afinidad con

deteccindelaluminiscenciabasadaentransferenciadeenergaresonante
luminiscente (LRET). Estas investigaciones se realizaron durante la
estancia de tres meses en el Departamento de Biotecnologa de la
Universidad de Turku (Finlandia) bajo la supervisin del Prof. Tero
Soukka. Con esta visita se cumple uno de los requisitos para optar a la
MencindeDoctoradoInternacional.Elestudiorealizadohadadoorigena
unartculocientficoenvasderedaccin:

J.GodoyNavajas,T.Riuttamki,T.Soukka.Evaluationofdifferent
donoracceptor pairs for the development of homogeneous
bioaffinity assays using upconversion luminescence resonance
energytransfer.

La transferencia de energa resonante (RET) o transferencia de

energaelectrnica(EET)esunprocesoquetienelugarentredosmolculas
omateriales.Unadeestasmolculasactacomodador,emitiendoenerga
aunadeterminadalongituddeonda,mientrasquelaotramolculaacta
como aceptor, absorbiendo la energa emitida. Cuando las dos molculas
son fluorescentes, a este mecanismo se le conoce como transferencia de
energa resonante de fluorescencia (FRET) o de luminiscencia (LRET). No
obstante, la energa no siempre se transmite por fluorescencia y no
necesariamente el aceptor emite fluorescencia ya que pueden utilizarse
tambininhibidoresdefluorescencia.Paraqueesteprocesotengalugarse
tienequecumplirunaseriederequisitos:(1)lamolculadadoradebetener
un elevado rendimiento cuntico; (2) debe existir un solapamiento
247

Captulo4

sustancialentreelespectrodeemisindeldadoryelespectrodeabsorcin
delaceptor;(3)esnecesariounadecuadoalineamientoentrelosmomentos
detransicindelaabsorcinylaemisin;y(4)ladistanciaqueseparaal
dadordelaceptordebesermuycorta,normalmentemenorde10nm[1].
LosUCPshansidousadosparaeldesarrollodediferentesensayos
basadosenlatecnologaLRETutilizandofluorforosconunalongitudde
ondadeabsorcinsimilaralaemisindelUCPencuestin.Porejemplo,
los UCPs se han usado como dadores, mientras que el fluorforo Oyster
556 se utiliz como aceptor para el desarrollo de un inmunoensayo
homogneo para la determinacin de estradiol [2]. Utilizando otros tipos
defluorforos,comosonlasficobiliprotenas(Figura1),sehadescritoun
ensayodetransferenciadeenergaconlosUCPscomodadoresmedianteel
sistemadeafinidadbiotinaestreptavina[3].Enestosestudiosdeafinidad,
losUCPsseunenaestreptavidina(SA)paraformarunconjugadoSAUCP
que acta como dador y un fluorforo orgnico se enlaza a biotina para
que acte como aceptor. Cuando el dador y el aceptor interaccionan, se
produce la transferencia de energa, cuya intensidad se utiliza como
parmetroanaltico.EstametodologaLRETpermiteexcitarenlazonadel
infrarrojocercano(paralosUCPsdopadosconYb(III),laexcitacinocurre
a 980 nm). En esta zona del espectro, la probabilidad de interferencias
debidasalamatrizdelamuestraesrelativamentebaja,loquecontribuyea
disminuir las seales de fondo y, por tanto, a mejorar los lmites de
deteccin. La emisin ocurre en la zona del visible, sobre 600 nm, si se
utiliza ficoeritrina como aceptor. Los ensayos de afinidad desarrollados
por el grupo del Prof. Soukka, basados en metodologas LRET, han
implicadoelusodeUCPsdopadosconEr(III)paraformareldador[2,3]

248

Upconvertingphosphors

Figura1.Fenmenodetransferenciadeenergaresonantedeluminiscencia
(LRET) donde el dador es un UCP, el aceptor es un fluorforo y la
interaccin entre ambos se lleva a cabo mediante el sistema de afinidad
biotinaestreptavidina.

Ms recientemente, se ha diseado un biosensor basado en el

fenmeno LRET usando UCPs como dadores y nanoesferas de polim
fenilendiamina(PMPD)comoaceptoresparaladeterminacindeADNen
muestras de suero humano [4]. Por otro lado, estos nanocristales pueden
combinarse con otro tipo de nanomaterial para obtener una eficiente
transferencia de energa. La transferencia de energa entre los UCPs y
nanopartculas de carbono ha sido la base para el diseo de un nuevo
biosensorhomogneoenladeterminacinde2metaloproteinasaensangre
[5].
El estudio que se incluye pretende expandir la aplicabilidad de
UCPs dopados con Ho(III) y Tm(III), susceptibles de ser utilizados
individualmente o en ensayos multiplex donde puedan funcionar como

249

Captulo4

dadores, adems de los convencionalmente utilizados, que son

nanocristalesdopadosconEr(III).Porotraparte,sehanensayadodiversos
fluorforos orgnicos pertenecientes a los ATTO y los Alexa Fluor, para
investigarsuusocomopotencialesaceptores.

BIBLIOGRAFA
(1)

J.Fan,M.Hu,P.Zhan,X.Peng.Energytransfercassettesbasedon

organicfluorophores:constructionandapplicationsinratiometricsensing.
Chem.Soc.Reviews(2013)42,2943.
(2)

K. Kuningas, T. Ukonaho, H. Pkkil, T. Rantanen, T. Lvgren, T.

Soukka. Upconversion fluorescence resonance energy transfer in a

homogeneous immunoassay for estradiol. Anal. Chem. (2006) 78, 4690
4696
(3)

K. Kuningas, T. Rantanen, T. Ukonaho, T. Lvgren, T. Soukka.

Homogeneous assay technology based on upconverting phosphors. Anal.

Chem.(2005)77,73487355
(4)

Y. Wang, Z. Wu, Z. Liu. Upconversion fluorescence resonance

energytransferbiosensorwitharomaticpolymernanospheresasthelabel
freeenergyacceptor.Anal.Chem.(2013)85,258264
(5)

Y. Wang, P. Shen, C. Li, Y. Wang, Z. Liu. Upconversion

fluorescence resonance energy transfer based biosensor for ultrasensitive

detection of matrix metalloproteinase2 in blood. Anal. Chem. (2012) 84,
14661473.

250

Upconvertingphosphors

This chapter tackles a systematic study of the usefulness of

upconverting phosphors (UCPs) in homogeneous affinity assays with

detection of luminescence resonance energy transfer (LRET). These
investigations were performed during the threemonth stay at the
Department of Biotechnology of the University of Turku (Finland)
underthesupervisionofProf.TeroSoukka.Thisisoneoftherequisites
for the International Doctorate Mention. The study performed has
givenrisetoascientificarticlecurrentlyinpreparation:

J. GodoyNavajas, T. Riuttamki, T. Soukka. Evaluation of

different donoracceptor pairs for the development of
homogeneous

bioaffinity

assays

using

upconversion

luminescenceresonanceenergytransfer.

The resonance energy transfer energy (RET) or electron energy

transfer (EET) is a process that happens between two molecules or
materials.Oneofthesemoleculesactsasdonor,whichemitsenergyat
aspecificwavelength,whereastheothermoleculeactsasacceptor,thus
absorbing the emitted energy. When both molecules are fluorescent,
thisphenomenonisknownasfluorescenceorluminescenceresonance
energy transfer (FRET or LRET). However, the energy is not always
transferred as fluorescence and the acceptor does not necessarily emit
fluorescence, since fluorescence quenchers can also be used. Some
requisites need to be met for this process to occur: (1) the donor
molecule should have a high quantum yield; (2) both donor emission
251

Chapter4

andacceptorabsorptionspectrahavetooverlapinahighextent;(3)an
adequate alignment of absorption and emission transition moments is
required;and(4)thedistancethatseparatesdonorfromacceptorneeds
tobeveryshort,usuallylowerthan10nm[1].
TheUCPshavebeenusedtodevelopdifferentassaysbasedon
LRETusingfluorophoreswithanabsorptionwavelengthsimilartothe
UCP emission wavelength. For instance, the UCPs have been used as
donors, while the Oyster 556 fluorophore was used as acceptor to
develop a homogeneous immunoassay for the determination of
estradiol [2]. Another energy transfer assay has been reported using
other types of fluorophores, such as phycobiliproteins as acceptors
(Figure 1) and UCPs as donors, using the biotinstreptavidin affinity
system[3].Inthesestudies,theUCPsbindstreptavidin(SA)togiverise
to a SAUCP conjugate, which acts as a donor and an organic
fluorophoreboundtobiotinistheacceptor.Whendonorandacceptor
interact, the energy transfer process occurs, which intensity is used as
analytical parameter. This LRET methodology allows the excitation in
the near IR region of the spectrum (Yb(III)doped UCPs are excited at
980nm).Inthisregionofthespectrum,theprobabilityofinterferences
owingtosamplematrixisrelativelylow,whichcontributestodecrease
background signals, and hence, to improve detection limits. When
phycoerythrin is used as acceptor, the emission happens in the visible
region, at about 600 nm. The affinity assays based on LRET
methodologies developed by the research team of Prof. Soukka have
involvedtheuseofEr(III)dopedUCPstogiverisetothedonor[2,3]
252

Upconvertingphosphors

Figure 1. Luminescence resonance energy transfer (LRET) where the

UCP acts a donor and the fluorophor is the acceptor, their interaction
occurringbymeansofbiotinstreptavidinaffinitysystem.

Morerecently,abiosensorbasedontheLRETphenomenonthat
involves the use of UCPs as donors and nanospheres of polym
phenylenediamine(PMPD)asacceptorsfortheDNAdeterminationin
human serum [4] has been designed. On the other hand, these
nanocrystalscancombineothertypesofnanomaterialstogiverisetoan
efficientenergytransfer.TheenergytransferbetweenUCPsandcarbon
nanoparticleshasbeenthebasisforthedesignofanewhomogeneous
biosensorforthedeterminationof2metalloproteinaseinblood[5].
The study included in this Dissertation is aimed to expand the
applicability of Ho(III) and Tm(III)doped UCPs, which can be used
individually or in multiplexed assays as donors together with the
conventionally used Er(III)doped UCPs. On the other hand, several

253

Chapter4

organicfluorophoresbelongingtoATTOandAlexaFluorfamilieshave
beenassayedinordertoinvestigatetheirpotentialuseasacceptors.

LITERATURE
(1)

J.Fan,M.Hu,P.Zhan,X.Peng.Energytransfercassettesbased

on organic fluorophores: construction and applications in ratiometric

sensing.Chem.Soc.Reviews(2013)42,2943.
(2)

K.Kuningas,T.Ukonaho,H.Pkkil,T.Rantanen,T.Lvgren,

T. Soukka. Upconversion fluorescence resonance energy transfer in a

homogeneousimmunoassayforestradiol.Anal.Chem.(2006)78,4690
4696
(3)

K. Kuningas, T. Rantanen, T. Ukonaho, T. Lvgren, T. Soukka.

Homogeneous assay technology based on upconverting phosphors.

Anal.Chem.(2005)77,73487355
(4)

Y. Wang, Z. Wu, Z. Liu. Upconversion fluorescence resonance

energy transfer biosensor with aromatic polymer nanospheres as the

Labelfreeenergyacceptor.Anal.Chem.(2013)85,258264
(5)

Y. Wang, P. Shen, C. Li, Y. Wang, Z. Liu. Upconversion

fluorescence resonance energy transfer based biosensor for

ultrasensitive detection of matrix metalloproteinase2 in blood. Anal.
Chem.(2012)84,14661473.

254

Upconvertingphosphors

Evaluationofdifferentdonoracceptorpairsforthe
developmentofhomogeneousbioaffinityassaysusing
upconversionluminescenceresonanceenergytransfer
J.GodoyNavajasa,T.Riuttamkib,T.Soukkab
DepartmentofAnalyticalChemistry.ResearchInstituteonFineChemistry.Campus
Rabanales.AnnextoMarieCurieBuilding.14071Crdoba.Spain
bDepartmentofBiotechnology.UniversityofTurku.Biocity6A.20520Turku.
Finland

Abstract

A systematic study on the suitability of different donoracceptor

pairs for the development of affinity assays with biotin as model analyte is
reported. Upconversion luminescence resonance energy transfer (LRET) has
been measured for several streptavidincoated upconverting phosphors
(UCPs)(donors)withdifferentbiotinylatedfluorescentacceptors.Thedonors
assayed were streptavidincoated UCPs of different chemical composition,
such as NaYF4, Yb(III)Er(III); NaYF4, Yb(III)Ho(III) and NaYF4, Yb(III)Tm(III).
Biotinylated derivatives of suitable fluorescent acceptors, namely
phycoerythrin (BPE), Rphycoerythrin (RPE), Alexa Fluor 488 (AF488), Alexa
Fluor546(AF546),AlexaFluor680(AF680)andATTO495,wereassayed.

The results obtained show that the LRET intensity depends on the

UCPconsidered.ThebrightestluminescencewasobtainedwithEr(III)doped
UCP,butthesensitivityoftheassays,intermsofbiotinIC50values,wasnot
influencedbyUCPcomposition.TheuseofTm(III)providesgoodopportunity
to develop LRET assays using biotinylated Rphycoerythrin (bioRPE) as the
acceptor. Thus, the present study opens the possible use of UCPs other than
Er(III)dopedonestodevelopsensitiveLRETaffinityassays.
Keywords:homogeneousaffinityassays,upconversionluminescence,resonance
energytransferassays,nanosphosphors.

255

Captulo4

Introduction
The analytical applications of antiStokes luminescence have
experienced a huge increase in the last decade, with the advent of
lanthanide upconverting phosphors (UCPs). These phosphors are
composed of inorganic host lattices, where NaYF4 is one of the most
commonly used, although the use of Gd and La lattices has also been
reported [17]. These inorganic matrices are usually doped with the
infrared emitting Yb(III) ion together with other lanthanide ions, such as
Er(III),Ho(III)andTm(III),amongothers.
ThephenomenonofantiStokesluminescencehappensbyirradiating
thesenanophosphorswithalasersourceintheinfraredregion,generallyat
980nm,andmeasuringthereaftertheemissionoftheotherlanthanideions
in the green to red region of the spectrum. This effect constitutes an
advantage compared to Stokes luminescence since many undesirable
phenomena that usually affect the performance of photoluminescence
emission techniques, such as the autofluorescence coming from sample
matrix when excited at short wavelengths in the UVvisible region, are
avoided.Samplematricesdonotabsorbatthelongexcitationwavelength
used to excite UCPs, so a high spectral selectivity is attained. Another
advantage of using these nanophosphors is the excellent photostability
owing to their inorganic nature and to the low power required for their
excitation,thusbeingtheselasersourcesmuchcheaperthanthepowerful
lasers required for twophoton excitation. Also, some excellent properties
that are common to conventional Stokes lanthanide sensitized
luminescence, such as narrow emission peaks, long luminescence lifetime

256

Upconvertingphosphors

andtheirlowtoxicitymakethemespeciallysuitableforbioanalyticaland
biomedicalapplications[2,8].Someapplicationsdescribetheiruseaslabels
in heterogeneous assays to determine prostate specific antigen (PSA) [8]
and biotin [9,10]. However, many analytical applications of UCPs have
been focused on the development of luminescence resonance energy
transfer (LRET) assays [1114], which have opened new possibilities for
homogeneous binding assays. LRET phenomenon relies on the
nonradiative energy transfer from one fluorescent molecule (donor) to an
acceptor,whichisalsoafluorophore.
The excellent features of UCPs above mentioned have enabled the
avoidance the influence of background signals from biological samples,
and hence, to obtain relatively low detection limits using homogeneous
assays. The use of UCPs in bioanalysis requires these nanocrystals to be
biocompatibleandreadilydispersedinwater,owingtotheirhydrophobic
shell,e.g.ofoleicacid,aftersomesynthesisprocedures[1,2].Thus,theuse
of functionalization procedures is needed, among which ligand exchange
[1114] and silanization [1,2] have been used for this purpose. Ligand
exchange procedures are simple to perform but it has been reported that
theligandsattachedtothesurfacecanbefinallylost.Thestabilityofsilica
encapsulatedUCPsisbetter,althoughithasbeenobservedinsomecasesa
decrease in the luminescence intensity, which has been ascribed to the
remainingfreeaminogroupsonthesurfaceandtoasubsequentdecrease
in the quantum yield [15]. Furthermore, the performance of LRET assays
relies on the use of streptavidincoated UCPs donors and biotinylated
acceptors, where similar functionalization procedures to those above
mentioned are needed. However, the comparison of the performance of

257

Captulo4

LRET assays using different UCP functionalization procedures has not

beenperformed,althoughthiscanbeafactorinfluencingthesensitivityof
thehomogeneousassaysdeveloped.
Theworkpresentedherereportsasystematicstudyofthebehaviour
of different UCPs according to the dopant element (Er(III), Ho(III) and
Tm(III)). Er(III)doped UCPs have already proven to give rise to very
luminescent donors in LRET assays. However, the use of Ho(III) and
Tm(III) may provide good options for multiplexing or to expand the
application field of UCP technology, since the emission wavelength of
Tm(III)ismuchshorterthanthoseofEr(III)andHo(III)phosphorsanditis
closer to the maximum excitation wavelength of many organic acceptors.
Bearingthisinmind,theperformanceofseveraldonoracceptorpairshas
been evaluated, using streptavidin coated nanophosphors doped with
either Er(III), Ho(III) and Tm(III) as the donors and organic dyes as the
acceptors. Phycobiliprotein dyes have been used as acceptors in some
LRET assays [11,13]. Although these compounds show an intense
fluorescence,theyarealsorelativelyexpensive,sothepotentialusefulness
of other fluorophores, such as Alexa Fluor or ATTO dyes has been
checked.Also,theinfluenceofUCPfunctionalizationproceduresbasedon
silylationandonligandexchangehasbeenstudiedforEr(III)andHo(III)
doped UCPs. This study was carried out to elucidate which is the most
adequateforthedevelopmentofhomogeneousLRETassaysandwhether
thetreatmentdependsonthechemicalcompositionoftheUCP.

258

Upconvertingphosphors

2.Experimentalprocedures
2.1.Reagents
Different infrared to visible nanosized UCPs, which were doped
withEr(III),Ho(III)andTm(III)andobtainedbyareprecipitationmethod
[16], were used to perform all the experiments. Fluorescent
phycobiliproteins, such as phycoerythrin and Rphycoerythrin (BPE)
were purchased from Cyanotech Corp (KailuaKona, HI), supplied in 100
mM sodium phosphate buffer, pH 7.0, containing 60% ammonium
sulphate and 0.02% (w/v) sodium azide, with protein concentration of 10
mg/mL.AlexaFluor488,AlexaFluor546andAlexaFluor680succinimidyl
esterswerepurchasedfromMolecularProbesInvitrogenPaysley,UK)and
ATTO 495 succinimidylester was obtained from Sigma. Streptavidin was
purchased from SpaBioSpa (Milan, Italy). Biotinamidohexanoic acid N
hydroxysuccinimide ester (bioLCNHS) was from Pierce (Rockford, IL).
Fluorescentprotein(bioBPE)wasbiotinylatedwitha25foldmolarexcess
of NHSLCbiotin according to a previously described procedure [11]. A
freshly prepared solution of NHSLCbiotin in dimethylformamide was
addedintoproteinsolutioncontaining8.5mg/mLphycobiliproteinsin50
mMcarbonatebuffer,pH9.3.Thereactionmixture,withatotalvolumeof
500Landcontainingabout2%(v/v)DMF,wasincubatedinrotationat17
rpm (Rotamix RK, HetoHolten A/S, Allerod, Denmark) protected from
lightfor3hatroomtemperature.Finally,itwaspurifiedusingNAP5and
NAP10 columns (Amersham Biosciences, Uppsala, Sweden). Polyacrylic
acid ammonium salt, Additol XW330 (MW 30000 50000) (Surface

259

Captulo4

Specialities) was used to prepare 5% Additol solutions in doubly de

ionizedwatertocoatUCPs.
Cyclohexane,
diamine

(TMED,

nhexanol,
97%

N(3trimethoxysilyl)propyl)ethylene

purity)

and

3(trihydroxysilyl)propyl

methylphosphonate (THPMP, purity 42%) (Sigma Aldrich), Triton X100

and tetraethyl orthosilicate (TEOS, 98% purity) (Acros Organics), were
used for the silylation of UCPs. Ammonium hydroxide, acetone and
absolute ethanol were used to purify the conjugated UCPs. Glutaric
anhydride (95% purity, SigmaAldrich) and dry pyridine were used to
change amino groups of silicaUCPs into carboxyl groups. N(3
dimethylamino)propyl)Nethylcarbodiimide hydrochloride (EDAC) and
Nhydroxysulfosuccinimide sodium salt (sulfoNHS) (Fluka) and a buffer
solution of morpholino ethanesulfonic acid (MES) (20 mM, pH 6.1) were
usedforconjugationreactionsbyusingcarbodiimidemediatedsynthesis.
Streptavidincoated normalcapacity microtitration wells, assay buffer (50
mMTrisHCl,pH7.8,containing9g/LNaCl,0.5g/LNaN3,5g/LBSA,0.1
g/L bovine globulin and 20 M DTPA), and wash solution (5 mM Tris
HCl,pH7.8,containing9g/LNaCl,0.05g/LTween20,and1g/LGermal
II) were obtained from Innotrac Diagnostics Oy (Turku). Delfia
enhancement solution (DES) and enhancer solution (DE) were purchased
fromPerkinElmerLifeandAnalyticalSciences(WallacOy,Turku).

2.2.Instrumentation
A Plate Chameleon (Hidex Oy, Turku) equipped with a 200 mW
infrared laser module (Roithner Lasertechnik) and RG850 nm longpass
excitation filter (Andover Corp., Salem, NH) was used. The

260

Upconvertingphosphors

photomultiplier tube in the plate fluorometer was replaced with

Hamamatsu R4632 (Hamamatsu Photonics K.K.). Emission light coming
fromdonorandacceptorattheircorrespondingwavelengthswascollected
for 2000 ms under continuous laser excitation at 980 nm. AntiStokes
photoluminescence emission from Ho(III) and Er(III) doped UCPs was
measured at 550 nm employing a bandpass emission filter of 535/50 nm
(center wavelength 535 nm, halfwidth 50 nm; peak transmittance 60%;
Bk Interferezoptik Electronik, Nabburg, Germany), combined with an
absorptive neutral density filter (optical density 2.0; i.e. average
transmittance 1%; Thorlabs, Newton, NJ). The LRET sensitized emission
wasmeasuredat600nmfor1000ms,byusingabandpassemissionfilter
of 600/40 nm (peak transmittance 80%; Chroma Technology Corp.). For
Tm(III)dopedUCPs,LRETwasmeasuredusingaemissionfilterof555/20
nm.

2.3. Measurement of NaYF4:Yb(III), Er(III), Ho(III), and Tm(III) luminescence

intensity

AnamountofUCP(ca.1mg)isplacedinanEppendorftubeanda

volume of 0.75% acetic acid is added to reach a UCP concentration of 6

mg/mL.Thetubeissonicatedfor20cycles,then,doublydeionizedwateris
addedtodilutethedispersionupto2mg/mLandthetubeisplacedina
thermal block at 77 C at the maximum power for 1 h and it is vortexed
once during incubation. After this, it is incubated for 30 minutes under
continuousrotationatroomtemperatureandsonicatedagain.Avolumeof
thisdispersionwasdiluted10timeswiththemeasurementbufferand150
L were dispensed in triplicate onto Maxisorp microplates and the

261

Captulo4

luminescencewasmeasuredusingChameleon5instrumentfittedwiththe
appropriatefiltersforeachUCPcomposition.

2.4.MeasurementofUCPconcentration
ThepreparationofUCPstandardsandUCPsampleswasdoneby
diluting thephosphor in the measurement buffer and by sonicating them
for1min.Then,aduplicateanalysiswasdoneinduplicatebyadding150
L to each well. The measurements were obtained for 2 s by using a 550
nm for Er(III) and Ho(III) doped UCPs and 470 nm for Tm(III)doped
UCPs.

2.5.UCPsuspensionandpreprecipitation
The dispersion of the synthesized nanophosphors is made more
reproducibleinsizebyasuspensionandpreprecipitationmethodtogeta
morecolloidalformoftheseUCPsandtoremovethelargestaggregatesin
order to discard them prior to the functionalization step. The suspension
wasperformedbyweighing15mgofUCPinanEppendorftubeand930
L of 50% diethyleneglycol in doubly deionized water were added. The
suspension was vortexed and sonicated using 20 + 20 cycles with 100%
amplitude. The bath was left in swirl for 3 min and the dispersion was
vortexedagainandsonicatedwith20+20cycles.TheEppendorftubewas
incubated again in rotation at 60 C for 1 h and then, sonicated in swirl
until the UCP was totally dispersed, and incubated for 1 h at room
temperature. The mixture was vortexed and sonicated for 20 + 20 cycles
againandincubatedonrotationovernight.

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Upconvertingphosphors

Thepreprecipitationprocesswasdonejustbeforecoatingandthe
UCPsuspensionwaslettostandfor30minand870Lofthesuspension
wastakenfromthetopandtransferredtoanothertubeandtherestofthe
dispersion,whichcontainedlargeparticles,wasdiscarded.

2.6.Functionalizationofthesurfacewithcarboxylicacidgroups
Coating with poly(acrylic acid): An amount of preprecipitated
UCPs (5 mg, 390 L), was taken and mixed with Additol 0.1% and the
reaction mixture was left to react overnight at 35 C in rotation.
Afterwards, the Additol excess was removed by centrifugating first for 7
min at 8000 rpm, then, the supernatant was replaced with MilliQ water
and the dispersion vortexed and sonicated for 1 min and subjected to
centrifugation again. This process was repeated four times, leaving the
thirdwashaliquotfor45minatroomtemperatureandrotation.Thewash
process was ended by suspending UCPs in MilliQ water, until a final
volumeof300L.
CoatingwithglutaricanhydrideafterUCPsilylation:Anamountof
preprecipitatedUCPs(6mg,470L)wastakenforsilylation.Theprocess
was done by using a reverse micelle microemulsion method, which was
performed first by mixing 11.25 mL of cyclohexane, 2.66 mL of Triton X
100 and 2.7 mL of nhexanol. The mixture was divided in two different
microemulsionfractionsA(7.5mL)andB(therestofthemicroemulsion).
The tube with microemulsion A wasmixed with 435 L UCP suspension
and 90 L of ammonium hydroxide were added and the tube was
sonicatedinabathfor10min.TothetubewithmicroemulsionB,350Lof
water were added to give rise to a waterinoil microemulsion, 9.4 L of

263

Captulo4

TEOS,37.5LofTMEDand37.5LofTHPMPweresubsequentlyadded
and the mixture vortexed after each addition. Both tubes A and B were
combined, vortexed and incubated for 2 h at room temperature with
sonication in the first 10 min and the rest of the time with continuous
vortexing.Afterwards,avolume(2.25mL)ofacetonewasaddedtobreak
the microemulsion and precipitate the UCPs, which were collected after
centrifugation at 2860 rpm for 1 min. The supernatant was transferred to
anothertubeandacetonewasaddedtoreachatotalvolumeof20mLand
centrifuged again (2860 rpm, 3 min), the supernatant was discarded and
the UCPpellet was suspended in a small volume (400 L) of doubly
deionized water. The UCP particles obtained after the two precipitation
processeswerecombinedinasingle2mLEppendorftube.Theprecipitate
waswashedtwicewithethanolandtwicewithdoublydeionizedwater,by
sonicating each washing for 1 min and centrifugating at 8000 rpm for 7
min.Thepelletwasfinallysuspendedindoublydeionizedwatertoobtain
afinalvolumeof500L.ThesefunctionalizedUCPswerestoredatroom
temperature in rotation and the concentration of the silicafunctionalized
NPs was measured following the procedure described in section 2.4. The
next step was to change the aminofunctionalized UCPs into carboxylic
functionalizedUCPs.

The introduction of carboxylate groups was done by reacting the

silylated amino UCPs with glutaric anhydride. The UCPs obtained in the
above mentioned procedure were centrifuged (12000 rpm, 7 min), the
supernatantliquidwasdiscardedand150Lofdrypyridinewasadded.
Then, glutaric anhydride was added in a 10fold mass ratio compared to
UCP mass, after being dissolved in 120 L of pyridine. The mixture was

264

Upconvertingphosphors

vortexedandsonicatedfor5minandlatertransferredtoabathfor2.5hat
50 C. Afterwards, another batch of glutaric anhydride was diluted and
added and the process was repeated as for the first batch. At the end, a
volume(400L)ofethanolwasaddedandcentrifugedat12000rpmfor7
min. Finally, the pellet was washed twice with doublydeionized water,
suspended in 450 L of doublydeionized water and stored at room
temperatureinslowrotation.

2.7.ConjugationofCOOHfunctionalizedUCPswithstreptavidin
The conjugate of streptavidin (SA) with the functionalized UCPs
wassynthesizedbyusingacarbodiimidereactioninthepresenceofsulfo
NHS and EDC. To start the process, functionalized UCPs were washed
with 1 mL of MES buffer (pH 6.1, 20 mM) and then, they were re
suspended in 500 L. On the other hand, 20 mM EDC and 30 mM sulfo
NHSweredissolvedbyweighingtheappropriateamountbroughttoroom
temperature.Afterwards,avolume(10L)ofsulfoNHSandavolume(10
L) of EDC were added to the UCP dispersion and the resulting mixture
wasincubatedfor45minatroomtemperatureinrotation.Afterthistime,
thedispersionwaswashedwithMESbuffer,centrifugedat12000rpmfor7
min and the dispersion was reconstituted with MES buffer. To the
synthesized sulfosuccinimidyl ester, 33 L of streptavidin (30 mg/mL)
were added to the mixture and incubated for 2.5 h at room temperature
and in rotation. The reaction was stopped by adding 12.8 mL of 2 M
glycine, pH 11, and the mixture was allowed to react for 30 min with
rotation. The conjugate obtained was purified by washing twice with
borate buffer (10 mM, pH 8.5) containing 0.1% Tween 20 and one with

265

Captulo4

storage buffer, which consisted of borate buffer (5 mM, pH 8.5), 0.2%

Tween 85, 0.5% BSA and 0.05%NaN3. The conjugate was stored at room
temperature in an Eppendorf tube in rotation and its concentration was
determined as described in the procedure for the measurement of UCP
concentration.

The amount of total SA and free SA was calculated by using a

heterogeneous competitive binding for streptavidin using a biotinTb

chelate conjugate [11]. Unconjugated SA was separated from UCP
suspension by centrifugation and collection of the supernatant. In the
assay, 050 ng of streptavidin in 75 L of assay buffer, or appropriate
dilutionsofphosphorsuspensionandsupernatant,weremixedwith75L
of4nMbiotinconjugateofTbchelateandthereactionswereincubatedfor
30 min at room temperature with slow shaking to capture all unbound
biotin conjugates of Tbchelate. Finally, the wells were washed twice and
the signal was developed and measured similarly to the heterogeneous
biotinbindingassay.

2.8.Homogeneousacceptortitration
The amount of biotinacceptor concentration necessary to saturate
SAUCPbindingsiteswasfoundusingahomogeneousacceptortitration,
which involves the use of biotin as the analyte, a donor (a SAconjugate)
and an acceptor (biotinylated fluorophores). The microtiter plates used
wereblackhalfarea96wellplates.
The assay was performed as follows: a volume (32 L) of biotin
12.5 M or same volume of reaction buffer was added to 24 L of a SA
UCP(0.05mg/mL,0.015mg/mlinthefinalreactionmixture).Themixture

266

Upconvertingphosphors

was incubated for 15 min at 23 C and at 900 rpm using a plate tape to
protecttheplatefromlightandtheluminescencewasmeasuredwiththe
Chameleon5instrument,usinganemissionfilterof550nm,for2000ms.
Afterwards, biotinylatedacceptor at different concentrations (0.25, 0.74,
2.2, 7,20 nM in the finalreaction mixture) wasadded and theincubation
was performed at 23 C at 900 rpm. Luminescence measurements were
takenat45minusing600and550nmasemissionfiltersfor2000msinthe
caseofEr(III)andHo(III)dopedUCPs,andof555and470nmforTm(III)
dopedUCPs.Signalsweretreatedasdescribedpreviouslyelsewhere[11].

2.9HomogeneousLRETaffinityassayforDbiotin
Thehomogeneouscompetitiveformatforbiotindeterminationwas
also assayed using Dbiotin concentrations in the range of 0 12.5 M in
theassaytube.TheconcentrationofSAUCPwas0.015mg/mLforEr(III)
and Ho(III) donors and 0.03 mg/mL for Tm(III) donor. The acceptor
concentrationsusedwere0.2and0.4nM,forbioBPE,1.33nMforbioRPE
and4nMforAF680.A12.5Mbiotinconcentrationwasusedtoverifythe
backgroundlevel.

3. ResultsandDiscussion
3.1.

Luminescentfeaturesofdonoracceptorpairs
The LRET study presented has been carried out using the

streptavidinbiotin affinity system, as model assay. Streptavidin was

coated to UCP donors, whereas biotin was coupled to fluorescent
acceptors.Figure1showsatypicalschemeforthissystem,inwhichcanbe
seenthatafterUCPexcitation,thereisanenergytransferprocessfromthe

267

Captulo4

donortotheacceptor,thusresultinginacceptoremission.Thisprocessis
subject to the following two conditions: 1) The excitation spectrum of the
acceptorhastooverlapdonoremission,and2)Thedistancebetweendonor
andacceptorfollowsFrsterequation.

Figure 1. General scheme of a homogeneous upconversion LRET affinity assay (

streptavidin coated UCP (donor),
(acceptor)

biotin D,

biotinylatedfluorophore

Figure2showstheexcitationandemissionspectraofthedifferent
donoracceptor pairs used in this study. Figure 2.A shows the up
conversion emission spectra of the Er(III)dopedUCP (donor) when
excited at 980 nm and the excitation and emission spectra of
phycoerythrin (BPE) (acceptor). As it can be seen, the overlap of UCP
emissionandBPEexcitationiscomplete.AcriticalaspecttoenhanceFRET
emission is the selection of the appropriate wavelengths of the emission
filter to measure acceptor emission without collecting the signal coming
fromacceptorselfexcitation.Forthisreason,althoughmaximumemission
ofBPEhappensat575nm,a600/40nmfilterwaschosentomeasureLRET
intensity.TheUCPemissionat550nmwasmeasuredbyusing535/50nm
filter. This combination of donoracceptor pairs has been previously used
for the development of several affinity assays [11, 13] and homogeneous
immunoassays [12] to determine estradiol. Other fluorescent acceptors,

268

Upconvertingphosphors

suchasAF546(ex556,em570nm)andAF680(ex680,em702nm),were
alsoassayed.AsitcanbeobservedinFig.2.B)the600/40nmfilterisstill
appropriatefortheEr(III)dopedUCPAF546pair,whilea750/40nmfilter
isusedwhenAF680isusedasacceptor.A665/10nmfilterwasusedinthis
instancetocollectUCPemission.
SimilarluminescencespectrawerealsorecordedforHo(III)doped
(Fig 2.D and 2.E) and Tm(III)doped (Figs 2.F2.H) UCPs as donors using
different fluorescent acceptors. For Ho(III)dopedUCPs, BPE and AF546
wereassayedaspotentialacceptors,usinginbothcases600/40nmfiltersto
measuretheLRETintensity.ThisispossibleowingtothefactthatHo(III)
dopedUCPshavetheirmaximumemissionat535nm.Inasimilarwayas
for Er(III)doped UCPs, the wide excitation spectra of BPE allows its
efficient excitation. The excitation of AF546 is less favoured because only
theemissionofthedonormatchestheexcitationoftheacceptorat50%of
itsmaximumintensity.However,itsemissionwavelengthenablestheuse
of600/40tomeasureLRETwithminimumbackground.
Tm(III)dopedUCPshavealoweremissionwavelengththanEr(III)
andHo(III)centeredat472nm(Figs.2.F2.H).Althoughthebestoverlapis
obtainedfortheTm(III)dopedUCPandAF488pair,comparedtotheuse
ofRPEandATTO495.RegardingthefiltersusedtomeasureLRET,theuse
of600/40nmfilterseemstobethebestoptiontocollecttheemissionfrom
RPE, although some emission from acceptor selfexcitation could still
happen.ForAF488,severalfilterssuchas520/10,555/20and600/40,could
be used to collect emission. As it can be seen, the emission intensity at
600/40isreallypoor.ThebestoptionforATTO495seemedtobethefilter

269

Captulo4

555/20,sincetheintensityobtainedwasaboutthreetimeshigherthanthat
obtainedwiththe600/40nmfilter.

500

550

600

650

200

700

400

450

WAVELENGTH (nm)

600

400

600/40

200

400

450

500

550

600

650

1000

600/40

400

450

500

550

600

WAVELENGTH (nm)

700

800

400

200

450

500

550

600

650

700

650

700

1000

G)

800

600

600/40

400

200

555/20

400

600

WAVELENGTH (nm)

E)

400

NORMALIZED LUMINESCENCE INTENSITY

NORMALIZED LUMINESCENCE INTENSITY

200

500

WAVELENGTH (nm)

600

700

600

700

F)

800

650

200

800

WAVELENGTH (nm)

1000

600

400

600/40

NORMALIZED LUMINESCENCE INTENSITY

NORMALIZED LUMINESCENCE INTENSITY

800

550

600

WAVELENGTH (nm)

D)

1000

500

800

400

450

500

550

600

650

700

WAVELENGTH (nm)

1000

H)

800

600

400

600/40

450

400

C)

1000

200

555/20

400

600

NORMALIZED LUMINESCENCE INTENSITY

600/40

200

800

750/40

400

NORMALIZED LUMINESCENCE INTENSITY

600

B)

1000

600/40

520/10

800

NORMALIZED LUMINESCENCE INTENSITY

NORMALIZED LUMINESCENCE INTENSITY

A)

1000

400

450

500

550

600

650

700

WAVELENGTH (nm)

Figure 2. Emission spectra obtained for different UCPs (solid line) and excitation and
emission spectra of different acceptors (dashed lines). In 2.A2.C, Er(III)doped UCP
emissionspectratogetherwithBPE(2.A),AF546(2.B)andAF680(2.C).In2.Dand2.E,
Ho(III)doped UCPs with BPE (2.D) and AF546 (2.E). In 2.F2.H, Tm(III)doped UCPs
withRPE(2.F),AF488,(2.G)andATTO495(2.H).

3.2.

Homogeneousacceptortitrationexperiments
The aim of these titration assays is to find the amount of

biotinylated acceptor required to saturate the binding sites of the UCPs.

Thelowertheconcentrationofbiotinylatedacceptor,themoresensitivethe

270

Upconvertingphosphors

homogeneous LRET determination will be. These experiments were

performed for biotinylated UCPs of different chemical composition. In
order to make the different measurements comparable, the streptavidin
coated UCPs (SAUCPs) were diluted to be at the same final UCP
concentration. The influence of surface functionalization procedure was
studiedbyperformingthetitrationofSAcoatedUCPdonorscomposedby
the same UCP that had been functionalized according to different
procedures,withthesameacceptor.Ithasbeenpreviouslydescribedthat
bioBPE provides excellent LRET signals with SAEr(III)doped UCPs as
donors [1114]. For this reason, Er(III)doped UCPs and Ho(III)doped
UCPs functionalized with Additol and those encapsulated with a silica
layerwereassayedaspotentialdonors,atanequivalentUCPconcentration
of0.015mg/mLwithvaryingbioBPEconcentrations(Figure3).Ascanbe
seen, the best luminescence intensity was obtained for a bioBPE
concentration lower than 1 nM and the highest values were obtained for
SAEr(III)doped UCPs. It can be also seen that the functionalization
proceduredidnotaffectthefinalLRETintensity,althoughitwasobserved
forbothtypesofUCPsthattheacceptorconcentrationrequiredforUCPs
coated with Additol was lower than that for those encapsulated into a
silicashell.ApossibleexplanationofthisfactcouldbethatSAsilicaEr(III)
dopedUCPcontainedabout2foldstreptavidinmoleculesattachedtothe
surface than SAAdditolEr(III)coated UCP donor. As it can be seen, the
LRET intensity when SAHo(III)doped donors were used was much
lower, so the feasibility of using other SAHo(III)doped UCP
concentrationswasexploredinafurtherexperiment.

271

Captulo4

800000

up conversion LRET (cts)

600000

400000

200000

4
3

1,2

[Bio-BPE] (nM)

Figure 3. Homogeneous acceptor titrations for streptavidinEr(III) and streptavidin

Ho(III) doped UCP donors obtained by ligand exchange with Additol (2,4) and by
silylation(1,3).In1,2[Ho(III)dopedUCP]=0.015mg/mL.In(3,4)[Er(III)dopedUCP]=
0.015mg/mL.LRETsignalswerecalculatedbysubtractingthebackgroundsignalofthe
assay(excessofDbiotin)fromthemaximumsignal(absenceofDbiotin),cts:counts.

InanattempttoincreasethesignalexhibitedbySAHo(III)doped
UCPs,higherequivalentUCPconcentrations(0.03and0.045mg/mL)were
assayed,usingbioBPEasacceptor(Figure4).However,theLRETsignals
obtainedwere5timeslowerthanthoseobtainedforSAEr(III)dopedUCP
donors.A0.4nMbioBPEand0.03mg/mLSAEr(III)UCPwerechosento
performhomogeneousassaysforbiotindetermination,sincetheyprovide
an adequate LRET signal with a bioBPE concentration relatively low,
reachinganadequatesensitivity.

272

Upconvertingphosphors

160000

up conversion LRET (cts)

120000

80000

40000

0
4
8
12
16
20

-1
[Bio-BPE]
x
10
(nM)

Figure4.InfluenceontheconcentrationofHo(III)dopedUCPontheluminescenceand
on the sensitivity of homogeneous acceptor titration curves. [donor] are: (1) 0.015
mg/mL, in (2) 0.03 mg/mL in (3) 0.045 mg/mL. LRET signals were calculated as
indicatedinFigure3.

As indicated above, the UCP silylation can produce more stable

SAUCP donors. Several assays were carried out to optimize this process
using a reversemicelle microemulsion procedure for the formationof the
silicashell.Theconcentrationofsurfactantusedandtheadditionorderof
thereagentscanaffecttheshapeandthethicknessofsilicashell[17].The
functionalization

was

performed

by

assaying

three

surfactant

concentrations and modifying the addition order of the microemulsion

components. Figure 5 shows the different curves obtained for the
homogeneous acceptor titration with the SAUCP donors. As it can be
seen,theadditionorderisacriticalvariable,whichcouldbeexplainedbya
differentstructureofthesilicalayerwithnoapparentchangesinthefinal
amount of SAEr(III)doped UCP. It has also been described that the free

273

Captulo4

aminogroupsfromsilicalayercanmodifyUCPintensity[15],whichcould
explain the different LRET intensity obtained for identical donor and
acceptorconcentrations.TheuseofsilicatoencapsulateUCPsseemstobe
a good alternative to the use of Additol since the decrease in the
luminescence intensity is relatively small. Thus, all the homogeneous
assays involving either Er(III), Ho(III) or Tm(III) nanosized UCPs were
performed after functionalization by silylation procedures to obtain the
donors.

1000000

up conversion LRET (cts)

800000

4
600000

3
2

400000

1
200000

0
5
10
15
20

-1
[Bio-BPE] x 10 (nM)

Figure 5. Homogeneous acceptor titration curves obtained after different silylation

procedures.VolumesofTritonX100usedwere:(1)1.3mL,(2)2,7mL,(3)5,3mLand
(4) adding Triton 2.7 mL of Triton X100 at the end. LRET signals were calculated as
indicatedinFigure3.

274

Upconvertingphosphors

BPEandRPEareveryfluorescentproteinscommerciallyavailable,
but they are relatively expensive. Thus, the feasibility of other alternative
fluorophores, with suitable wavelengths to produce LRET was checked.
Figure6showsthetitrationcurvesobtainedforSAEr(III)doped(6.A)and
SAHo(III)doped (6.B) UCP donors with bioBPE and bioAF546 as
acceptors. The LRET intensity was much lower for bioAF546 and the
increaseofSAHo(III)dopedUCPconcentrationdidnotimprovetheLRET
signal unlike it happened for the pairs with SAEr(III)doped UCPs and
bioBPE as donor and acceptor, respectively. The same behaviour was
observedforSATm(III)donors,inwhichtheacceptorsusedwerebioRPE,
bioAF488andbioATTO495(Fig.6.C).AllofthemprovidedLRETsignal,
but the highest intensity was obtained using bioRPE, so this pair was
chosen to develop further homogeneous affinity assays for biotin with
Tm(III)dopedUCPsasdonors.

3.3DeterminationofbiotinusinghomogeneousLRETassays
Several donoracceptor pairs were used to perform homogeneous
LRET assays for biotin. Figure 7 shows the curves for biotin obtained for
the donoracceptor pairs studied. These pairs were chosen amongst those
which provided better LRET intensities with minimal acceptor
concentrations.

275

10

15

20

25

276

u p co n versio n L R E T (cts)

15

20

[acceptor] (nM)

10

10

25

[Bio-BPE] x 10-1 (nM)

12

30

14

[acceptor] (nM)

20000

40000

60000

u p c o nv ersio n LR E T (c ts )

40

2
1

10

80

[bio-RPE] x 10-1, nM

60

[acceptor] (nM)

20

20000

40000

60000

80000

100000

120000

12

100

14

u p co n versio n L R E T (cts )

u p co n vers io n L R E T (c ts )

u p c o n ve rs io n (L R E T ) (c ts )

Figure6.Homogeneoustitrationcurvesobtainedfordifferentdonoracceptorpairs.In6.A)[Er(III)dopedUCP=0.015
mg/mL and acceptors were: (1) bioBPE; (2) AF680 and (3) AF546. In 6.B) [Ho(III)doped UCP] were (1) 0.030 mg/mL
withbioBPEasacceptor,(2)0.015mg/mLwithbioAF546asacceptorandin(3)0.030mg/mLwithAF546asacceptor.In
(C) [Tm(III)doped UCP] was used as donor and the conditions were (1) [donor] = 0.0075 mg/mL and bioAF488 as
acceptor,in(2)[donor]=0.0075mg/mLandbioATTO495asacceptor,in(3)[donor]=0.045mg/mLandbioAF488as
acceptor, in (4) [donor] = 0.045 mg/mL and bioATTO495 as acceptor, in (5) [donor] = 0.015 mg/mL and bioRPE as
acceptor.LRETsignalswerecalculatedasindicatedinFigure3.

100000

80000

100000

C)

500000

100000

200000

300000

120000

200000

300000

400000

500000

1000000

B)

400000

500000

1500000

A)

2000000

2500000

Captulo4

Upconvertingphosphors

4000000

250000

A)

B)

up conversion LRET (cts)

up conversion LRET (cts)

3000000

2000000

1000000

200000

150000

100000

50000

0
0,1

10

100

1000

10000

0,1

[Biotin] (nM)

10

100

1000

10000

[Biotin] (nM)

120000

C)

up conversion LRET (cts)

100000

80000

60000

40000

20000

0,1

10

100

1000

10000

[Biotin] (nM)

Figure7.HomogeneousLRETaffinityassaysforDbiotinwithdonoracceptor
pairsgivingthebestresults.InA)withEr(III)dopedUCP(0.015mg/mL)and
(1)0.4nMBPEand(2)4nMAF680.In(B)Ho(III)dopedUCP(0.030mg/mL)
and0.2nMBPE.In(C)Tm(III)dopedUCP(0.030mg/mL)and1.33nMRPE.
LRETsignalswerecalculatedasindicatedinFigure3.

As it can be also seen from the Figure, the highest luminescence

intensitieswereobtainedforEr(III)dopedUCPswithBPEandAF680nm.
However, the Table 1 shows the different assays performed according to

277

Captulo4

the results obtained for homogeneous acceptor assays and the IC50
obtainedforbiotin.

Table 1. IC50 values for biotin assays using different donor-acceptor pairs
IC50 for biotin (nM)

Acceptor
Donor
Er(III)-doped UCP
(0.015 mg/mL)

Bio-BPE

Bio-BPE

Bio-RPE

Bio-AF680

(0.2 nM)

(0.4 nM)

(1.33 nM)

(4 nM)

---

8.57*

---

14.37

5.7

---

---

---

---

7.20

---

---

---

---

3.35

---

Ho(III)-doped
UCP, 0.015
mg/mL)
Ho(III)-doped
UCP, 0.015
mg/mL)
Tm(III)-doped
UCP
(0.030 mg/mL)
*The concentration of bio-BPE was 0.3 nM

These IC50 values were calculated after adjusting the normalized

intensitiesofthecalibrationcurvesforbiotinto4parameterlogisticcurves,
whichregressioncoefficients(r)werehigherthan0.99inallinstances.The
lowest IC50 was obtained for the pair SATm(III)UCP with bioRPE in
spiteofitslowLRETintensity.ThehighestIC50wasobtainedfortheSA
Er(III)doped UCP functionalized with silica and bioAF680, which was
about14.4nM.Thevaluesobtainedarecomparable,andinsomeinstances
better than those obtained by some previously reported methods [11,12]

278

Upconvertingphosphors

using Er(III)doped UCPs coated with Additol as donors and bioBPE as

acceptor.

4.

Conclusions
AsystematicstudyonthesuitabilityofdifferentUCPdonoracceptor

pairshasbeenperformedusingbiotinasmodelanalyte.TheuseofEr(III)
doped UCPs has resulted in higher LRET intensities, the maximum of
which have been obtained using bioBPE as acceptor. Although BPE and
RPE have shown to provide the best LRET intensities, the use of other
fluorophores, such as AF680 allows determinations of biotin with IC50
valuesinthenMrange.
The results obtained show the potential feasibility of multiplexed
assaysusingdifferentLRETdonoracceptorpairs.

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(1)

Gnach A., Bednarkiewicz A. Lanthanidedoped upcoverting

nanoparticles:meritsandchallenges.NanoToday(2012)7,532536.

(2)

Wang F., Banerjee D., Liu Y., Chen X., Liu X. Analyst (2010) 135,
18391854.

(3)

MisiakM.,ProrokK.,CichyB.,Berdnarkiewicz,StrekW.Thulium
cocentration quenching in the upconverting aTm3+/Yb3+ NaYF4
colloidalnanocrystals.Opt.Mat.(2013)35,11241128.

(4)

Jiang T., Song W., Liu S. Qin W., Synthesis and upconversion
luminescene properties study of NaYbF4:Tm3+ crystals with
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(5)

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Koepke C., Piatkowski D., Wisniewski K., Naftaly M. On

competitionbetweentwotypesofantiStokesemissionintheHo3+
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(7)

WnukA.,KaczkanM.,FrukaczZ.,PrackaI.,ChadeyronG.,Joubert
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(8)

Ukonaho T., Rantanen T., Jmsen L. Kuningas K., Pkkil, T.

Lvgren,T.Soukka.Comparisonofinfraredexcitedupconverting
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Kuningas K., Rantanen T., Lvgren T., Soukka T. Enhanced

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(10)

Kuningas K., Rantanen T., Karhunen U., Lvgren T., Soukka T.

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(11)

Kuningas K., Rantanen T., Ukonaho T., Lvgren T., Soukka T.

Homogeneousassaytechnologybasedonupconvertingphosphors.
Anal.Chem.(2005)77,73487355.

(12)

Kuningas K., Ukonaho T.,Pakkil H., Rantanen T., Rosenberg J.

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Rantanen T., Jarvenpa M.L., Vuojola J., Kuningas K., Soukka T.

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Hyppnen I., Hls J., Kankare M., Lastusaart M., Pihlgren L.,
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281

CAPTULO 5
CHAPTER 5

Discusin de resultados

Discussion of the results

Discusinderesultados

Introduccin
En este captulo se discuten las caractersticas de los mtodos
desarrolladosenestaMemoria,realizandounestudiocomparativodesus
ventajas y limitaciones frente a mtodos previamente descritos en la
bibliografa.Tambinsediscutelautilidaddelasmetodologaspropuestas
mediantesuaplicacinalanlisisdealimentos.
El captulo se ha dividido en tres apartados: 1)Nanopartculas de
sliceenanlisisdealimentos,2)Nuevasestrategiasparaladeterminacin
de antioxidantes en alimentos y 3) Nuevas investigaciones en el uso de
upconverting phosphors en sistemas de transferencia de energa de
resonancialuminiscente.
1. Nanopartculasdesliceenanlisisdealimentos.
Como ya se ha indicado, el uso de nanomateriales est teniendo un
importanteaugeendistintasreasdelaQumicaAnaltica[15].Dentro
de la gran diversidad de nanomateriales existentes en la actualidad, la
utilizacin de nanopartculas de slice (SiO2NPs) se ha extendido
rpidamente a lo largo de la ltima dcada gracias a sus excelentes
propiedades que las hacen viables para su uso como marcadores en el
campodelbioanlisis.Enelsiguienteapartadosediscutirnlosresultados
obtenidosconestasNPsentresdelosartculosincluidosenestaMemoria.

Sntesis y caracterizacin de nanopartculas dopadas con

fluorforosdelargalongituddeonda

285

Captulo5

El uso de SiO2NPs para formar marcadores tiene inters en los

mtodoscondeteccinpticadebidoprincipalmentealatransparenciade
laslicealaradiacinvisible,ademsdeotraseriedeventajasdescritasen
laIntroduccindeestaMemoria.
En los dos mtodos generales para la sntesis de SiO2NPs, el
mtodoStberyeldeformacindemicroemulsindemicelasinversas,se
utilizan precursores de slice tales como el tetrametoxisilano (TMOS) y el
tetraetoxisilano (TEOS), los cuales, tras reacciones de hidrlisis y
condensacindanlugaraNPsesfricasdispersas[6].Laeleccindeunou
otro mtodo va a depender de las propiedades fsicas y qumicas de las
especiesquesepretendenencapsularenestasNPs[2,4].
En la Memoria presentada se describe el procedimiento para
sintetizar SiO2NPs con emisin a larga longitud de onda, basado en la
formacin de una microemulsin de micelas inversas, dopando las NPs
condosfluorforosdelargalongituddeonda(LWFs):violetadecresiloy
azulnilo.Paraellosehanoptimizadotodaslasvariablesimplicadasenel
procesodesntesisysehancomparadosuscaractersticas(tamaodeNP,
cantidad de nanomaterial, intensidad de fluorescencia, etc) con las que
presentanlasobtenidasmedianteelmtodoStber.
La formacin de la microemulsin utilizada para la sntesisde las
NPsdopadasconlosfluorforosanteriormenteindicadosimplicaelusode
un tensoactivo, una disolucin acuosa y un disolvente orgnico, mientras
que para la formacin de la base estructural de las NPs es necesario un
precursor de slice. Dependiendo de las cantidades relativas de estos

286

Discusinderesultados

componentessepuedeobtenerunamicroemulsindeaguaenaceite(rica
en disolvente orgnico) o bien una microemulsin de aceiteenagua (rica
en agua). De acuerdo a lo descrito en la bibliografa, se ensay el uso de
ciclohexano, 1hexanol y Tritn X100 para formar la microemulsin, y
mientrasqueelprecursordesliceseleccionadofueelTEOS[2,4,6,810].
Sin embargo, las condiciones experimentales anteriormente descritas no
fueronsatisfactoriasparalasntesisdeNPsdopadasconazulniloovioleta
de cresilo. Los estudios realizados sobre la posible composicin de la
microemulsin pusieron de manifiesto que las NPs sintentizadas en
ausencia de ciclohexano presentaban similares caractersticas a las
obtenidasenpresenciadeestedisolvente,enloreferenteanmerodeNPs
y tamao, pero la intensidad de fluorescencia obtenida fue ligeramente
mayor.ComopuedeobservarseenlasimgenespresentadasenlaFigura
1, obtenidas mediante microscopa electrnica de transmisin (TEM), la
cantidad y tamao de las NPs en ausencia de ciclohexano (Figura 1a) fue
comparable con las NPs obtenidas utilizando 7,5 mL de ciclohexano
(Figura1c).Sinembargo,elusodevolmenesintermediosdeciclohexano
(Figura1b)proporcionaNPsdemayortamaoyunaelevadadistribucin
de tamaos. Segn estos resultados, se prescindi del ciclohexano en el
proceso de sntesis de las NPs dopadas con azul nilo y violeta de cresilo,
utilizndose agua, Tritn X100 y 1hexanol para la formacin de la
microemulsin.
Tras estudiar la influencia de la cantidad de Tritn X100, se
observ que el nmero de NPs dopadas con azul nilo aumentaba con la
cantidad de tensoactivo utilizada, mientras que el tamao de las NPs

287

Captulo5

decreca. Los mejores resultados fueron obtenidos utilizando 510520 mg

deTritnX100(Figura1f),yaquemenorescantidadesdeestetensoactivo
proporcionaronNPsdetamaosuperioralos200nmdedimetro(Figuras
1dy1e).Encambio,cuandolacantidaddeTritnX100eraprximaa1,0g
seobtenanNPsmuypequeasyparcialmenteagregadas.
Tambin se estudi la posibilidad de encapsular el fluorforo en las
SiO2NPs mediante el proceso de formacin de microemulsin de micelas
inversasconunaligeramodificacinadicional.Estamodificacinconsiste
en un paso previo de reaccin, donde el fluorforo reacciona con un
precursor de slice, 3aminopropiltrietoxisilano (APS), para que, una vez
unidocovalentementeaesteprecursor,ambosseincluyanenelprocesode
polimerizacin de las NPs. El objetivo de este procedimiento fue evitar
posibles prdidas de fluorforo a travs de la matriz de la slice,
reduciendo tambin el nmero de lavados necesarios para eliminar el
excesodefluorforo,etapapreviaalautilizacindelasNPs.Noobstante,
comopuedeobservarseenlaFigura1g,lacantidaddeNPsresultantefue
considerablemente menor a la obtenida sin la formacin del precursor
APSazulnilo,ademsdepresentarunaelevadadistribucindetamaos.
Porlotanto,sedescartlaposibilidaddeutilizarestetipodeNPsparala
obtencindemarcadoresutilizablesenposterioresensayos,puestoqueno
mejoraban las caractersticas de las NPs frente a las obtenidas sin incluir
esta modificacin. Este comportamiento se puede atribuir a la
estabilizacin de los fluorforos, cargados positivamente, con los grupos
silanoles, cargados negativamente, caractersticos de la matriz de slice.

288

Discusinderesultados

Adems,serequiereunaetapaadicionalenelprocesodesntesis,conun
mayorconsumodereactivosytiempo.

Figura 1. Imgenes obtenidas mediante microscopa electrnica de transmisin (TEM)

deNPsdopadasconazulniloutilizandodiferentesvolmenesdeciclohexano(a,byc),
diferentes concentraciones de Tritn X100 (d, e y f) y NPs dopadas con (3
aminopropiltrietoxisilano)APSAzulNilo(g).Condicionesexperimentales:100L(0,44
mmol)TEOS,0,2mLde1x103(0,2mol)azulnilo,3mL(0,024mol)hexanol,70L(0,9
mmol) NH4OH. En (Figuras 1a c): 510 520 mg (0,79 0,82 mmol) Tritn X100. En
Figura 1a 11,3 mL (0,63 mol)agua, 0 mL ciclohexano, en Figura 1b, 7,5mL (0,42 mol)
agua, 3,75 mL (0,034 mol) ciclohexano, en Figura 1c, 3,8 mL (0,21 mol) agua, 7,5 mL
(0,068mol)ciclohexano.EnFigura1df,11,3mL(0,63mol)deagua,enFigura1d,0
mg,enFigura1e,310,9mg(0,48mmol),enfigura1f,510,9(0,79mmol)deTritnX100.
Barra de escala: 1 m. En figura 1g, condiciones experimentales: 510 520 mg (0,79
0,81 mmol) de Tritn X100, 10,3 mL (0,57 mol) de agua, 100 L (0,44 mmol) TEOS,
1,2mLprecursordereaccin,3mL(0,024mol)hexanol,70L(0,9mmol)deNH4OH.
Barradeescala:2m.

289

Captulo5

Tambin se estudiaron las proporciones ptimas entre diferentes

reactivos, observndose que la relacin Tritn X100/hexanol es una
variable crtica en el proceso de sntesis. Como puede observarse en la
Figura 2, una cantidad de 170 mg de Tritn X100 por mL de hexanol
proporcionaNPsconunaintensidaddefluorescenciamxima.

Figura 2. Influencia de la relacin Tritn X100/hexanol en la intensidad de

fluorescenciadelasNPsazulnilo.Condicionesexperimentales:11,3mL(0,63
mol) de agua, 100 L (0,44 mmol) TEOS, 0,2 mL de 1x103 M (0,2 mol) azul
nilo,3mL(0,024mol)hexanol,70L(0,9mmol)NH4OH.

Lacantidaddeazulniloutilizadaenelprocesodesntesisesotra

variable crtica. Como puede observarse en la Figura 3, la intensidad de

fluorescenciamximaseobtuvocon1,8moldeazulnilo.Ladisminucin

290

Discusinderesultados

deestaintensidaddefluorescenciaconcantidadesdefluorforomayores,
puede atribuirse a la formacin de agregados moleculares de azul nilo,
cuyafluorescenciaesmenorqueladelmonmero[7].

Figura3.Influenciadelacantidaddeazulniloadicionadaenlaintensidadde
fluorescencia de las NPs dopadas con este fluorforo. Condiciones
experimentales:510520mg(0,790,81mmol)deTritnX100,11,3mL(0,63
mol)deagua,100L(0,44mmol)TEOS,3mL(0,024mol)hexanoly70L(0,9
mmol)NH4OH.

Trasestudiarlainfluenciadetodaslasvariablesinvolucradasenel

proceso de sntesis mediante el mtodo de microemulsin de micelas

inversas, las NPs obtenidas fueron comparadas con las sintetizadas
mediante el mtodo Stber. En la Tabla 1 se recogen diferentes
caractersticas de las NPs dopadas con violeta de cresilo obtenidas por

291

Captulo5

ambosmtodos.Conelfindecompararlascaractersticasluminiscentesde
ambos tipos de NPs se prepararon suspensiones de igual concentracin,
observndosequeeldimetromediodelasNPsobtenidasporelmtodo
de microemulsin es ligeramente mayor que el de las obtenidas por el
segundo mtodo, aunque las diferencias no son excesivamente elevadas.
Encambio,sexistendiferenciasdestacablesenlosvaloresdeintensidades
defluorescencia.Esteestudioserealizpreparandosuspensionesenmedio
acuoso de las NPs obtenidas mediante ambos mtodos, encontrando que
las NPs del mtodo optimizado presentaban una intensidad de
fluorescencia12vecesmayorquelasobtenidasenelmtodoStber,loque
puedeatribuirsealamenordegradacindelvioletadecresiloenelmedio
alcalinoutilizadoduranteelprocesodesntesis,graciasalaproteccinque
leproporcionaelmediomicelar.
Tabla1.ComparacindelaspropiedadesdelasNPsdopadasconvioleta
decresilo.

Mtododemicroemulsin
demicelasinversas
0.1

Stbermethod

Cantidaddepartculas,mg

0.02

0.02

Dimetro,nm

178

133

2.94x109

7.06x109

125563

10138

RelacinIntensidad/Npartculas

4.27x105

1.44x106

Intensidadespecfica

1.45x107

1.17x106

Concentracin,mg/mL

NmerodePartculas
Intensidada

0.1

Las medidas fueron realizadas utilizando microplacas (200 L) y filtros de

excitacinyemisinde531/25y620/8nm(longituddeondanominal/pasode
banda),respectivamente.
a

292

Discusinderesultados

La comparacin de los espectros de emisin de las NPs dopadas

con cada uno de los fluorforos con los obtenidos para los mismos
fluorforosendisolucinyparalasNPssindopante,mostrquelaslice
por s sola no presenta emisin fluorescente a las longitudes de onda
tpicasdelosfluorforosyqueelmximodeemisindelasNPsdopadas
conestasoxacinassufreunligerodesplazamientohipsocrmicodeunos9
nmfrentealosfluorforosendisolucin.Parallevaracabolosestudiosde
fotodescomposicin,seirradiaronlosfluorforosendisolucinylasNPs
dopadasduranteunahoraalaslongitudesdeondademximaexcitacin
y emisin. La intensidad de fluorescencia disminuy un 75 % para el
violeta de cresilo y un 62 % para el azul nilo, ambos en disolucin,
mientras que la fluorescencia de las NPs prcticamente no se alter. El
mismo estudio se realiz con las SiO2NPs preparadas en presencia de la
mezcla azul nilo y APS, obteniendo una disminucin de fluorescencia de
aproximadamenteel10%.Laestabilidaddelfluorforoencapsuladoenlas
NPstambinseestudialolargodeltiempo,comprobndosequelaseal
fluorescentepermanecaconstantedurantealmenosunmes.
En resumen, se puede afirmar que el mtodo modificado de
microemulsindemicelasparalasntesisdeNPsdopadascondosLWFs
(azul nilo y violeta de cresilo) proporciona unas NPs de similares
caractersticas (tamao, cantidad de material, dimetro medio) que las
obtenidas mediante el mtodo Stber, considerado como mtodo de
referencia. Sin embargo, la intensidad de fluorescencia de las NPs
sintetizadas en el primer caso es mucho mayor que la obtenida por el

293

Captulo5

mtodo Stber, atribuibles a la estabilidad y proteccin que les confiere a

losLWFslamicroemulsindondetienelugarlapolimerizacindelaslice.

Utilizacin de nanopartculas de slice en inmunoensayo

heterogneoparaladeterminacindemacromolculasymolculas
pequeas
La determinacin de biomolculas constituye un campo de

aplicacin de la Qumica Analtica de gran inters. Se han desarrollado

numerosos mtodos cromatogrficos y electroforticos para la
determinacin de macromolculas y molculas pequeas [11 15].
Normalmente, este tipo de tcnicas requiere que el analito presente una
propiedad medible como, por ejemplo, absorbancia o fluorescencia. En
casocontrario,esimprescindibleelusodeunaetapadederivatizacinque
complica y aumenta la duracin del anlisis [16, 17]. Se han desarrollado
diferentes mtodos cromatogrficos acoplados a espectrometra de masas
para la determinacin de biomolculas, los cuales requieren
instrumentacinsofisticadaycostosa[1820].Adems,enalgunoscasos,
es necesario incluir etapas previas para la eliminacin de la matriz de la
muestra,quealarganelprocesoyaumentanlamanipulacinporpartedel
analista. Los mtodos de screening desempean dos funciones muy
importantes en el anlisis de biomolculas: por un lado, el nmero de
muestrassusceptiblesdesersometidasamtodosconfirmatoriosesmenor
y, por otro lado, se reduce el coste del anlisis. Los inmunoensayos,
extensamente utilizados en anlisis cuantitativo y semicuantitativo,
presentanunaseriedeventajasparasuusocomomtodosdescreening
enladeterminacindesustanciasantignicas,yaseanmolculaspequeas

294

Discusinderesultados

(haptenos) o macromolculas. Se han descritos varios inmunoensayos

heterogneos que utilizan marcadores enzimticos (ELISA) comerciales y
no comerciales [21 25]. Un porcentaje destacable de estos mtodos hace
usodemedidasfotomtricas,obtenindoselmitesdedeteccinprximosa
ng/mL, mientras que para obtener menores lmites de deteccin es
necesarioelusodesistemasdedeteccinquimioluminiscente[26]obien,el
usodequelatosdeeuropio(III)paraformarelmarcador[27,28].
La utilizacin de NPs inorgnicas funcionalizadas para obtener
marcadoreseninmunoensayoesuncamporelativamentenuevoydegran
proyeccin gracias a las verstiles propiedades fsicas y qumicas que
presentanestosmateriales,mejorandofrecuentementelascaractersticasde
losensayos[2931].EntrelasNPsinorgnicas,lasSiO2NPsdopadasson
una opcin muy til debido a las propiedades anteriormente indicadas.
Adems, presentan la capacidad de encapsular una gran variedad de
compuestos,quequedanprotegidosfrentealosagentesambientales,gran
reasuperficialyfcilfuncionalizacindesusuperficie.Estaspropiedades
han dado lugar a numerosos inmunoensayos para la determinacin de
macromolculas y haptenos [32, 33]. Por ejemplo, se ha desarrollado un
mtodo para la determinacin de E. Coli utilizando NPs dopadas con el
complejo rutenio (II) tris(bipiridina) (Ru(bpy)32+) inmovilizando el
anticuerpo en la superficie de la NP, lo que ha permitido llegar a
determinarunasolabacteriasinrequerirlaamplificacindelaseal[34].
De manera similar, se ha determinado albmina de suero bovino (BSA)
mediante un ensayo de afinidad biotinaestreptavidina utilizando NPs
dopadas con rodamina 6G para formar el marcador [35], as como la

295

Captulo5

determinacin de fetoproteina (AFP) y antgeno de superficie de la

hepatitisB(HBsAg)utilizandoSiO2NPsdopadasconfluorescena[36,37].
EstasSiO2NPstambinpuedenencapsularotrasespeciescomoquelatosde
terbio y europio, que han sido utilizados como marcadores para la
determinacin de HBsAg, antgeno carcinoembrinico (CEA) y antgeno
prostticoespecfico(PSA)[3840].Delosejemploscitados,sededuceque
lasSiO2NPsdopadashansidoprincipalmenteutilizadasenanlisisclnico,
pero su uso para desarrollar inmunoensayos aplicables al anlisis de
alimentosestclaramentejustificado.
En los inmunoensayos presentados en esta Memoria se utilizan
SiO2NPsdopadasconazulniloparaobtenerelmarcador,aplicndolasala
determinacin de protenas de soja (macromolculas) y del antibitico de
uso veterinario monensn, cuya presencia en los alimentos de origen
animal est regulada a nivel europeo. Los resultados obtenidos han
demostradoqueelusodeestasNPsproporcionaalgunasventajasrespecto
aotrostiposdeinmunoensayos,comosonlmitesdedeteccinmsbajos
y,enelcasodelenzimoinmunoensayo,unmenornmerodeetapasyaque
no es necesaria la adicin de un sustrato para medir la actividad de la
enzima.
La incorporacin de azul nilo a las SiO2NPs ha tenido como
objetivo el uso de medidas de fluorescencia a larga longitud de onda, las
cuales proporcionan una selectividad espectral adecuada al eliminar las
seales de fondo producidas por la matriz de la muestra. Como se ha
indicado anteriormente, el uso de SiO2NPs podra estar limitado por la
posibleprdidadefluorforoatravsdelamatrizdeslice.Noobstante,

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Discusinderesultados

como ya se ha comentado, las SiO2NPs dopadas con azul nilo muestran

una excelente estabilidad debida a las interacciones electrostticas del
fluorforocargadopositivamenteconlosgrupossilanolesdelaslice.
A continuacin se discuten los resultados obtenidos en los
inmunoensayosdesarrolladosenestaMemoria.

Determinacin de protenas de soja en alimentos mediante

inmunoensayoheterogneo.
En los ltimos aos, el consumo de alimentos enriquecidos con

protenasdesojasehaincrementadosustancialmentedebidoalosaspectos
beneficiosos para la salud que se han descrito para estas protenas, tales
como un descenso del colesterol en sangre, prevencin de enfermedades
como el cncer, la diabetes o la obesidad, as como proteccin frente a
enfermedades que daan el intestino o el rin. Sin embargo, tambin se
ha descrito que el consumo de estas protenas puede originar reacciones
alrgicas.
En esta Memoria se describe el desarrollo de un inmunoensayo
heterogneo competitivo para la determinacin de protenas de soja en
muestras de alimentos utilizando SiO2NPs dopadas con azul nilo para
formarelmarcador,estudindosedosmodalidadesdeinmunoensayo,con
capturadeanalitoyconcapturadeanticuerpo,comoseesquematizaenlas
Figuras 4 y 5. Las NPs fueron unidas a protenas de soja y a anticuerpos
antiprotena de soja para estudiar su viabilidad como trazadores en los
dosformatosdeinmunoensayo.Elprimerodeellos(Figura4),concaptura
deanalito,implicalainmovilizacindeanticuerposantiIgGdeconejoen

297

Captulo5

lasuperficiedelospocillosseguidadelainmovilizacindelosanticuerpos
antiprotenadesojayelposteriorusodelasNPsunidasalaprotenade
soja como marcador. Se utilizaron anticuerpos antiIgG debido al menor
costequesuponenfrentealusodeanticuerposantiprotenadesoja,para
recubrirlatotalidaddelospocillos.Elusodedichosanticuerpossobrelos
que se unen los anticuerpos frente al analito disminuye la concentracin
necesariadeestosltimosy,porlotanto,elcostetotaldelinmunoensayo.
La superficie de los pocillos que forman la microplaca fue tratada con
albmina de suero bovino (BSA) a diferentes concentraciones para evitar
uniones no especficas entre el marcador y los anticuerpos antiIgG
inmovilizados previamente. No obstante, los ensayos realizados
demostraron que el procedimiento no fue suficientemente adecuado ya
que,peseadichorecubrimientoconBSA,anseproducandichasuniones
noespecficas.

Figura 4: Inmunoensayo heterogneo competitivo con captura de analito

utilizandocomomarcadorSiO2NPsdopadasconazulnilounidasaprotenade
soja.

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Discusinderesultados

Para solucionar este problema, se opt por hacer uso del formato
de inmunoensayo con captura de anticuerpo (Figura 5). Para ello, las
protenas de soja se inmovilizaron sobre la superficie de la microplaca,
adicionando posteriormente BSA para evitar posibles uniones no
especficasconelmarcador,formadoporanticuerposantiprotenasdesoja
marcados con las NPs. A continuacin, tras adicionar la muestra y el
marcador a los pocillos, se produjo el inmunoensayo basado en la
competencia entre la protena de soja presente en la muestra y la
inmovilizadaenlaplacaporenlazarsealmarcador.Despusdeincubar,se
elimina la mezcla de reaccin, se lavan los pocillos para eliminar las
especies no unidas a la superficie y se llevan a cabo las medidas de
fluorescenciaenlasuperficiedelospocillos.

Figura 5: Inmunoensayo heterogneo competitivo con captura de

anticuerpo utilizando como marcador SiO2NPs dopadas con azul nilo
unidasaanticuerposantiprotenasdesoja.

Lasmedidasserealizaronaex620yem680nm,obtenindoseun
intervalolinealde0,110mg/Lyunlmitededeteccinde0,05mg/L,el

299

Captulo5

cual es 10 veces menor que el obtenido con el kit comercial ELISA [41].
Adems, a diferencia del mtodo ELISA, en este caso no es necesaria la
adicin de sustrato enzimtico, reducindose as el nmero de etapas del
ensayoyladuracindelanlisis.
Laprecisin,expresadacomodesviacinestndarrelativa,ensayada
a dos concentraciones diferentes, 0,5 y 5 mg/L, fue de 9,6 % y 6,1%
respectivamente.Laselectividaddelmtododependeprincipalmentedela
selectividad de los anticuerpos antiprotena de soja. De acuerdo con las
especificaciones indicadas por el proveedor de estos anticuerpos, estos
presentan cierta reactividad cruzada con extractos de harina de arroz y
ovoalbmina tras su tratamiento con urea caliente. En cambio, no
interaccionan con extractos de carne, maz o casenas, as como arroz o
patata. Adems, en un estudio previo en el que se utiliza el mismo
anticuerpo antiprotena de soja, se describe que las concentraciones de
BSA, globulinas, mioglobina y hemoglobina toleradas son 80 veces
mayoresquelaconcentracindeanalito[42].
El mtodo fue aplicado al anlisis de muestras de leche, yogur y
zumo de frutas que contenan soja. Estas muestras tambin fueron
analizadasconunkitcomercialELISA[41]y,enamboscasos,lasmuestras
fueron sometidas a un tratamiento de desnaturalizacinrenaturalizacin
de acuerdo con las recomendaciones procedentes del distribuidor de los
anticuerposantiprotenadesoja.Losresultadosobtenidossemuestranen
la Tabla 2, los cuales fueron sometidos al test de la t por parejas con un
nivel de confianza del 95 %, sin encontrarse diferencias estadsticamente
significativasentreelmtodopropuestoyelkitcomercial.

300

Discusinderesultados

Tabla2.Determinacindeprotenasdesojaenmuestrasdealimentos
Muestra

MtodoPropuestoa,b

Lechedesoja
Zumodefrutaysoja

KitcomercialELISAa,b

746

843

8,40,7

10,50,3

644

603

Yogurdesoja

MediaDE(n=3)
Unidades:lechedesojaeng/L;zumodefrutasysojayyogurdesojaeng/Kg

Para llevar a cabo el estudio de recuperaciones, se aadieron tres

cantidades diferentes de protenas de soja a las muestras, obtenindose

unosvaloresderecuperacinentre81,5%y111,0%,comosemuestraenla
Tabla 3, lo que pone de manifiesto la utilidad del mtodo para realizar
estosanlisis.

Tabla3.Recuperacionesdeprotenasdesojademuestrasdealimentos
Aadidoa,b

Encontradoa,b

Recuperacin(%)

Lechedesoja

24

272

111,0

72

694

95,7

123

11010

89,4

Zumodefrutasysoja

2,7

2,20,2

81,5

8,1

7,70,6

95,1

13,5

121

88,9

Yogurdesoja

23

212

91,3

65

614

93,2

113

957

84,1

Muestra

MediaDE(n=3)
Unidades:lechedesojaeng/L;zumodefrutasysojayyogurdesojaeng/Kg

301

Captulo5

Determinacin de monensn en muestras de leche mediante

inmunoensayoheterogneo.

El monensn es un antibitico de uso veterinario con actividad

antibacteriana y coccidiosttica, usado tradicionalmente para prevenir la

coccidiosis en pollos [43]. Este antibitico es utilizado tambin como
promotor del crecimiento, ya que previene enfermedades que retrasan el
crecimientode los pollos como diarreas hemorrgicas y prdidas de peso
asociadas. Aunque el uso de promotores del crecimiento comenz a
prohibirseapartirde2006,algunosdeestosanpuedenseradministrados
bajo regulacin de la Unin Europea [44]. Adems, el tratamiento con
monensn ha sido ampliado por la Unin Europea a terneros,
establecindoselmitesmximosderesiduoparaesteantibitico.
Lapreocupacinporlaseguridadalimentariahallevadoabuscary
desarrollar nuevos mtodos analticos de screening y confirmatorios
para detectar la presencia de residuos de antibiticos en alimentos, los
cuales, al llegar al consumidor, pueden dar lugar a un incremento de la
resistenciabacterianaaantibiticos.
En esta Memoria se ha presentado un mtodo que constituye el
primer inmunoensayo para la determinacin de monensn utilizando
SiO2NPsdopadasconazulniloparaformarelmarcador.Sehademostrado
su utilidad para mejorar las caractersticas analticas, tales como el lmite
dedeteccin,respectoalosinmunoensayosdescritosanteriormenteparala
determinacindeestefrmacoutilizandomarcadoresenzimticos[22,45
48].

302

Discusinderesultados

Como se ha indicado anteriormente, el uso de SiO2NPs dopadas

con azul nilo puede mejorar los lmites de deteccin gracias a la
selectividadespectralquepresentanlosLWFsyaqueseminimizalaseal
de fondo procedente de la matriz de la muestra. Adems, la excelente
estabilidad del fluorforo encapsulado en las NPs frente a agentes
ambientalesyalaradiacinexcitante,evitandosufotodescomposicin,as
comolaestabilidadtemporaldeestasNPsdopadas,justificanlautilizacin
de este nanomaterial para formar marcadores y aplicarlos en
inmunoensayos. Otra ventaja del mtodo propuesto es el uso de
microplacas con pocillos de 100 L, a diferencia de las microplacas
tradicionales con pocillos de 300 L. De esta manera, se reducen los
volmenes de reactivos utilizados durante el desarrollo del mtodo,
disminuyndose as el coste total del inmunoensayo y los volmenes de
residuos generados. Se utilizan este tipo de placas ya que las medidas se
realizanensuperficieslidayelfondodeestospocillosestaunamayor
altura que en las placas tradicionales, de forma que la distancia entre la
superficie slida y el detector disminuye, minimizando prdidas de seal
luminiscenteymejorandolasensibilidad.
Al igual que en el apartado anterior, se estudiaron dos posibles
formatos competitivos para este inmunoensayo heterogneo, con captura
de anticuerpo y con captura de analito. El primero de ellos se bas en la
inmovilizacin en la microplaca de un conjugado BSAmonensn y un
marcadorformadoporNPssobrelasqueseinmovilizelanticuerpoanti
monensn.Encambio,elsegundoformatonecesitunmarcadorformado
por NPsmonensn, sintetizado via carbodiimida usando EDAC

303

Captulo5

(clorhidrato

de

N(3dimetilaminopropil)Netilcarbodiimida),

la

inmovilizacin del anticuerpo antiIgG de oveja sobre los pocillos y la

posterior unin delanticuerpo antimonensn sobre esta superficie slida.
Enambosensayosseobtuvounadiferenciaapreciableentrelosvaloresde
intensidaddefluorescenciaobtenidosenausenciayenpresencia(0,2g/L)
de monensn. Sin embargo, esta diferencia de seal fue tres veces mayor
utilizando el formato de captura de anticuerpo, por lo que se seleccion
esteformatoparaladeterminacindemonensnenmuestrasdeleche.
El mtodo desarrollado presenta un intervalo lineal dinmico de
0,05 5 g/L y unos lmites de deteccin y cuantificacin de 0,015 y 0,05
g/L,respectivamente,quecorrespondena0,12y0,40g/Kgenmuestras
de leche. Este ltimo valor es 5 veces menor que el lmite mximo de
residuo (MRL) establecido por la Comisin de Regulacin de la Unin
Europea en la normativa 37/2010/EC en muestras de leche. Adems, el
lmite de deteccin es cuatro veces menor que el descrito en el kit ELISA
con deteccin quimioluminiscente [32]. La precisin, expresada como
desviacin estndar relativa, fue ensayada a dos concentraciones de
monensn, 0,2 y 1 g/L, obtenindose unos porcentajes de 5,8 % y 4,0 %,
respectivamente.
El mtodo se aplic al anlisis de muestras de leche entera, semi
desnatada y desnatada, realizando un tratamiento previo sencillo basado
en una etapa de desproteinizacin y la posterior evaporacin del
disolvente. Para validar el mtodo se llev a cabo un estudio de
recuperaciones(Tabla4),obteniendovaloresentre83,3%y107,5%,loque
demuestrasuutilidadparaelanlisisdeestasmuestras.

304

Discusinderesultados

Tabla 4. Recuperaciones obtenidas para monensn aadido a muestras de

leche
Muestra
Monensn

Aadidoa,b
(g/Kg)
1,5

Encontradoa,b
(g/Kg)
1,60,1

Recuperacin
(%)
106,7

1,960,09

98,0

4,10,2

102,5

1,5

1,50,1

100,0

2,00,1

100,0

3,40,3

85,0

1,5

1,250,07

83,3

1,70,1

85,0

4,30,3

107,5

Lechedesnatada

Lechesemidesnatada

Lecheentera

MediaDE(n=3)

En resumen, los estudios realizados utilizando SiO2NPs dopadas

con un LWF para obtener los correspondientes marcadores, ponen de

manifiesto su aplicabilidad para la determinacin de macromolculas
(protenas)ymolculaspequeas(antibiticos)enanlisisdealimentos.Se
consiguen menores lmites de deteccin que en ELISA y se evita la etapa
finalparalamedidadelaactividaddelaenzima.Asimismo,losmtodos
propuestoshancontribuidoaexpandirelusodelananotecnologaenesta
reaanaltica.

305

Captulo5

2. Nuevas estrategias para la determinacin de antioxidantes en

alimentos

Los procesos de oxidacin que se producen en el organismo

implican la formacin de radicales libres responsables del desarrollo de
algunas enfermedades, tales como enfermedades cardiovasculares,
Alzheimer y cncer. Este fenmeno redox es producido por especies
reactivas de oxgeno (ROS), capaces de daar las membranas celulares,
aunque este efecto puede ser reducido por la ingesta de compuestos
antioxidantes.Lacapacidadantioxidantedealgunosalimentosvienedada
principalmenteporlapresenciadepolifenoles,comolosflavonoides,yde
lasvitaminasCyE,entreotros[49].
EnestaMemoriasehadesarrolladounmtodoparaladeterminacin
delacapacidadantioxidantedealgunosalimentos,enelquesedemuestra
la utilidad de medidas de fluorescencia a larga longitud de onda, y otro
mtodoparaladeterminacindepolifenolesenelqueseproponeelusode
nanomaterialescomoactivadoresdelaenzimalaccasa.Acontinuacin,se
discutenlascaractersticasdeestosmtodosysuaplicabilidad.

Determinacin fluorimtrica a larga longitud de onda de la

capacidad antioxidante de alimentos utilizando azul nilo como
reactivo

Desdeelpuntodevistaanaltico,existendostiposdemtodospara
ladeterminacindelacapacidadantioxidantedelosalimentos.Elprimer
grupoimplicareaccionesdetransferenciadeelectrones,lascualespueden

306

Discusinderesultados

seguirsemediantealgncambiodecolorodefluorescenciadetectableen
el medio de reaccin. El segundo grupo se basa en reacciones de
transferencia de protones, en las que el antioxidante y un sustrato
compitenporlosradicaleslibresgenerados.ElmtodoORAC(capacidad
deabsorcinderadicalesdeoxgeno),pertenecienteaestesegundogrupo,
utiliza un generador de radicales libres que ataca directamente a un
cromforooaunfluorforo,experimentandostosunadisminucindesu
absorbancia o de su fluorescencia, respectivamente. Para este ensayo se
hanutilizadocromforos,talescomoelrojodepirogalol[50],yenmucha
mayorextensindiferentestiposdefluorforostalescomolaficoeritrina
y la fluorescena [51 57]. A pesar de la buena sensibilidad que ofrecen
estos compuestos, el desplazamiento Stokes que muestran es bastante
estrecho (alrededor de 27 nm), lo cual favorece los fenmenos de
dispersin de la radiacin. Adems, el coste de la ficoeritrina es
relativamente elevado y suele sufrir fenmenos de fotodescomposicin,
mientras que la emisin de la fluorescena normalmente solapa con las
sealesdefondoprocedentesdelamatrizdelamuestra.ElusodeLWFs
puede evitar estos inconvenientes, aunque su potencial en ensayos de
actividadantioxidantenohasidoinvestigadohastaahora.
En esta Memoria se describe un mtodo analtico para la
determinacin de la capacidad antioxidante de algunos alimentos
medianteelensayoORACutilizandoazulnilocomoreactivofluorescente,
diclorhidrato de 2,2azobis(2amidinopropano) (AAPH) como generador
deradicaleslibres,ycido6hidroxi2,5,7,8tetrametilcroman2carboxlico
(Trolox) como analito de referencia. Las medidas de fluorescencia se

307

Captulo5

llevaronacaboenmodocinticoregistrandosuvariacinconeltiempoy
utilizandocomoparmetroanalticoelreanetabajolacurvanormalizada,
AUCneta = (AUCmuestra AUCblanco)/AUCblanco), obtenida para diferentes
concentracionesdeanalito.
Como

se

ha

comentado

anteriormente,

los

fluorforos

tradicionalmente utilizados para este tipo de ensayos han sido la

ficoeritrina y la fluorescena, los cuales ven inhibida su prdida de
fluorescencia en este tipo de ensayos en presencia de compuestos
antioxidantes, pero su utilizacin presenta las limitaciones ya indicadas.
Con objeto de evitar estas limitaciones, se estudi la posibilidad de usar
otrostiposdefluorforosqueemitanalargalongituddeonda,comoson
las oxacinas y las tiacinas, siendo el azul nilo, azure A y azure B los
fluorforosseleccionados.Parallevaracaboesteestudio,seregistraronlas
curvascinticasparalostresfluorforosenpresenciayausenciadeTrolox.
ComopuedeobservarseenlaFigura6,enlostrescasosexistendiferencias
entre la curva del blanco y la del estndar, aunque se obtuvo una mayor
diferenciaconelfluorforoazulnilo.LainfluenciadelpHfueestudiadaen
el intervalo de 4 11, utilizando las disoluciones reguladoras acetato,
fosfato,boratoycarbonato,aunaconcentracinde0,075M,paramantener
constanteelvalordepHdeseado.

308

Discusinderesultados

18000

18000
16000

a)

Intensidadfluorescenciarelativa
Relative fluorescence intensity

Intensidadfluorescenciarelativa
Relative fluorescence intensity

16000
14000
12000
10000

8000
6000
4000
2000

b)

14000
12000
10000

8000
6000

4000
2000

0
0

20

40

60

80

100

20

40

60

80

100

Tiempo
(min)
Time (min)

Time(min)
(min)
Tiempo

18000

Relative
Intensidadfluorescenciarelativa
fluorescence intensity

16000

c)

14000
12000

10000
8000

6000

4000

1
2000
0

20

40

60

Tiempo
(min)
Time (min)

80

10

Figura6:CurvasobtenidasparaelensayoORACconlosfluorforos(a)azul
nilo,(b)azureA,y(c)azureB:(1)blancoy(2)enpresenciadeTrolox(2M).

Como puede observarse en la Figura 7, el rea bajo la curva

normalizadanoesapreciableparavaloresinferioresa5,8ysuperioresa8,
obtenindose los mejores resultados en el intervalo de 6 a 7. El
comportamiento del sistema a valores de pH bajos puede deberse al pKa
delTrolox(3,89),cuyasolubilidaddisminuyealaproximarseaestevalor.

309

Captulo5

0.10
0,10

reabajo lacurvanormalizada
AUC

A)
0.08
0,08

0.06
0,06

0.04
0,04

0.02
0,02

0,00
0.00

5.5
5,5

6.0
6,0

6.5
6,5

7.0
7,0

pH

7.5
7,5

8.0
8,0

8.5
8,5

Figura 7. Influencia del pH en el mtodo ORACazul nilo. Condiciones

experimentales:[azulnilo]=120nM;[tampn]=0,075M;[AAPH]=0,024
M,[Trolox]=2Mytemperatura=27oC.

La influencia de la concentracin del fluorforo se estudi en el

intervalo de 60 a 370 nM (Figura 8), encontrndose que el sistema era
prcticamente independiente de esta variable en el intervalo de 80 a 130
nM,porloqueseeligiunaconcentracinptimadefluorforode89nM.
CabeindicarqueseestudilaposibilidaddeutilizarSiO2NPsdopadascon
azul nilo como alternativa al uso del fluorforo en disolucin, pero los
resultados no fueron satisfactorios, como era previsible, debido a la
proteccin que ejercen las NPs sobre la fluorescencia del azul nilo
encapsulado.SeevalulaconcentracindeAAPH,enunintervalode6a
24 mM, encontrndose que las diferencias del rea bajo la curva entre el

310

Discusinderesultados

blanco y en presencia de una concentracin determinada de Trolox

disminuyen al aumentar la concentracin de AAPH. Sin embargo, las
curvas obtenidas a bajas concentraciones de AAPH mostraron escasa
reproducibilidad,porloqueseoptporseleccionarunaconcentracinde
AAPHde12mMcomoptima.

B)

0.12
0,12

0.08
0,08

AUC

reabajo lacurvanormalizada

0.16
0,16

0,04
0.04

0,00
0.00

0
0

100
100

200
200

[NB], nM
[azulnilo](nM)

300
300

400
400

Figura 8. Influencia de la concentracin de azul nilo sobre el mtodo ORAC

azulnilo.Condicionesexperimentales:[AAPH]=0,012M;[Trolox]=2M;pH
=6,9;[fosfato]=0,085M;ytemperatura=37oC.

Elintervalolinealdinmicoobtenidofuede0,88MdeTroloxy
el lmite de deteccin fue de 0,45 M, el cual es menor o similar a los
obtenidos en otros mtodos ORAC donde se utiliza la fluorescena como
fluorforo. La precisin del mtodo se ensay a dos concentraciones

311

Captulo5

diferentes de Trolox, 1 y 5 M, obtenindose una desviacin estndar

relativade5,6%y2,9%,respectivamente.
Elmtodopropuestoseaplicalanlisisdemuestrasdediferentes
tipos de vinos y zumos de frutas, comparando los resultados con los
obtenidos en el mtodo ORAC convencional utilizado como mtodo de
referencia. El tratamiento de las muestras fue igual para ambos mtodos,
siendo slo necesario ajustar el pH hasta la neutralidad y diluir las
muestrashastaadecuarlaconcentracindeantioxidantealintervalolineal
dinmicodelacurvadecalibrado.
En la Tabla 5 se muestran los resultados obtenidos con ambos
mtodos.Elensayoestadsticodelatporparejasdemostrquenoexistan
diferencias significativas entre ellos, lo cual confirma la utilidad del
mtodopropuestoenestaMemoria.
Tabla5.Capacidadantioxidantedemuestrasdealimentosanalizadasporlos
mtodosORACazulniloyORACfluorescena
Muestra
ORACazulniloa,b
ORACfluorescenaa,b

Vinoblanco(Manzanilla)

1,500,09

1,30,2

Vinosemiseco

7,70,9

4,40,4

Vinotinto

15,30,4

10,40,9

2,2220,001

1,930,09

Zumodepia

2,70,3

2,3990,006

Zumodemanzana

2,70,3

3,50,4

Zumodemelocotn

MediaDE(n=3)
ExpresadacomomiliequivalentesdeTroloxenlamuestra

312

Discusinderesultados

Sellevacabounestudioderecuperacionesparavalidarelmtodo

mediante la adicin de tres concentraciones diferentes de Trolox a cada

una de las muestras analizadas y sometindolas al mismo tratamiento
mencionado anteriormente. En la Tabla 6 se muestran los resultados
obtenidos en el estudio de recuperacin, oscilando estos resultados entre
72,7%y113,6%,conunosvaloresmediosde92,0%y92,9%paramuestras
devinosydezumos,respectivamente.Estavalidacininternaconfirmala
utilidaddelmtododesarrolladoparaelanlisisdemuestrasreales.
Tabla6.Valoresderecuperacinobtenidosparalasdiferentesmuestras
analizadas

Estudioderecuperaciones
Muestra
Vinoblanco

Aadidoa
2,2
8,9
11,1

Encontradoa,b
2,00,2
6,70,9
9,20,8

Recuperacin(%)
92,0
75,2
82,9

Vinosemiseco

4,4
8,8
13,2

3,60,2
8,10,3
12,90,6

81,8
92,1
97,0

Vinotinto

17,8
35,6
44,4

182
384
444

101,1
106,7
99,1

Zumodemelocotn

2,2
4,4
5,5

2,10,1
3,50,4
4,30,2

95,5
79,5
78,2

Zumodepia

2,2
4,4
5,5

2,50,2
4,50,3
5,80,4

113,6
102,3
105,5

Zumodemanzana

4,4
3,20,2
8,8
7,90,5
11,1
111
aUnidadesenmiliequivalentesdeTroloxenlamuestra
bMediaDE(n=3)

313

72,7
89,6
99,1

Captulo5

Losresultadosobtenidosmuestranlautilidaddelafluorescenciaa

largalongituddeondaparaladeterminacindelacapacidadantioxidante
de alimentos utilizando el fluorforo azul nilo. El uso de este reactivo en
lugardeotrosfluorforospreviamenteutilizadosconestepropsito,como
sonlafluorescenaylaficoeritrina,suponeunaalternativaparareducir
posiblessealesdefondoprocedentesdelamatrizdelamuestra,lascuales
pueden aparecer al excitar a longitudes de onda ms cortas. Adems, el
relativamente ancho desplazamiento Stokes que presenta el azul nilo
minimiza los efectos de dispersin de la radiacin, muy comunes al usar
fluorforos con desplazamientos Stokes relativamente estrechos. Por
ltimo,laprdidadefluorescenciadebidaalafotodescomposicindelazul
nilo es menor que en el caso de la ficoeritrina, siendo tambin su coste
inferior.

Determinacin automtica de polifenoles en vinos utilizando

laccasaynanopartculasdexidodeterbio

Los polifenoles constituyen un importante grupo de compuestos

antioxidantes naturales presentes en alimentos que, tras numerosos

estudios clnicos, han mostrado su eficacia para la prevencin de algunas
enfermedadescrnicas.Comosehadescritoenelapartadoanterior,seha
desarrollado una gran diversidad de mtodos analticos cromatogrficos
[58, 59] y electroforticos [60, 61] para la determinacin de estos
antioxidantes naturales. Sin embargo, debido al elevado nmero de
compuestosfenlicosquepuedenencontrarseenmuestrasalimenticias,es
frecuente el uso de mtodos analticos que determinan el contenido

314

Discusinderesultados

fenlico total as como la capacidad antioxidante total. Estos parmetros

han sido ampliamente determinados mediante mtodos fotomtricos o
fluorimtricos haciendo uso de generadores de radicales libres o bien
reacciones enzimticas, que compiten con los antioxidantes con el fin de
inhibir la prdida de fluorescencia o absorbancia que tendra lugar en
ausenciadelosmismos[56,6267].
Lalaccasaesunaenzimafeniloxidasaquehasidoutilizadaparala
determinacin del contenido fenlico total y la capacidad antioxidante,
utilizando diferentes condiciones experimentales. Como ya se ha
comentado anteriormente en esta Memoria, esta enzima cataliza la
oxidacin de compuestos fenlicos a quinonas o radicales mediante
reduccindeloxgenodisueltoenelmedio,locualhasidoutilizadoparael
desarrollo de sensores ampermetricos [68 70] as como mtodos con
deteccinfotomtrica[7173]yfluorimtrica[74].
EnestaMemoriasemuestralautilidadanalticadelusocombinado
denanopartculasdexidodeterbio(Tb4O7NPs),laccasa,yelfluorforo8
hidroxipireno1,3,6trisulfonatotrisdico(HPTS)paraladeterminacinde
polifenolesenmuestrasdevino.Aligualqueenelmtodoexpuestoenel
apartadoanterior,lasmedidasdefluorescenciasellevaronacaboenmodo
cintico registrando su variacin con el tiempo y utilizando como
parmetro analtico el rea bajo las curvas obtenidas a diferentes
concentracionesdecidoglico,utilizadocomoestndardecalibracin.
En primer lugar, se investig el comportamiento de diversos
fluorforos como posibles sustratos de la laccasa.Con tal fin se ensay la
capacidad de Styryl 7, 2Di1ASP, azure B, azul de toluidina, azul nilo,
fluorescena y HPTS para competir con los compuestos fenlicos por los

315

Captulo5

sitios activos de la enzima. Aunque el verde de indocianina ha sido

descrito anteriormente como sustrato para la laccasa [74], no pudo
ensayarse debido a que el lector de placas utilizado no tiene capacidad
para medir la fluorescencia a la larga longitud de onda que presenta este
compuesto. El estudio realizado demostr que slo el fluorforo HPTS
experimentaba una disminucin relativamente rpida y notable de su
fluorescenciaenpresenciadelalaccasa.
Como puede observarse en la Figura 9, el fluorforo HPTS tiene un
grupo fenlico en su estructura, lo cual puede explicar este
comportamiento, dando lugar a su oxidacin enzimtica por el oxgeno
disuelto.

HO

O
ONa

NaO
O

ONa
O

Figura9.Estructuradelfluorforo8hidroxipireno1,3,6trisulfonato
trisdico(HPTS).

Con objeto de mejorar las caractersticas del sistema, se estudi el

posibleefectoactivadordediferentestiposdeNPsenlareaccincatalizada
por la laccasa. Para este estudio se utilizaron NPs de diamante, Eu2O3,
Tb4O7, Ag y magnticas recubiertas de oro, en el intervalo de
concentraciones 0,1 2 mg/mL, encontrndose que slo las Tb4O7NPs
proporcionaban un aumento notable de la velocidad de reaccin del
sistema.ComopuedeobservarseenlaFigura10,lasreasbajolacurvaen
presencia y en ausencia de cido glico fueron menores al utilizar estas

316

Discusinderesultados

NPs, lo cual proporciona un aumento en la velocidad de muestreo.

Adems, se estudi la influencia de estas NPs sobre la intensidad de
fluorescencia del fluorforo en ausencia de laccasa, permaneciendo dicha
fluorescencia constante con el tiempo. En cambio, las NPs de diamante y
las magnticas recubiertas de oro dieron lugar a una prdida de
fluorescenciadelfluorforoenausenciadelaccasa,lasAgNPsinhibanel
efectocatalticodelalaccasaylasEu2O3NPsnoalterabanlascaractersticas
delsistema.

Intensidad de Fluorescencia Normalizada

1,2

1,0

14

0,8

0,6

0,4

0,2

0,0

10

15

20

Tiempo (min)

Figura10.CurvascinticasobtenidasparaelsistemaHPTSlaccasaenausencia
(curvas 1 y 2) y en presencia (curvas 3 y 4) de 2 M de cido glico, y en
presencia(curvas1y3)yenausencia(curvas2y4)de1mg/mLdeTb4O7NPs.
[HPTS]=10M,[laccasa]=1U/mL,pH=7,0,[fosfato]=0,4M,temperatura=
370C.

TambinseestudilaposibilidaddeutilizarionesTb(III)enlugar

delasTb4O7NPsparaaumentarlavelocidaddereaccindelsistema.Con

317

Captulo5

estefinseutilizaronconcentracionesequivalentesdeTb(III)alasutilizadas
enformadeNPs,obtenindosereasbajolacurvanetasmenores,locual
ratifica la utilidad de estas Tb4O7NPs como activadoras en el sistema
enzimtico.

Las variables implicadas en el sistema fueron optimizadas

medianteelmtodounivarianteutilizandoelreabajolacurvaneta(AUC)
como parmetro analtico. Sin embargo, fue necesario alcanzar una
solucin de compromiso entre este parmetro y el tiempo necesario para
llevar a cabo la medida, con el fin de obtener una sensibilidad y una
duracindelensayoadecuadas.

La influencia del pH en el sistema se estudi entre 4,0 9,0

utilizando diferentes disoluciones reguladoras, obtenindose que la

intensidad de fluorescencia prcticamente desapareca a valores de pH
cidos, mientras que dicha seal era muy elevada a pH 8,0 9,0, pero la
laccasanopresentabaactividadsobreelfluorforoaestosvaloresdepH.
Sinembargo,avaloresprximosalaneutralidad,7,07,5,laintensidadde
fluorescenciadelHPTSerabastanteelevadayseinhibaenpresenciadela
enzima. Por lo tanto, el valor de pH seleccionado para desarrollar el
mtodofue7,0,utilizandounadisolucindefosfato,deconcentracin0,4
M, para ajustar el pH. El estudio de la influencia de la concentracin de
HPTS en elsistema demostr que sta era independiente de esta variable
en el intervalo de concentraciones 8 12 M, seleccionndose el valor
intermedio,10M,comoconcentracinptima.

LaactividaddelalaccasaylaconcentracindeTb4O7NPssondos

variables crticas que afectan notablemente al valor neto de AUC y a la

velocidaddereaccin.Ambasvariablesfueronestudiadasenlosintervalos

318

Discusinderesultados

de 0,5 2 U/mL y 0,2 2 mg/mL para la actividad de la laccasa y la

concentracin de NPs, respectivamente. Para valores elevados de ambas
variables, el aumento de la velocidad de reaccin era muy considerable,
proporcionandoAUCnetasmuypequeas,locualibaendetrimentodela
sensibilidad del mtodo. En cambio, para valores muy bajos de estas
variables, estas reas netas, y por ende, la sensibilidad del mtodo,
aumentaban considerablemente, pero la duracin del ensayo era
excesivamentelarga.Porlotanto,losvaloresseleccionadoscomoptimos,
con el fin de obtener valores adecuados de sensibilidad con tiempos de
ensayorelativamentecortos,fueron1U/mLdelaccasay1mg/mLdeNPs.
Comosehadescritoanteriormente,elparmetroanalticoutilizado
en este mtodo fue el AUC, obtenida a partir de las curvas cinticas
normalizadasparadiferentesconcentracionesdecidoglico.LaFigura11
muestra las curvas registradas a ex 450 nm y em 535 nm, bajo las
condicionesexperimentalesptimas.
El intervalo lineal dinmico de la curva de calibrado fue 0,5 12
M de cido glico, obtenindose un lmite de deteccin de 0,14 M, el
cual es unas tres veces menor que el obtenido (0,4 M) en ausencia de
Tb4O7NPs.EstadiferenciademuestraelefectopositivodelasTb4O7NPs,no
sloenlavelocidaddereaccin,sinotambinenellmitededeteccin.El
lmite de deteccin obtenido en presencia de Tb4O7NPs es menor que los
lmites de deteccin descritos utilizando biosensores amperomtricos, los
cuales oscilaban entre 0,19 y 50 M [69, 70]. Aunque se ha obtenido un
lmite de deteccin de 0,04 M para cido glico utilizando verde de
indocianina [74], dicho mtodo implica el anlisis secuencial de cada
muestra, lo que conlleva una velocidad de muestreo mucho menor. La

319

Captulo5

precisin del mtodo, expresada como desviacin estndar relativa, fue

ensayadaparadosconcentracionesdiferentesdecidoglico,0,7y5M,
obtenindosevaloresde6,3y2,5%,respectivamente.

Intensidad de Fluorescencia Normalizada

1,0

0,8

16

0,6

0,4

0,2

0,0
0

10

20

30

40

Tiempo (min)

Figura 11. Curvas cinticas obtenidas con diferentes concentraciones de

cidoglico:(1)0M,(2)0,5M,(3)1M(4)2M(5)5My(6)10M.
Condicionesexperimentales:[HPTS]=10M,[laccasa]=1U/mL,[NPs]=1
mg/mL,pH=7,0,[fosfato]=0,4M,temperatura=37oC.

Para aplicarel mtodo al anlisis demuestras reales, previamente

se realiz un estudio de selectividad, comprobando la influencia de otras

sustancias reductoras no fenlicas que pueden estar presentes en las

320

Discusinderesultados

muestras de vinos utilizadas para demostrar la aplicabilidad del mtodo.

La Tabla 7 recoge los resultados obtenidos para varios posibles
interferentes. El principal interferente fue el cido ascrbico cuya
concentracin tolerada fue igual a la concentracin molar del analito. Sin
embargo, hay que tener en cuenta que la concentracin mxima de cido
ascrbico que puede hallarse como aditivo en vinos es de 0,85 mM [75],
menorquelaconcentracindepolifenolesquenormalmenteexisteenvino.
Laconcentracindesulfitotoleradaes20vecesmayorquelaconcentracin
de analito y, aunque los sulfitos pueden ser eliminados de la muestra
medianteuntratamientoprevio[70],noesnecesariadichaetapayaquela
presencia de sulfitos est regulada en funcin del contenido total de
sustanciasreductoras[76].

Tabla7.Influenciadealgunosagentesreductoresenelmtodopropuesto
para la determinacin de antioxidantes fenlicos a una concentracin de
cidoglicode2M
Compuestos
Relacinmaximatolerada
interferente/analito*
cidomlico
100
cidoctrico

100

Sacarosa

100

Glucosa

25

Sulfitosdico

20

cidoascrbico

*Laconcentracinmximaensayadafue100vecesmayorqueladelanalito
El mtodo propuesto fue aplicado al anlisis de muestras de vino
tinto,blancoydulce,lascualessolorequirieroncomotratamientopreviola
dilucinadecuadaparaquelaconcentracindepolifenolespresentealser

321

Captulo5

analizadas estuviera dentro del intervalo lineal dinmico del mtodo,

utilizando cido glico como estndar y la AUC neta como parmetro
analtico. La Tabla 8 muestra el contenido en polifenoles de las muestras
analizadasmedianteelmtodopropuestoyelmtododeFolinCiocalteu,
elcualsueleserconsideradocomomtododereferencia.

Tabla8.Concentracindeantioxidante(expresadocomoequivalente
milimolardecidoglico)
Muestra
Mtodopropuestoa,b MtodoFolinCiocalteua,b
Vinotinto(Rioja)

7,00,4

11,50,1

Vinotinto(Cuenca)

9,50,5

14,040,18

Vinoblanco(Rueda)

1,000,06

1,920,04

1,000,03

2,0280,005

1,270,09

3,640,05

Vinoblanco
(Valdepeas)
Vinodulce(Montilla)
a

MediaDE(n=3)
Unidades:equivalentemMdecidoglico

Como puede observarse en la Tabla 8, los valores obtenidos

medianteelmtododeFolinCiocalteusonmayoresquelosobtenidospor
elmtododelalaccasa,loqueseatribuyealacapacidaddeestemtodo
para reaccionar con sustancias reductoras no fenlicas. Los valores
obtenidosporelnuevomtodosonsimilaresalosdescritoshaciendouso
deotrosmtodosparaelanlisisdeestetipodemuestras[68,69].
Se llev a cabo un estudio de recuperaciones para validar el
mtodo mediante la adicin de tres concentraciones diferentes de cido
glico a cada una de las muestras analizadas y sometindolas al mtodo

322

Discusinderesultados

determinativo propuesto. Como puede observarse en la Tabla 9, los

porcentajesderecuperacinobtenidososcilaronentre81,0%y108,3%,con
unosvaloresmediosde90,9%,92,4%y99,5%paralasmuestrasdevino
tinto,blancoydulce,respectivamente.

Tabla9.Valoresderecuperacinparalasmuestrasdevinoanalizadas.
Muestra
Aadidoa
Encontradoa,b Recuperacin(%)
Vinotinto(LaRioja)

4,30,5

108,3

12

10,00,5

84,1

20

161

81,0

3,20,2

80,0

12

11,70,3

97,4

20

18,90,8

94,5

0,25

0,240,03

97,2

0,75

0,770,04

102,8

1,25

1,080,03

86,7

Vinoblanco

0,25

0,230,03

92,0

(Valdepeas)

0,75

0,700,04

92,2

1,25

1,00,1

83,3

1,010,09

101,5

3,00,3

102,0

4,70,3

95,0

Vinotinto(Cuenca)

Vinoblanco(Rueda)

Vinodulce(Crdoba)

MediaDE(n=3)
Unidades:equivalentesmMdecidoglico

Los resultados obtenidos demuestran la utilidad del uso

combinado de la enzima laccasa, Tb4O7NPs y el fluorforo HPTS para la
determinacindepolifenolesenmuestrasdevino.Lapresenciadelaccasa

323

Captulo5

y NPs permite aumentar la velocidad de muestreo del mtodo y mejorar

los valores de sensibilidad, siendo estos superiores a los obtenidos en la
mayora de los mtodos descritos anteriormente [68, 69]. Como aspecto
msnovedosodelainvestigacinrealizada,cabedestacarquesedescribe
por primera vez el efecto activador de las Tb4O7NPs en un sistema
enzimtico.Adems,elusodellectorautomticodemicroplacas,permite
el procesamiento simultneo de las muestras consiguiendo una velocidad
de muestreo de 35 muestras a la hora, analizando cada muestra por
triplicado.

3. Nuevas investigaciones en el uso de upconverting phosphors

ensistemasdetransferenciadeenergaresonanteluminiscente
Esteestudioseharealizadodurantelaestanciadetresmesesenel
Departamento de Biotecnologa de la Universidad de Turku (Finlandia).
Debidoalacortaduracindedichaestancia,lainvestigacinsecentren
laevaluacinsistemticadediferentesdadoresyaceptoresparaeldiseo
de un sistema de transferencia de energa resonante luminiscente (LRET)
haciendo uso del fenmeno antiStokes en ensayos de afinidad biotina
estreptavidina. Este estudio tuvo como objetivo la seleccin del sistema
msadecuadoparasuposterioraplicacinanaltica.
Como se ha descrito en la Introduccin de esta Memoria, los
upconverting phosphors (UCPs) son un tipo de NPs inorgnicas que
puedenserdopadasconioneslantnidosparallevaracaboelfenmenode
emisin de fluorescencia antiStokes. En esta investigacin se presenta el
estudio sistemtico de diferentes UCPs dopados con Er(III), Ho(III) y

324

Discusinderesultados

Tm(III), utilizados como dadores, y varios fluorforos, utilizados como

aceptores, para el desarrollo de nuevas metodologas basadas en el
fenmenoLRET.Paraqueseproduzcaestefenmenosonimprescindibles
doscondiciones:1)elespectrodeexcitacindelaceptortienequesolapar
conelespectrodeemisindeldador,y2)ladistanciaentredadoryaceptor
tienequecumplirlaecuacindeFrster.
Entre

la

gran

variedad

de

fluorforos

existentes,

las

ficobiliprotenas,talescomolaficoeritrina(BPE)ylaRficoeritrina(RPE),
han sido utilizadas anteriormente como aceptores en diferentes ensayos
LRET [77 79]. Su uso se debe principalmente a su elevada emisin de
fluorescencia, aunque tienen el inconveniente de un coste relativamente
elevado. Por este motivo se ha estudiado la posibilidad de utilizar otros
tipos de fluorforos de menor coste para el desarrollo de ensayos de
afinidad LRET, como los Alexa Fluor o los ATTO. Para llevar a cabo este
estudio sistemtico entre dadores y aceptores, los UCPs fueron marcados
conestreptavidina(SA)mientrasquelosfluorforosfueronmarcadoscon
biotina(Bio).
EnlaFigura12semuestranlosespectrosdeemisindelosdadores
y los espectros de excitacin de los aceptores. Las Figuras 12.A12.C
correspondenalacombinacindelUCPdopadoconEr(III)conlaprotena
fluorescenteBPEylosfluorforosAlexaFluor546(AF546)y680(AF680),
observndose un buen solapamiento entre los espectros de emisin y
excitacindedadoryaceptor,respectivamente.

325

Captulo5

Similares espectros fueron obtenidos al utilizar los UCPs dopados

con Ho(III) (Figura 12.D12.E) ensayados con BPE y AF546. Al igual que
ocurrealusarelEr(III),elanchoespectrodeexcitacindelaBPEpermite
excitarla eficientemente mientras que al usarse AF546, esta transferencia
deenergapresentaunmenorrendimiento.
Los UCPs dopados con Tm(III) se combinaron con RPE, Alexa
Fluor 488 (AF488) y el fluorforo ATTO495 (Figura 12.F12.H). En este
caso, el dador presenta un mximo de emisin a una longitud de onda
menorqueelEr(III)yelHo(III),conunbuensolapamientotericoconel
fluorforo AF488, aunque, como podr observarse posteriormente, no
siempre un adecuado solapamiento terico da lugar en la prctica a una
transferenciadeenergaeficaz.
ComoyaseindicenlaIntroduccindeestaMemoria,laausencia
de grupos funcionales en este tipo de nanomateriales inorgnicos hace
imprescindible llevar a cabo previamente su recubrimiento y
funcionalizacinpara,posteriormente,unirlosabiomolculas.Despusde
esteproceso,losdiferentesdadoresyaceptoresfueronmarcadosconSAy
Bio, respectivamente, para llevar a cabo la optimizacin de las
concentraciones de cada uno de ellos y, finalmente, obtener las curvas de
calibrado para la Bio, utilizada como analito modelo, en las mejores
condiciones.ParaevitarelelevadocostedelasficobiliprotenasBPEyRPE,
seestudilaalternativadeutilizarotrosfluorforosparadisearelsistema
LRET, comparando los resultados con los obtenidos usando estas
protenas.

326

400

200

550

600

650

400

700

450

600

400

200

400

450

500

550

600

650

500

550

600

650

700

800

400

200

450

500

550

600

650

700

700

G)

1000

800

600

600/40

400

200

555/20

200

LONGITUD DE ONDA (nm)

600

LONGITUD DE ONDA (nm)

600

400

INTENSIDAD LUMINISCENCIA NORMALIZADA

400

600/40

INTENSIDAD LUMINISCENCIA NORMALIZADA

600

450

500

LONGITUD DE ONDA (nm)

800

400

700

800

700

F)

650

E)

1000

LONGITUD DE ONDA (nm)

1000

600

200

600/40

INTENSIDAD LUMINISCENCIA NORMALIZADA

800

600/40

INTENSIDAD LUMINISCENCIA NORMALIZADA

D)

550

400

LONGITUD DE ONDA (nm)

LONGITUD DE ONDA (nm)

1000

500

600

400

450

500

550

600

LONGITUD DE ONDA (nm)

650

700

H)

1000

800

600

400

600/40

500

200

C)

800

200

555/20

450

400

INTENSIDAD LUMINISCENCIA NORMALIZADA

400

600

520/10

800

1000

750/40

600

INTENSIDAD LUMINISCENCIA NORMALIZADA

800

B)

1000

600/40

INTENSIDAD LUMINISCENCIA NORMALIZADA

A)

1000

600/40

INTENSIDAD LUMINISCENCIA NORMALIZADA

Discusinderesultados

400

450

500

550

600

LONGITUD DE ONDA (nm)

650

700

Figure 12: Espectros de emisin de los UCPs y aceptores utilizados en este

estudio. En 12.A12.C, espectro de emisin de UCP dopado con Er (III) junto
conBPE(12.A),AF546(12.B)yAF680(12.C).En12.Dy12.E,elUCPutilizado
eseldopadoconHo(III)juntoconBPE(12.D)yAF546(12.E).En12.F12.H,los
UCPs dopados con Tm (III) son combinados con RPE (12.F), AF488 (12.G) y
ATTO495(12.H).

En la Figura 13, se muestran las curvas de calibracin obtenidas

parabiotinaconUCPscomodadoresdopadosconEr(III)SA(13.Ay13.B),

327

Captulo5

Ho(III)SA (13.C) y Tm(III)SA (13.D), combinados con diferentes

fluorforos. En todos los casos se obtuvo emisin luminiscente, pero los
mejores resultados se lograron con las combinaciones Er(III)BPE, Er(III)
AF680, Ho(III)BPE y Tm(III)RPE, las cuales se utilizaron para obtener
dichascalibraciones.

4000000

600000

A)

B)

up conversion LRET (cts)

up conversion LRET (cts)

500000

3000000

2000000

1000000

400000
300000
200000
100000
0

0,1

250000

10
100
[Biotina] (nM)

1000

0,1

10000

100

1000

10000

1000

10000

120000

D)
100000

up conversion LRET (cts)

up conversion LRET (cts)

10

[Biotina] (nM)

C)
200000

150000

100000

50000

80000

60000
40000

20000

0
0,1

10

100

1000

10000

0,1

10

100

[Biotina] (nM)

[Biotina] (nM)

Figura 13. Ensayos homogneos LRET de afinidad para biotina con los pares
dadoraceptorqueproporcionanlosmejoresresultados.A)UCPEr(III)(0,015
mg/mL)y0,4nMBPE.B)UCPEr(III)(0,015mg/mL)y4nMAF680.C)UCP
Ho (III) (0,030 mg/mL) y 0,2 nM BPE. D) UCPTm (III) (0,030 mg/mL) y 1,33
nMRPE.Lasintensidadesdeluminiscenciafueroncalculadasrestandolaseal
de fondo (exceso de biotina) a la seal mxima (ausencia de biotina), cts:
cuentas.

328

Discusinderesultados

Como se observa en la Figura 13, la mayor intensidad de

fluorescenciafueobtenidaconelUCPsEr(III)juntoconBPEyAF680.Sin
embargo, como puede apreciarse en la Tabla 10, donde se resumen los
diferentes ensayos llevados a cabo para la valoracin homognea de
biotinaascomolosvaloresdeIC50obtenidos,losmenoresvaloresdeeste
parmetro correspondieron al par UCPTm(III)SA y RPE, pese a
registrarselaintensidaddeluminiscenciamsbaja.ElmayorvalordeIC50
fueobtenidoparaUCPEr(III)SAyBioAF680,elcualfueprximoa14,4
nM. Los valores obtenidos son similares y, en algunos casos, mejoran los
descritos previamente en los mtodos propuestos utilizando UCPsEr(III)
recubiertodeaditolcomodadoryBPEcomoaceptor[77,78]
Tabla10.ValoresdeIC50paralosensayosdebiotinautilizandodiferentes
paresdadoraceptor.
IC50parabiotina (nM)

Aceptor
Dador

BioBPE

BioBPE

BioRPE

BioAF680

(0,2nM)

(0,4nM)

(1,33nM)

(4nM)

8,57*

14,37

5,7

7,20

3,35

UCPdopadocon
Er(III)
(0,015mg/mL)
UCPdopadocon
Ho(III)(0,015mg/mL)
UCPdopadocon
Ho(III)(0,015mg/mL)
UCPdopadocon
Tm(III)
(0,030mg/mL)
*LaconcentracindeBioBPEfuede0,3nM

329

Captulo5

En resumen, del estudio sistemtico realizado para evaluar la

posibleutilidaddediferentesUCPscomodadoresdeenergaydiferentes
fluorforoscomoaceptoresdeenergaensistemasLRET,cabedestacarlo
siguiente: 1) El dador UCPEr(III) proporciona los mejores resultados al
utilizarBioBPEcomoaceptor;2)LautilizacindelfluorforoAF680como
aceptor alternativo a las ficobiliprotenas permite tambin obtener una
elevadasealfluorescente,aunqueelvalordeIC50alcanzadoparabiotina
es mayor que los obtenidos en presencia de las ficobiliprotenas. No
obstante, la utilizacin de dicho fluorforo tambin permite la
determinacin de biotina a nivel nanomolar, evitando el mayor coste que
suponeelusodelasficobiliprotenas.

ComoresumendelasinvestigacionesdescritasenestaMemoria,se
presentan en el siguiente esquema las principales ventajas y limitaciones
delasmetodologasanalticasdesarrolladasenestaMemoria:
Ventajas

Limitaciones

SiO2NPsdopadasconLWFs
Sntesisreproduciblesmedianteelmtodode No

existen

formacin de microemulsiones de micelas

comercialmente

inversas

disponibles

NPs

Aumento de estabilidad frente a fluorforos Las condiciones de

endisolucin
Diversas

opciones

sntesisdependende
de

funcionalizacin

caractersticas

disponibles por la variedad de reactivos

fsicoqumicas del

organosilano

fluorforo

330

las

Discusinderesultados

Los inmunoensayos heterogneos propuestos

mejoranlosELISAsyaque:

Se

obtienen

menores

lmites

de

menos

etapas,

se

deteccin.

Se

requieren

simplificanlosensayos.

Semejoralaselectividadespectral

Ensayosparadeterminacindesustanciasantioxidantes
EnsayoORACalargalongituddeonda

Disminucin de los fenmenos de Carencia

de

dispersindelaradiacin

mtodos

Mejoraselectividadespectral

normalizados

Determinacin de polifenoles mediante

laccasayTb4O7NPs

Lmites de deteccin menores que los

obtenidosmediantebiosensores

ElusodeTb4O7NPspermite:

La enzima no se

o Reducir la cantidad de enzima

puedereutilizar

requerida
o Acortarlostiemposdeensayo

Tb4O7NPsdisponiblescomercialmente

EstudiosistemticodeUCPsenfenmenosLRET
La

luminiscencia

antiStokes

utiliza No

pueden

excitacin en el IR que minimiza la

utilizar (espectro)

interferenciadelamatrizdelamuestra

fluormetros

Es posible utilizar diferentes pares dador

aceptorparasuusoenensayosmultiplex
Se ha demostrado lautilidad de fluorforos
alternativos a las ficobiliprotenas para ser
utilizadoscomoaceptores

331

se

convencionales

Captulo5

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342

in

Discussionoftheresults

Introduction
The features of the methods developed in this Dissertation are

discussed in this chapter. A comparative study of their advantages and

disadvantages to the methods previously described on the literature is
carried out. Also, the usefulness of the proposed methodologies by their
applicationtofoodanalysisisdiscussed.
Thischapterisstructuredinthreesections:1)Silicananoparticlesin
food analysis, 2) New strategies for the determination of antioxidants in
foodsand3)Newinvestigationsintheuseofupconvertingphosphorsin
luminescenceresonanceenergytransfersystems.
1. Silicananoparticlesinfoodanalysis.
As it has been previously described, the use of nanomaterials is
having a huge growth in different areas of Analytical Chemistry [1 5].
Among the wide range of nanomaterials currently available, the use of
silicananoparticles(SiO2NPs)hasrapidlygrowninthelastdecadedueto
theirexcellentproperties,whichareespeciallyappropriatefortheiruseas
labels in bioanalysis. The results achieved with these NPs in the three
articlesincludedinthisDissertationwillbediscussedinthenextsection.

Synthesisandcharacterizationoflongwavelengthfluorophore
dopedsilicananoparticles

The use of SiO2NPs as alternative labels in methods with optical

detectionisveryinterestingduetotransparencyofsilicatovisiblelight,as

343

Chapter5

well as other advantages already described in the Introduction of this

Dissertation.
Two general routes for the synthesis of silica NPs, namely Stber
method and reversemicelle microemulsion method, involve the use of
different silica precursors, such as tetramethoxysilane (TMOS) and
tetraethoxysilane (TEOS). These precursors undergo hydrolysis and
polycondensation reactions, giving rise to the formation of monodisperse
spherical SiO2NPs [6]. The choice of the most appropriate synthesis
procedure will depend on the physical and chemical properties of the
speciestobeencapsulated[2,4].
The synthesis of fluorescent SiO2NPs doped with two long
wavelength fluorophores (LWFs), cresyl violet and nile blue, is described
in this Dissertation using the reversemicelle microemulsion method. All
thevariablesinvolvedinthissynthesisprocedurehavebeenoptimizedfor
thispurposeandtheirfeatureshavebeencompared(NPsize,nanomaterial
amount,fluorescenceintensity,etc)withthoseoftheNPsobtainedbythe
Stbermethod.
The formation of the microemulsions involves the use of a
surfactant,anaqueoussolution,anorganicsolvent,andasilicaprecursor,
which is added to perform the SiO2NP synthesis. Depending on the
relative amounts of the components, waterinoil microemulsions (rich in
organic solvent) or oilinwater microemulsions (rich in water) can be
formed. Cyclohexane, 1hexanol, Triton X100 and water were chosen to
formthemicroemulsion,accordingtotheprocedurespreviouslyreported,

344

Discussionoftheresults

andTEOSwasselectedasthesilicaprecursor[2,4,6,810].However,the
useoftheexperimentalconditionspreviouslydescribeddidnotgiveriseto
satisfactoryresultsforthesynthesisofthecresylvioletandnilebluedoped
silicaNPs.Severalassayswerecarriedouttooptimizethemicroemulsion
composition.ThisstudyshowedthattheNPssynthesizedintheabsenceof
cyclohexane had similar features (number and size) as those obtained
using cyclohexane, but their fluorescence intensity was slightly higher.
Figure 1 shows transmission electronic microscopy (TEM) images of the
NPs obtained in the presence of different volumes of ciclohexane. As can
be seen, the amount and sizes of the NPs synthesized in the absence of
cyclohexane(Figure1a)werecomparabletothoseachievedusing7.5mLof
cyclohexane (Figure 1c). However, the use of intermediate volumes of
cyclohexane(Figure1b)providedlargerNPswithwidersizedistributions.
Accordingtotheseresults,theuseofcyclohexanewasprecludedandonly
water,TritonX100and1hexanolwereselectedasthecomponentsofthe
microemulsion.
ThestudyoftheinfluenceofTritonX100amountshowedthatthe
numberofnilebluedopedNPsincreasesandtheirsizedecreasesasTriton
X100amountincreases.Thebestresultswereobtainedusing510520mg
ofTritonX100(Figure1f)becauseloweramountsgaveNPsofsizeshigher
than200nm(Figure1dand1e).VerysmallandpartiallyaggregatedNPs
wereobtainedwhentheTritonX100amountwascloseto1.0g.
The feasibility of a slight modification of the reversemicelle
microemulsion procedure for fluorophoredoped SiO2NPs by introducing
theuseofsilicaprecursorswasalsostudied.Thismodificationincludesa

345

Chapter5

previousreactionstepinwhichthecovalentlinkingbetweentheprimary
aminogroupsofthefluorophoreand3aminopropyltriethoxysilane(APS)
wasassayedinordertopreventthedyeleakagefromthesilicamatrix.

Figure 1. TEM images of nile bluedoped NPs synthesized using different

cyclohexanevolumes(a,bandc),differentTritonX100concentrations(d,e,f)and
thosefornileblueAPSdopedsilicaNPs(g).Experimentalconditions:100L(0.11
mmol)TEOS,0.2mLof1x103M(0.2mol)nileblue,3mL(0.024mol)hexanol,70
L(0.9mmol)NH4OH.In(Figure1ac):510520mg(0.790.82mmol)TritonX
100.InFigure1a11.3mL(0.63mol)water,0mLcyclohexane,inFigure1b,7.5mL
(0.42mol)water,3.75mL(0.034mol)cyclohexane,inFigure1c3.8mL(0.21mol)
water,7.5mL(0.068mol)cyclohexane.InFigure1df11.3mL(0.63mol)ofwater,
inFigure1d0mg,inFigure1e310.9mg(0.48mmol),inFigure1f510.9(0.79mmol)
ofTritonX100.Scalebar:1m.InFigure1gexperimentalconditions:510520mg
(0.790.81mmol)ofTritonX100,10.3mL(0.57mol)ofwater,100L(0.44mmol)
TEOS, 1.2 mL precursor reaction mixture, 3 mL (0.024 mol) hexanol, 70 L (0.9
mmol)ofNH4OH.Scalebar:2m.

346

Discussionoftheresults

Nevertheless, as can be seen in Figure 1g, the NPs amount collected was
substantially lower than that obtained in the absence of APS. Also, these
NPs showed a wide size distribution. Thus, the use of these NPs as
potential labels for further assays was discarded as they do not improve
the features of the NPs obtained without this modification. Also, this
synthesis procedure requires an additional step and an increased reagent
andtimeconsumption.
It was checked that the Triton X100/hexanol ratio is a critical
variable,asFigure2shows.Asitcanbeseen,aratioof170mgTritonX
100/mL hexanol provided the maximum fluorescence intensity of the
synthesizedmaterial.

Figure2.InfluenceofTritonX100/hexanolratioonthefluorescenceintensity
ofnilebluedopedNPs.Experimentalconditions:11.3mL(0.63mol)ofwater,
100L(0.44mmol)TEOS,0.2mLof1x103M(0.2mol)nileblue,3mL(0.024
mol)hexanol,70L(0.9mmol)NH4OH.

347

Chapter5

The amount of fluorophore used is another critical variable. The

influence of this variable on the fluorescence intensity is shown in the
Figure3,inwhichcanbeseenthatthemaximumvaluewasobtainedusing
1.8 molofnile blue. The decrease in the fluorescence intensity at higher
values is ascribed to the formation of fluorophore aggregates, which are
lessfluorescentthanthemonomers[7].

Figure 3. Influence of the amount of nile blue added on the fluorescence

intensity of fluorophoredoped NPs. Experimental conditions: 510 520 mg
(0.790.81mmol)TritonX100,11.3mL(0.63mol)water,100L(0.44mmol)
TEOS,3mL(0.024mol)1hexanoland70L(0.9mmol)NH4OH.

348

Discussionoftheresults

Afterstudyingofallthevariablesinvolvedinthesynthesisprocess,

the NPs obtained by means of the reversemicelle microemulsion method

were compared to the NPs synthetized by the Stber method. Table 1
shows the comparison of the cresyl violetdoped silica NPs obtained by
both methods, using the same NP concentration. It was found that the
average diameter of the NPs obtained by the microemulsion method was
slightly greater, whereas the number of NPs is higher using the Stber
method,althoughthedifferenceswerenotverysignificantinanyinstance.
However, the fluorescence intensity and the specific intensity of the NPs
obtained by the modified microemulsion method were about 12 times
higherthanthecorrespondingvaluesobtainedfortheNPssynthesizedby
the Stber procedure. These results can be ascribed to the fact that cresyl
violet is less degraded under the alkaline conditions of the synthesis
procedureowingtotheprotectiveeffectofthemicelles.
Table1.ComparisonofthepropertiesofcresylvioletdopedNPsobtained
bytheproposedreversemicellemicroemulsionandStbermethods.

Reversemicelle
Stbermethod
microemulsionmethod
Concentration,mg/mL
0.1
0.1
Particleamount,mg

0.02

0.02

Diameter,nm

178

133

2.94x109

7.06x109

125563

10138

Intensity/particleratio

4.27x105

1.44x106

Specificintensity

1.45x107

1.17x106

Particlenumber
Intensitya

Measurements performed in microplates (200 l) using excitation and

emissionfiltersof620/8and680/10nm,respectively
a

349

Chapter5

The spectral features of the synthesized NPs were compared to

those shown by the dyes in solution and by bare SiO2NPs. The emission
maxima of the oxazinedoped SiO2NPs showed a hypsochromic shift of
about9nm,whencomparedtotheemissionmaximaofthefluorophoresin
solution,whereasthebareSiO2NPsdidnotshowanyfluorescentsignal.

The synthesized NPs in aqueous solution were irradiated at their

correspondingmaximumexcitationwavelengthforonehourtoinvestigate
their potential photobleaching and the results were compared to those
obtainedforthedyesinsolution.Thefluorescenceintensityofcresylviolet
andnileblueinsolutionshowedafluorescencedecreaseofabout72%and
65%,respectively,butthefluorescenceintensityofbothcresylvioletand
nile bluedoped NPs remained almost constant. The same study was
carriedoutfortheSiO2NPspreparedbyusingthenileblueAPSmixture,
obtaining a decrease of about a 10 % of the initial fluorescence intensity.
Also,thestabilityofthefluorescentSiO2NPswascheckedovertimeandit
was found that the luminescence intensity was constant for at least one
month.

Insummary,itcanbeconfirmedthatthemodifiedreversemicelle

microemulsionmethodtosynthesizetwotypesofLWFsdopedsilicaNPs,
usingnileblueandcresylviolet,providesNPswithsimilarcharacteristics
(size, amount and average diameter) as those obtained by means of the
Stber method, which is considered as a reference method. However, the
fluorescenceintensityoftheNPssynthetizedbythefirstmethodishigher
than that of NPs obtained by the Stber method. This behavior can be

350

Discussionoftheresults

ascribed to the stability and protection that the microemulsion used to

performthesynthesisconferstotheLWFs.

Utilization

of

silica

nanoparticles

in

heterogeneous

immunoassay for the determination of macromolecules and

smallmolecules
The determination of biomolecules is an application field of great
interest in Analytical Chemistry. A high number of chromatographic and
electrophoretic methods have been developed for the determination of
macromolecules and small molecules [11 15]. Usually, these techniques
requirethattheanalyteshowsameasurableproperty,suchasabsorbance
or fluorescence. Otherwise, the use of a derivatization step is required,
which lengthens and complicates the analysis [16, 17]. Although
chromatographicmethodswithmassspectrometrydetectionareavailable,
they require sophisticated and expensive instrumentation [18 20]. Also,
these methods usually involve timeconsuming sample treatment steps to
remove the sample matrix. The use of screening methods plays two
essential roles: in one hand, the number of samples subjected to
confirmatoryanalysisislowerand,ontheother,thecostoftheanalysisis
reduced. Immunoassays, which can be used for quantitative or semi
quantitative purposes, have shown their usefulness as screening methods
for the determination of antigenic substances, either small molecules
(haptens)ormacromolecules.Numerouscommercialandnoncommercial
enzymelinked immunosorbent assays (ELISAs) have been described for
thispurpose[2125].Ahighpercentageofthesemethodsinvolvetheuse
of photometric measurements, reaching detection limits close to ng/mL,

351

Chapter5

but lower values canbeobtainedusing chemiluminescencedetection [26]

or timeresolved luminescence detection with europium(III) chelates as
labels[27,28].
TheuseoffunctionalizedinorganicNPsaslabelsinimmunoassays
is a relatively new trend justified by their versatile physicochemical
properties,whichallowtheimprovementoftheseassaysfeatures[2931].
AmongtheseNPs,dopedSiO2NPsareausefuloptionowingtotheirabove
mentionedcharacteristics.TheseSiO2NPscanencapsulateawidevarietyof
compounds, which are protected from environmental factors. Also, they
show large surface area and easy surface functionalization. These
properties have given rise to a great variety of immunoassays devoted to
thedeterminationofmacromoleculesandhaptens[32,33].Forexample,a
method for the determination of E. Coli using tris(bipyridine)ruthenium
(II)doped NPs has been developed by immobilizing the antibody on the
NP surface, which has allowed the determination of only one bacteria
withoutusingofanenhancementsignalprocedure[34].Similarly,bovine
serumalbumin(BSA)hasbeendeterminedbyastreptavidinbiotinaffinity
assay using rhodamine 6Gdoped SiO2NPs as labels [35]. Also, the
determination of fetoprotein (AFP) and hepatitis B surface antigen
(HBsAg)havebeenreportedbyusingfluoresceindopedSiO2NPs[36,37].
These SiO2NPs can also encapsulate other species such as terbium and
europium chelates, which have been used as labels to determine HBsAg,
carcinoembrionic antigen (CEA) and prostatespecific antigen (PSA) [38
40].FromtheseexamplesitcanbeinferredthatdopedSiO2NPshavebeen

352

Discussionoftheresults

mainly used in clinical analysis, which clearly justifies the investigations

performedtodevelopimmunoassaysforfoodanalysis.
The immunoassays described in this Dissertation have used nile
bluedoped SiO2NPs as labels for the determination of soy proteins
(macromolecules) and the veterinaryantibiotic monensin, which presence
in food is regulated by the European Union. The results obtained have
demonstrated that the use of these NPs provides some advantages over
other immunoassays, such as lower detection limits and, in the case of
ELISAs, fewer steps because the use of a substrate to measure the
enzymaticactivityisnotrequired.
Nile bluedoped SiO2NPs were chosen to use longwavelength
fluorescence measurements, which provide a suitable spectral selectivity,
avoiding the background signal from the sample matrix. As mentioned
above, a potential limitation of doped SiO2NPs is the potential leakage of
thefluorophorefromthesilicamatrix.However,nilebluedopedSiO2NPs
showanexcellentstabilityduetotheelectrostaticinteractionsbetweenthe
positivelychargedfluorophoreandthenegativelychargedsilanolgroups.
The results obtained in the immunoassays developed in this
Dissertationarediscussedbelow.

Determinationofsoyproteinsbyheterogeneousimmunoassay

In recent years, the global consumption of soy foodstuffs has

increased because of their reported beneficial effect on nutrition and
health, such as decrease of plasma cholesterol, prevention of cancer,

353

Chapter5

diabetes and obesity, and protection against bowel and kidney diseases.
However,ithasalsobeendescribedthatsoyproteinscanproduceallergic
responsesinsomepeople.
A competitive heterogeneous immunoassay for the determination
of soy proteins in food samples, using nile bluedoped SiO2NPs as labels,
has been developed in this Dissertation. For this purpose, two different
formatseitherinvolvinganalytecaptureorantibodycapturewereassayed,
respectively (Figures 4 and 5). These NPs were coupled to either soy
proteinsortoantisoyproteinantibodiestostudythepotentialuseofthe
corresponding tracers for the development of the two immunoassay
formats.Theanalytecaptureformat(Figure4)involvestheimmobilization
of antisoy protein onto the wells, after the previous immobilization of
antirabbit IgG antibodies, and the use of the tracer composed of soy
protein linked to nile bluedoped SiO2NPs. The use of antirabbit IgG
antibodies to immobilize the antisoy protein antibodies reduces the
consumption of these antibodies and, therefore, the total cost of the
immunoassay.However,althoughthewellsurfacewascoatedwithBSAat
different concentrations, nonspecific interactions of the tracer with the
antirabbit IgG antibodies were still observed, so that this approach was
unsuitable.

354

Discussionoftheresults

Figure4.Competitiveheterogeneousimmunoassaywithantigencaptureusing
nilebluedopedSiO2NPsaslabellinkedtosoyprotein.

The antibody capture format was assayed to overcome this

problem (Figure 5). Thus, soy proteins were immobilized first onto the
wells,andtheremainingsitesofthewellwereblockedwithBSA.Then,a
preincubated mixture of soy protein standard or sample together with a
conjugate antisoy proteinnile bluedoped silica NPs (tracer) was added,
competing both immobilized and in solution soy proteins for the active
sitesoftheconjugate.Afterincubationandwashingsteps,thefluorescence
fromtheboundtracerfractionwasmeasuredontothewellsurface.

355

Chapter5

Figure 5. Competitive heterogeneous immunoassay with antibody capture

usingnilebluedopedSiO2NPsaslabellinkedtoantisoyproteinantibodies.

Fluorescence intensity measurements were performed at the

maximum excitation and emission wavelengths of the tracer, ex 620 and

em680nm,respectively.Themethodpresentedadynamicrangeof0.1
10 mg/L and the detection limit was 0.05 mg/L, which is about ten times
lowerthanthatprovidedbytheELISAkitusedasreferencemethod[41].
Also, as the addition of an enzymatic substrate is not required, the assay
steps and the analysis time are reduced. The precision, expressed as the
percentageofrelativestandarddeviationandassayedattwodifferentsoy
protein concentrations, 0.5 and 5 mg/L, gave values of 9.6 % and 6.1 %,
respectively.

The selectivity of the method mainly depends on antisoy protein

antibodiesselectivity.Accordingtotheantibodyspecificationsheet,these
antibodiespresentsomecrossreactivitytowardshotureaextractsofwheat
and ovalbumin, but no crossreactivity is observed in extracts form meat,

356

Discussionoftheresults

corn or casein, and rice or potato flour. Additionally, in a previous work

thatinvolvedtheuseofthesameantisoyproteinantibodies,itwasfound
thatbovineserumalbumin,globulins,myoglobinandhemoglobinwere
toleratedinexcesshigherthan80foldtheanalyteconcentration[42].

Themethodwasappliedtotheanalysisofmilk,yoghourtandfruit

juice samples containing soy proteins. These samples were also analyzed
using a commercial ELISA kit [41]. Samples were subjected to a
denaturationrenaturation treatment according to the procedure indicated
by the antisoy protein antibody manufacturer. Table 2 shows the soy
protein content found by the reported immunoassay and by the ELISA
method, which were compared by a paired ttest carried out at a 95 %
significance level, finding that there were not statistically significant
differencesamongthem.

Table2.Determinationofsoyproteincontentinfoodsamples
Sample
Soymilk
Fruitandsoyjuice
Soyyoghourt

Reportedmethoda,b

CommercialELISAa,b

746

843

8.40.7

10.50.3

644

603

MeanSD(n=3)
Units:fruitandsoyjuice,soyyoghourt,g/Kg;soymilk,g/L

357

Chapter5

A recovery study was also carried out to validate the method

(Table 3). It was performed by adding three different amounts of soy
proteintoeachsampleandsubtractingtheresultsobtainedfromsimilarly
treatednonspikedsamples.Thevaluesobtainedrangedfrom81.5%and
111.0%.

Table3.Recoveryofsoyproteinaddedtofoodsamples
Addeda,b

Founda,b

Recovery(%)

Soymilk

24
72
123

272
694
11010

111.0
95.7
89.4

Fruitandsoyjuice

2.7
8.1
13.5

2.20.2
7.70.6
121

81.5
95.1
88.9

Sample

Soyyoghourt
23
212

65
614

113
957
aMeanSD(n=3)
bUnits:fruitandsoyjuice,soyyoghourt,g/kg;soymilk,g/L

91.3
93.2
84.1

Determinationofmonensinbyheterogeneousimmunoassayin
milksamples

Monensin is a veterinary drug that exhibits both antibacterial and

anticoccidial activities and has been traditionally used to prevent
coccidiosisinpoultry[43].Thisantibiotichasbeenconsideredasagrowth
promoter, since this disease prevents the growth of poultry owing to the
bloody diarrhea and weight loses associated. Although the use of many

358

Discussionoftheresults

veterinary antibiotics as growth promoters has been banned since 2006,

they can be still administered to some species according to the European
Union regulation [44]. Also, the treatment with monensin has been
extendedtocalvesfortheEuropeanUnionwithmaximumresiduelimits.
The concern on food safety has led to the search for reliable
analytical methods to screen and confirm the presence of antibiotic
residues in foodstuffs, which contribute to increase bacterial resistance to
antibiotics.
Thefirstimmunoassayformonensindeterminationusingnileblue
doped SiO2NPs as labels has been presented in this Dissertation. The
usefulnessofthisapproachtoimprovetheanalyticalfeatures,suchasthe
detectionlimit,oftheimmunoassayspreviouslydescribedusingenzymatic
labelsforthedeterminationofthisdrugisshown[22,4548].
As it has been previously indicated, the use of nile bluedoped
SiO2NPs as labels provides the spectral selectivity required to avoid
interferences from the sample matrix. In addition, the encapsulated
fluorophore inside the NPs shows an excellent stability to the action of
environmental agents and the incident light, avoiding photobleaching
phenomena. These features justify the use of these NPs as labels in
immunoassays.Anotheradvantageofthenewmethodistheuseof96well
microplates with 100L wells, which reduces the sample and tracer
volumes required in comparison to the conventional 300L wells, which
contributestominimizethecostsassociatedwiththeimmunoassayandthe
amount of waste generated. This plate type facilitates the use of front

359

Chapter5

surface measurements as the distance between the well bottom and the
detector is shorter than that for conventional plates, which minimizes
fluorescencesignallossesandimprovesthesensitivity.
As in the previous section, two heterogeneous immunoassays,
involvingantibodyorantigencaptureformat,wereassayedtochoosethe
bestoptionformonensindeterminationusingnilebluedopedSiO2NPsas
labels. The first format was based on the immobilization of a BSA
monensin conjugate and the use of an antimonensin antibodyNP tracer.
The second assay needed a monensinNP tracer, synthesized via a
carbodiimide reaction using EDAC (N(3methylaminopropyl)N
ethylcarbodiimide hydrochloride), and the immobilization of anti
monensin antibodies by coating the wells with antisheep IgG. A
significantdifferencebetweenthefluorescenceintensityvaluesobtainedin
theabsenceandinthepresence(0.2g/L)ofmonensinwasreachedinboth
assays,butthisdifferencewasabout3foldhigherfortheantibodycapture
format.Thus,thisformatwaschosentodevelopthenewimmunoassayfor
monensindetermination.
The method presents a dynamic range of 0.05 5 g/L and the
detection and quantification limits are 0.015 and 0.05 g/L, respectively,
whichwouldcorrespondto0.12and0.40g/Kginthemilksamples.The
lattervalueisabout5timeslowerthanthemaximumresiduelimit(MRL)
set by the 37/2010/EC Commission Regulation for monensin in milk
samples. Also, the detection limit is four times lower than that reported
using ELISA with chemiluminescence detection [32]. The precision,
expressedasthepercentageofrelativestandarddeviationandassayedat

360

Discussionoftheresults

twodifferentmonensinconcentrations,0.2and1g/L,gavevaluesof5.8
and4.0%,respectively.
The method was applied to the analysis of skimmed, semi
skimmedandwholemilksamples.Thesampletreatmentwasquitesimple
as it only required the deproteinization of samples and a further solvent
evaporation step. A recovery study was also carried out to validate the
method(Table4),obtainingvaluesintherangeof83.3107.5%.
Table4.Recoveriesobtainedformonensinaddedtomilksamples
Sample
Monensn

a,b
a,b

Found
Recovery
Added
(g/Kg)
(g/Kg)
(%)
Skimmedmilk
1.5
1.60.1
106.7

2
1.960.09
98.0

4
4.10.2
102.5
Semiskimmedmilk

1.5
2
4

1.50.1
2.00.1
3.40.3

100.0
100.0
85.0

Wholemilk

1.5
2
4

1.250.07
1.70.1
4.30.3

83.3
85.0
107.5

MeanSD(n=3)

In summary, the results obtained show the usefulness of LWF

dopedSiO2NPsaslabelstodeterminemacromolecules(proteins)andsmall
molecules (antibiotics) in food analysis. This approach provides lower
detectionlimitsthanELISAandavoidsthelaststepfortheenzymeactivity
measurement. Likewise, the proposed methods have contributed to
expandtheuseofnanotechnologyinthisanalyticalarea.

361

Chapter5

2. Newstrategiesforthedeterminationofantioxidantsinfoods
Oxidation processes involving free radicals contribute to the
development of many illnesses, such as cardiovascular disease,
Alzheimersdisorderandcancer.Thisredoxphenomenonisproducedby
reactive oxygen species (ROS), which damage cell membranes, but this
effect can be minimized by the intake of antioxidant compounds. The
antioxidant capacity of foods is mainly ascribed to polyphenols, such as
flavonoids,andvitaminsCandE,amongothers[49].
Amethodforthedeterminationoftheantioxidantcapacityofsome
food samples has been described in this Dissertation, which shows the
usefulness of longwavelength measurements. Another method for the
determination of polyphenols using nanomaterials as laccase enzyme
activators is also included. The features of both methods and their
applicabilityarediscussedbelow.

Longwavelength

fluorimetric

determination

of

food

antioxidantcapacityusingnileblueasreagent
From an analytical point of view, there are two types of methods
for assessing food antioxidant capacity. The first group involves a single
electrontransfer reaction, which can be followed by a change in the
solution color or fluorescence. The second group is based on the use of a
hydrogen atom transfer reaction, where the antioxidant and a substrate
compete for the free radicals generated. The oxygen radical absorbance
capacity (ORAC) assay, which belongs to the second group, involves the
useofafreeradicalgeneratorthatdirectlyattackseitherachromophoreor

362

Discussionoftheresults

afluorophore,leadingtothedecreaseoftheirabsorbanceorfluorescence,
respectively.Thechromophorepyrogallolred[50]andthefluorophores
phycoerythrin and fluorescein [51 57], have been used to develop this
assay.Despitethehighsensitivitythattheabovementionedfluorophores
offer, these compounds feature a relatively short Stokes shift (about 27
nm), which favors scattering phenomena. Also, phycoerythrin is
relatively expensive and suffers from photobleaching, while fluorescein
emission is usually overlapped by the static background signals from the
sample matrix. The use of longwavelength fluorophores can avoid these
problems, but their potential application to the determination of the
antioxidantcapacityhasnotbeenstudieduptonow.
An analytical method has been described in this Dissertation for
food antioxidant capacity determination by the ORAC assay using nile
blue

as

fluorescent

probe,

2,2azobis(2methylpropionamidine)

dihydrochloride (AAPH), as free radical generator, and 6hydroxi2,5,7,8

tetramethylcroman2carboxylic acid (Trolox) as model analyte have been
used for this purpose. Fluorescent measurements were carried out in the
kineticmodebymonitoringthechangeoffluorescenceovertimeandusing
the normalized net area under the curve, AUCnet = (AUCsample
AUCblanck)/AUCblanck), obtained for different analyte concentrations, as the
analyticalparameter.
Asithasbeenalreadymentioned,thetraditionalfluorophoresused
fortheseassayshavebeenphycoerythrinandfluorescein,whichshowa
delay in their fluorescence quenching in the presence of antioxidant
compounds,buttheirusehasthelimitationsaboveindicated.Thepotential

363

Chapter5

use of long wavelength fluorescent dyes as alternative reagents to avoid

theselimitationswasassessed.Severaloxazineandthiazinedyes,namely,
nile blue, azure A, and azure B, were assayed for this purpose. Figure 6
shows the curves obtained for each fluorophore, in the presence and
absence of Trolox. As can be seen, the three fluorophores gave rise to
significantAUCnetvalues,butthebestresultswereobtainedwithnileblue.

18000

18000
16000

a)

Relative fluorescence intensity

Relative fluorescence intensity

16000
14000
12000
10000

8000
6000
4000
2000

b)

14000
12000
10000

8000
6000

4000
2000

0
0

20

40

60

80

100

20

40

Time (min)

60

80

100

Time (min)

18000

Relative fluorescence intensity

16000

c)

14000
12000

10000
8000

6000
4000

2000
0
0

20

40

60

Time (min)

80

10

Figure6:CurvesobtainedfortheORACassayusing(a)nileblue,(b)azureA,
and(c)azureBfluorophores:(1)blankand(2)2MTrolox.

364

Discussionoftheresults

The influence of the pH was investigated in the range of 4 11,

using0.075Macetate,phosphate,borate,andcarbonatebuffersolutionsto
keepthepHconstantinthebufferingregionofeachsolution.AsFigure7
shows, the net AUC values were very low at pH below 5.8 and above 8,
obtainingthebestresultsinthepHrangeof6.07.0.Thebehaviorofthe
systematlowpHvaluescouldbeascribedtothefactthatthepKaofTrolox
isabout3.89,showingalowsolubilityatthispH.

0.10

A)
0.08

AUC

0.06

0.04

0.02

0.00
5.5

6.0

6.5

7.0

pH

7.5

8.0

8.5

Figure 7: Influence of pH on the ORACnile blue method. Experimental

conditions:[nileblue]=120nM;[buffer]=0.075M;[AAPH]=0.024M;[Trolox]
=2M;temperature=37C.

The influence of the fluorophore concentration was studied in the

rangeof60370nM(Figure8),obtainingthatthesystemwaspractically
independentofthisvariableintherangeof80130nM,choosing89nM

365

Chapter5

nile blue concentration for the development of the method. It is worth

indicating that the potential use of nile blue doped SiO2NPs as an
alternative to the fluorophore in solution was studied but, as it was
foreseen, unsatisfactory results were obtained, owing to the protection
provided by the NPs on the fluorescence of the encapsulated dye. The
effectoftheAAPHconcentrationonthesystemwasevaluatedintherange
of 6 to 24 mM, finding that the AUC values of both analyte and blank
solutionsdecreasewhentheAAPHconcentrationincreases.However,the
curves obtained at low AAPH concentrations were less defined and
showed a low reproducibility. Thus, a 12 mM AAPH concentration was
chosen.

0.16

B)

AUC

0.12

0.08

0.04

0.00

100

200

[NB], nM

300

400

Figure8:InfluenceofnileblueconcentrationontheORACnilebluemethod.
Experimental conditions: [AAPH] = 0.012M; [Trolox] = 2 M; pH = 6.9;
[phosphate]=0.085M;temperature=37C.

366

Discussionoftheresults

Thedynamicrangeofthecalibrationgraphwas0.88MTrolox

and the detection limit was 0.45 M, which is lower or similar to those
obtained in other ORAC methods involving fluorescein. The precision of
the method was assessed at two different Trolox concentrations, 1 and 5
M,obtainingrelativestandarddeviationsof5.6and2.9%,respectively.

The new method was applied to the analysis of wines and

commercial fruit juices and the results were compared to those obtained
withthetraditionalORACmethod.Thetreatmentofthesampleswasthe
sameforbothmethods,whichjustrequiredtoadjustthepHuntilneutral
valuesandthefurthersampledilutiontomatchthedynamicrangesofthe
corresponding calibration curves. Table 5 lists the content found in the
samplesusingbothmethods.Thepairedttestwasappliedtotheresultsat
the95%significancelevel,anditwasfoundthattherewerenotsignificant
differences in the results provided by both methods, which confirms the
practicalusefulnessofthenewmethoddescribedinthisDissertation.
Table5.AntioxidantcapacityoffoodsamplesanalyzedbyORACnileblue
andORACfluoresceinmethods
Sample
ORACnilebluea,b ORACfluoresceina,b
Whitewine(Manzanilla)

1.500.09

1.030.2

Semidrywine

7.70.9

4.40.4

Redwine

15.30.4

10.40.9

2.2220.001

1.930.09

Pineapplejuice

2.70.3

2.3990.006

Applejuice

2.70.3

3.50.4

Peachjuice

Meanstandarddeviation(SD)(n=3)
ExpressedasmillimolarTroloxequivalentsofsample

a
b

367

Chapter5

A recovery study was also carried out to validate the method. It

wasperformedbyaddingthreedifferentamountsofTroloxtoeachsample
and subtracting the results obtained from similarly treated unspiked
samples.Table6showstherecoverypercentages,whichrangedfrom72.7
%to113.6%.Themeanrecoveryvaluesobtainedwere92.0and92.9%for
wineandjuicesamples,respectively.Thisinternalvalidationalsoconfirms
theusefulnessofthedevelopedmethodfortheanalysisofrealsamples.
Tabla6.Recoveryvaluesobtainedforthedifferentsamplesanalyzed

Recoverystudy
Addeda

Founda,b

Recovery(%)

Whitewine

2.2
8.9
11.1

2.00.2
6.70.9
9.20.8

92.0
75.2
82.9

Semidrywine

4.4
8.8
13.2

3.60.2
8.10.3
12.90.6

81.8
92.1
97.0

Redwine

17.8
35.6
44.4

182
384
444

101.1
106.7
99.1

Peachjuice

2.2
4.4
5.5

2.10.1
3.50.4
4.30.2

95.5
79.5
78.2

Pineapplejuice

2.2
4.4
5.5

2.50.2
4.50.3
5.80.4

113.6
102.3
105.5

4.4
3.20.2
8.8
7.90.5
11.1
111
aUnitsinmillimolarTroloxequivalentsofsample
bMeanSD(n=3)

72.7
89.6
99.1

Sample

Applejuice

368

Discussionoftheresults

The results obtained show the feasibility of long wavelength

fluorimetryforthedeterminationoftheantioxidantcapacityinfoodusing
the fluorophore nile blue. The use of this reagent instead of other
fluorophorespreviouslyreportedforthispurpose,suchasfluoresceinor
phycoerythrin,isausefulalternativetoavoidpotentialbackgroundsignals
fromthesamplematrix,whichcanappearatlowerwavelengths.Also,the
relatively wide Stokes shift of nile blue reduces the scattering signals.
Finally, the probability of photobleaching for nile blue is lower than that
forphycoerythrin,aswellasitscost.

Automaticdeterminationofpolyphenolsinwineusinglaccase
andterbiumoxidenanoparticles

Polyphenols are an important group of natural antioxidant

compoundspresentinthehumandietthat,afternumerousclinicalstudies,
haveshowntheirusefulnessinthepreventionofvariouschronicdiseases.
As it has been above mentioned, numerous chromatographic [58, 59] and
electrophoretic [60, 61] methods have been developed for the
determination of these naturalantioxidants. However, the high variety of
phenoliccompoundsthatcanbefoundinfoodsamplesjustifiestheuseof
analytical methods to determine total phenolic content and total
antioxidant capacity. Both parameters have been widely determined by
using photometric or fluorimetric methods involving free radical
generatorsorenzymaticreactions,whichcompetewiththeantioxidantin
order to inhibit the fluorescence or absorbance decrease observed in
absenceoftheseantioxidants[56,6267].

369

Chapter5

Laccaseisaphenoloxidaseenzymethathasbeendescribedforthe
determination of both phenolic content and antioxidant activity, using
different experimental conditions. As it has been already mentioned, this
enzyme catalyzes the oxidation of phenolic compounds to quinones or
radicals by reducing thedissolvedoxygen to water, which has beenused
todevelopamperometricsensors[6870]aswellasphotometric[7173]
andfluorimetric[74]methods.
Theusefulnessofterbiumoxidenanoparticles(Tb4O7NPs),laccase
and the fluorophore 8hydroxypyrene1,3,6trisulfonic acid trisodium salt
(HPTS)forthedeterminationofpolyphenolsinwinesamplesisdescribed
in this Dissertation. As for the method reported in the previous section,
fluorescence measurements were carried out in the kinetic mode, by
measuring the change of fluorescence over time and using as analytical
parameter the net AUC values obtained for different concentrations of
gallicacid,whichwasusedasthecalibrationstandard.
Firstly, several potential fluorescent laccase substrates (Styryl 7, 2
Di1ASP, azure B chloride, toluidine blue O, nile blue chloride, sodium
fluorescein and HPTS) were assayed to study their capability to compete
with phenolic antioxidants for the active sites of the enzyme. Although
indocyaninegreenhasbeendescribedasalaccasesubstrate[74],itwasnot
assayed because its long excitation and emission wavelengths were not
adequateforthemicroplatereaderusedinthisstudy.Theresultsobtained
from the study of the behavior of the different fluorophores assayed
proved that only HPTS showed a relatively fast and remarkable decrease
initsfluorescenceintensityinthepresenceoflaccase.

370

Discussionoftheresults

As can be seen in Figure 9, the fluorophore HPTS has a phenolic

group in its structure, which can explain this behavior, giving rise to its
enzymaticoxidationbythedissolvedoxygen.

HO

O
ONa

NaO

ONa
O

Figure 9. Structure of the fluorophore 8hydroxypyrene1,3,6trisulfonate

trisodium(HPTS)

Another investigation carried out to improve the features of the

system was the study of the potential activator effect of different NPs on
the catalytic behavior of laccase. Several NPs, such as diamond, Eu2O3,
Tb4O7,AgandmagneticgoldNPswereassayedintheconcentrationrange
of 0.1 2 mg/mL, finding that only Tb4O7NPs notably increased the
reaction rate of the system. As can be seen in Figure 10, both blank and
analyte AUC decreased, which allows an increase in the sample
throughput.Also,theeffectofTb4O7NPsonthesystemwascheckedinthe
absenceoflaccase,findingthattheNPsdidnotmodifythefluorescenceof
HPTS.However,diamondandmagneticgoldNPscausedthefluorescence
quenching of HPTS, AgNPs inhibited the catalytic effect of laccase, while

371

Chapter5

nochangeinthekineticbehaviorofthesystemwasfoundinthepresence
ofEu2O3NPs.

Figure10.KineticcurvesobtainedfortheHPTSlaccasesystemintheabsence
(curves1and2)andinthepresence(curves3and4)of2Mgallicacid,andin
thepresence(curves1and3)andintheabsence(curves2and4)of1mg/mL
Tb4O7NPs.[HPTS]=10M,[laccase]=1U/mL,pH=7.0,[phosphate]=0.4M,
temperature=37C.

The potential use of terbium(III) ions in solution, instead of

Tb4O7NPs,wasalsostudiedusingaconcentrationequivalenttothatofthe
NPs, but lower net AUC values were obtained. These results show the
usefulnessoftheseNPsasactivatorsoftheenzymaticsystem.

Thevariablesaffectingthesystemwereoptimizedbytheunivariate

method using the net AUC as the analytical parameter. However, it was
372

Discussionoftheresults

necessarytofindacompromisedsolutionbetweenthisparameterandthe
time required for each measurement, in order to obtain an adequate
sensitivitybutavoidingtheexcessivedurationoftheassay.

TheinfluenceofthepHonthesystemwasstudiedintherangeof

4.0 9.0 using different buffer solutions. The fluorescence of HPTS was
very low at acid pH values and very high in the range of 8.0 9.0, but
laccase did not show any enzymatic activity at the higher pH values.
However,theHPTSfluorescenceintensitywashighenoughatpH7.07.5
anddecreasedinthepresenceoflaccase.Thus,apHof7.0wasselectedfor
themethoddevelopment,usinga0.4Mphosphatebuffersolutiontoadjust
this pH value. The study of the influence of the HPTS concentration
demonstratedthatthesystemwasindependentofthisvariableintherange
of 8 12 M, and the intermediate value (10 M) was chosen as the
optimumconcentration.

ThelaccaseactivityandtheTb4O7NPsconcentrationaretwocritical

variables that have a remarkable effect on both the net AUC and the
reactionrate.Bothvariableswerestudiedintherangesof0.52U/mLand
0.2 2 mg/mL for laccase activity and Tb4O7NPs concentration,
respectively.Highvaluesofbothvariablesgaverisetoveryhighreaction
rates and low net AUC values, which negatively affected the method
sensitivity. On the contrary, very low values of these variables caused a
notable increase of the net AUC and, therefore, the improvement of the
method sensitivity, but the duration of the assay was excessively long.
Thus, the values selected to obtain appropriate net AUC and duration of
theprocesswere1U/mLlaccaseactivityand1mg/mLNPsconcentration.

373

Chapter5

As it has been above mentioned, the analytical parameter used in

thismethodwasthenetAUC,obtainedfromthenormalizedkineticcurves
for different gallic acid concentrations. Figure 11 show the kinetic curves
obtained to ex 450 nm y em 535 nm under the optimum experimental
conditions.

Normalized fluorescence intensity

1.0

16

0.8

0.6

0.4

0.2

0.0

10

20

30

40

Time (min)

Figure 11. Kinetic curves obtained for different gallic acid concentrations: (1)
(1) 0 M, (2) 0.5 M, (3) 1 M (4) 2 M (5) 5 M y (6) 10 M. Experimental
conditions: [HPTS] = 10 M, [laccasa] = 1 U/mL, [NPs] = 1 mg/mL, pH = 7.0,
[phosphate]=0.4M,temperature=37oC.

Thedynamicrangeofthecalibrationgraphwas0.512Mgallic
acidandthedetectionlimitwas0.14M,whichisaboutthreetimeslower
than that obtained in the absence of Tb4O7 NPs (0.4 M). This difference
shows the positive effect of the NPs on the method, improving both the
reaction rate and the detection limit. The detection limit reached in the
presence of the NPs is lower than those described for gallic acid using

374

Discussionoftheresults

some amperometric biosensors, which were ranged between 0.19 and 50

M[69,70].Althoughadetectionlimitforgallicacidof0.04Mhasbeen
obtainedusingindocyaninegreen[74],thismethodinvolvesthesequential
analysisofeachsample,obtaininglowersamplethroughput.Theprecision
ofthemethod,expressedasthepercentageofrelativestandarddeviation,
wasassessedat0.7and5Mgallicacidconcentrations,providingvalues
of6.3and2.5%,respectively.

Theselectivityofthemethodwascheckedbyassayingsomenon

phenolicreducingsubstances,whichcouldbepresentinthewinesamples
used to demonstrate the applicability of the method. Table 7 shows the
results obtained after assaying different potential interfering compounds.
The most serious interference was caused by ascorbic acid, which was
tolerated at the same molar concentration than that of the analyte.
However,thisinterferencewouldbenegligiblebecausethemaximumlimit
allowedforascorbicacidwhenusedasanadditiveinwineis0.85mM[75],
which is lower than the usual content of polyphenols in wines. Sulfites
weretoleratedinaconcentration20foldthatoftheanalyte.Althoughthis
interference could be removed using a pretreatment step [70], it was not
necessary as the presence of sulfites is controlled according to their total
contentinreducingsubstances[76].

375

Chapter5

Table7.Influenceofsomereducingagentsontheproposeddetermination
ofphenolicantioxidantsat2Mgallicacid
Compound

Maximumtolerated
interferent/analyteratio*
100

Malicacid
Citricacid

100

Sucrose

100

Glucose

25

Sodiumsulfite

20

Ascorbicacid

*Maximumratioassayedwas100

The method was applied to the analysis of red, white and sweet
wines. The sample treatment only required the dilution of the sample to
match the dynamic range of the calibration curve, using gallic acid
standards and the net AUC as analytical parameter. Table 8 shows the
content of polyphenols found in these samples analyzed by the new
methodandbytheFolinCiocalteumethod,whichisusuallyconsideredas
areferencemethod.
Table8.Antioxidantconcentration(expressedasmillimolarequivalentsof
gallicacid)foreachsample
Sample

Proposedmethod FolinCiocalteumethoda,b

Redwine(Rioja)

7.00.4

11.50.1

Redwine(Cuenca)

9.50.5

14.040.18

Whitewine(Rueda)

1.000.06

1.920.04

Whitewine
(Valdepeas)

1.000.03

2.0280.005

Sweetwine(Montilla)

1.270.09

3.640.05

MeanSD(n=3)
Units:mMgallicacidequivalentsofsample

376

Discussionoftheresults

As it can be seen from the Table 8, the values provided by the

FolinCiocalteumethodwerehigherinallinstancesthanthoseprovidedby
the laccasemethod, which isascribed to the capability of the later toalso
detect nonphenolic substances. The values obtained by the new method
are similar to those obtained using other methods for the analysis of this
typeofsamples[68,69].
A recovery study was performed by adding three different
amountsofgallicacidtoeachsampleandsubtractingtheresultsobtained
fromsimilarlytreatedunspikedsamples.AsitcanbeseenintheTable9,
the recovery percentages obtained ranged from 81.0 to 108.3 % and the
meanrecoveriesforred,whiteandsweetwineswere90.9,92.4and99.5%,
respectively.
Table4.Recoverystudyforthewinesamplesanalyzed
Founda,b
Sample
Addeda
Redwine(LaRioja)
4
4.30.5
12
10.00.5
20
161
Redwine(Cuenca)
4
3.20.2
12
11.70.3
20
18.90.8
Whitewine(Rueda)
0.25
0.240.03
0.75
0.770.04
1.25
1.080.03
Whitewine(Valdepeas)
0.25
0.230.03
0.75
0.700.04
1.25
1.00.1
Sweetwine(Montilla)
1
1.010.09
3
3.00.3
5
4.70.3
bMeanSD(n=3)
aUnits:mMgallicacidequivalentsofsample

377

Recovery(%)
108.3
84.1
81.0
80.0
97.4
94.5
97.2
102.8
86.7
92.0
92.2
83.3
101.5
102.0
95.0

Chapter5

The results obtained evidence the usefulness of the combined use

of laccase, Tb4O7NPs and the fluorophore HPTS for the determination of

polyphenolsinwinesamples.Itisnoteworthytomentionthattheactivator
effectofTb4O7NPsonanenzymaticsystemisdescribedforthefirsttime.
TheuseoflaccaseandNPsincreasesthesamplethroughputofthemethod
andimprovesthesensibility,beingthesevaluesbetterthanthoseobtained
for other methods previously described [68, 69]. Also, the use of an
automatic microplate reader enables the simultaneous processing of the
samples, reaching an overall sample throughput of about 35 samples h1,
whenanalyzedintriplicate.
3. New investigations in the use of upconverting phosphors in
luminescenceresonanceenergytransfer

This study was developed during the threemonth stay at the

DepartmentofBiotechnologyoftheUniversityofTurku(Finland).Dueto
the short duration of this period, the research was focused to the
systematic evaluation of several donors and acceptors to design a
luminescence resonance energy transfer system (LRET) using the anti
Stokes phenomenon in biotinstreptavidin affinity assays. This study
aimed the selection of the most appropriate system for its subsequent
analyticalapplication.

As it has been described in the Introduction of this dissertation,

upconverting phosphors (UCPs) are composed of inorganic host lattices

usually doped with lanthanide ions to achieve the phenomenon of anti
Stokes fluorescence emission. The research presented here reports a

378

Discussionoftheresults

systematic study of the behavior of different UCPs doped with Er(III),

Ho(III) and Tm(III), used as donors, and several fluorophores, used as
acceptors,todevelopnewmethodologiesbasedintheLRETphenomenon.
Twomainrequirementshavetobefulfilledforthisprocesstohappen:1)
The excitation spectrum of the acceptor should overlap with the donor
emission spectrum; 2) The distance between donor and acceptor should
followtheFrsterequation.
Among

the

wide

variety

of

existing

fluorophores,

phycobiliproteins, such as phycoerythrin (BPE) and Rphycoerythrin

(RPE)havebeenpreviouslyusedasacceptorsindifferentLRETassays[77
79]. Their use is mainly due to their excellent fluorescence emission,
althoughtheirpriceisrelativelyhigh.Forthisreason,thepossibilitytouse
less costly fluorophores, such as Alexa Fluor or ATTO, to develop LRET
affinityassayshasbeenstudied.Thesefluorophorescouldalsobeusedto
develop multiplexed assays, since they feature different excitation and
emission wavelengths. To perform this systematic study between donors
and acceptors, streptavidin (SA) was used to coat UCP donors, whereas
biotin(Bio)wascoupledtofluorescentacceptors.

Figure12showstheexcitationandemissionspectraofthedifferent

donoracceptor pairs used in this study. Figures 12.A12.C show the

combination between the UCP doped with Er(III) and the fluorescent
proteinBPEandAlexaFluor546(AF546)and680(AF680)fluorophores.As
it can be seen, there is an excellent overlap between the emission and
excitationspectraofdonorsandacceptors,respectively..

379

Chapter5

Similar spectra were obtained when the UCPs doped with Ho(III)

(Figure12.D12.E)wereassayedwithBPEandAF546.Inasimilarwayto
the Er(III)doped UCP behavior, the wide excitation spectrum of BPE
allowsitsefficientexcitationwhiletheenergytransferusingAF546shows

200

700

400

450

400

200

600

650

400

200

550

600

WAVELENGTH (nm)

700

800

600

400

200

400

450

500

550

600

650

700

650

700

G)

1000

800

600

400

600/40

600

500

600

WAVELENGTH (nm)

WAVELENGTH (nm)

800

450

500

800

700

F)

700

E)

1000

WAVELENGTH (nm)

1000

200

200

400

450

500

550

600

WAVELENGTH (nm)

650

700

H)

1000

800

600

400

600/40

550

650

400

200

555/20

500

600

NORMALIZED LUMINESCENCE INTENSITY

450

550

600

600/40

600

500

800

WAVELENGTH (nm)

800

400

650

600/40

NORMALIZED LUMINESCENCE INTENSITY

600

D)

1000

550

200

WAVELENGTH (nm)

400

500

400

555/20

450

NORMALIZED LUMINESCENCE INTENSITY

NORMALIZED LUMINESCENCE INTENSITY

400

600

520/10

800

C)

1000

750/40

400

B)

1000

600/40

600

NORMALIZED LUMINESCENCE INTENSITY

800

NORMALIZED LUMINESCENCE INTENSITY

600/40

A)

1000

600/40

NORMALIZED LUMINESCENCE INTENSITY

NORMALIZED LUMINESCENCE INTENSITY

aloweryield.

400

450

500

550

600

650

700

WAVELENGTH (nm)

Figure 12. Emission spectra from UCPs and acceptors used in this study. In
12.A12.C, Er(III)doped UCP emission spectra together with BPE (12.A),
AF546 (12.B) and AF680 (12.C). In 12.D and 12.E, Ho(III)doped UCPs with
BPE (12.D) and AF546 (12.E). In 12.F12.H, Tm(III)doped UCPs with RPE
(12.F),AF488(12.G)andATTO495(12.H).

380

Discussionoftheresults

TheTm(III)dopedUCPswerecombinedwithRPE,AlexaFluor488

(AF488) and ATTO495 fluorophores (Figure 12.F12.H). In this case, the

donor has a lower emission wavelength than Er(III) and Ho(III), with a
good theoretical overlap with the AF488 fluorophore, although, as it will
be discussed below, an appropriate theoretical overlap does not always
provideasuitableenergytransferprocess.
As has been described in the Introductionof this Dissertation, the
absenceoffunctionalgroupsinthistypeofinorganicnanomaterialsmakes
itessentialtoperformacoatingandfunctionalizationprocess,beforethey
can be linked to biomolecules. After this step, the different donors and
acceptors were labeled with SA and Bio, respectively, to carry out the
optimization of their concentrations and, finally, to obtain the calibration
curves under the best conditions for biotin, which was used as model
analyte.

The possibility of designing LRET systems using alternative

fluorophorestothephycobiliproteinsBPEandRPE,inordertoavoidtheir
relatively high cost, was studied. Figure 13 shows the titration curves
obtained with Er(III)SA (13.A), Ho(III)SA (13.B) and Tm(III)SAdoped
UCPs as donors, combined with different fluorophores. Luminescence
emissionwasobtainedinallinstances,butthebestresultswereachieved
with the Er(III)BPE, Er(III)AF680, Ho(III)BPE and Tm(III)RPE pairs,
whichwereusedtoobtainthetitrationcurvesforbiotin.Asitcanbeseen
from Figure 13, the highest luminescence intensities were obtained for
Er(III)dopedUCPswithBPEandAF680.

381

Chapter5

600000

4000000

up conversion LRET (cts)

up conversion LRET (cts)

500000

3000000

2000000

1000000

0
1

10
100
[Biotin] (nM)

1000

300000
200000
100000

0,1

10000

10

100

1000

10000

120000

250000

D)

C)
100000

200000

150000

100000

50000

80000

60000

40000

20000

[Biotin] (nM)

up conversion LRET (cts)

up conversion LRET (cts)

400000

0,1

B)

A)

0,1

10

100

1000

10000

0,1

10

100

1000

10000

[Biotin] (nM)

[Biotin] (nM)

Figure13. HomogeneousLRETaffinity assaysfor biotin with donoracceptor

pairsgivingthebestresults.InA)withEr(III)dopedUCP(0.015mg/mL)and
(1)0.4nMBPEand(2)4nMAF680.In(B)Ho(III)dopedUCP(0.030mg/mL)
and0.4nMBPE.In(C)Tm(III)dopedUCP(0.030mg/mL)and1.33nMRPE.
LRET signals were calculated by subtracting the background signal of the
assay (excess of biotin) from the maximum signal (absence of biotin), cts:
counts.

382

Discussionoftheresults

However, according to the results shown in Table 10, which

summarizesthedifferentassayscarriedoutandtheIC50valuesobtained
forbiotin,thelowestvalueforthisparameterwasachievedwiththepair
SATm(III)UCPwithRPE,inspite ofitslowLRETintensity.Thehighest
IC50 was obtained for the SAEr(III)doped UCP with AF680, which was
about14.4nM.Thevaluesobtainedarecomparableand,insomeinstances,
better than those obtained by other previously reported methods [77, 78]
usingEr(III)dopedUCPsasdonorsandBPEasacceptor.
Table10.IC50valuesforbiotinassaysusingdifferentdonoracceptorpairs

Acceptor
Donor

IC50forbiotin(nM)
BioBPE
(0.2nM)

BioBPE
(0.4nM)

BioRPE
(1.33nM)

BioAF680
(4nM)

8.57*

14.37

5.7

7.20

3.35

Er(III)doped
UCP
(0.015mg/mL)
Ho(III)doped
UCP,0.015
mg/mL)
Ho(III)doped
UCP,0.015
mg/mL)
Tm(III)doped
UCP
(0.030mg/mL)
*TheconcentrationofBioBPEwas0.3nM

In summary, it is possible to draw several conclusions after the

systematicstudycarriedouttoevaluatethepotentialusefulnessofseveral

383

Chapter5

UCPsasdonorsandseveralfluorophoresasacceptorsinLRETsystems:1)
The donor UCPEr(III) provides the best results when it is used together
with BioBPE as acceptor; 2) The use of the fluorophore AF680 as
alternativeacceptortophycobiliproteinsprovidesahighfluorescentsignal,
althoughtheIC50valuereachedforbiotinishigherthanthatobtainedin
presenceofthephycobiliproteins.Nevertheless,theuseofthisfluorophore
also allows the determination of biotin at nanomolar concentration level,
avoidingtherelativelyhighcostofthesephycobiliproteins.
As a final summary of this chapter, the main advantages and
limitations of the new analytical methodologies developed in this
Dissertationareincludedinthefollowingtable:

Advantages

Limitations

LWFdopedSiO2NPs
Synthesis procedures using reverse
micelle

microemulsion

method

are

commerciallyavailable
A previous study of the

reproducible
Increased stability compared to the
fluorophoresinsolution

synthesis

conditions

needs

be

to

done

Many functionalization procedures are

according to physical

feasible owing to the great variety of

chemical properties of

organosilanereagents

theLWF

Improvementofspectralselectivity
The

heterogeneous

improve

some

immunoassays

features

of

ELISAs

methodsusedforthispurposesince:

Lowerdetectionlimitsareobtained

Lesserstepsarerequired,sotheyare
simplertoperform.

384

These NPs are not

Discussionoftheresults

Newmethodsforantioxidantdetermination
LongwavelengthORACassay

Decrease

of

light

scattering Lack of standardised

phenomena

methods

Improvementofspectralselectivity

Determination of polyphenols using

laccaseandTb4O7NPs

Lower detection limits than those

The enzyme cannot be

providedbybiosensors
TheuseofTb4O7NPsalsoallows:

reused

o A decrease of the amount of

enzymerequired
o Shorterassaytimes
Tb4O7NPsarecommerciallyavailable
SystematicstudyofUCPsinLRETphenomena
Interference of sample matrices is Conventional spectro
decreased owing to excitation in the IR

fluormeterscannotbe

regionofspectrum

used

It is posible to obtain LRET from

different donoracceptor pairs, so the
feasibility of multiplexed systems can
expand the application field of this
technique
Fluorophores

other

than

phycobiliproteinscanbeusedtodevelop
LRET assays, which can decrease the
costsoftheseassays

385

Chapter5

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in

CONCLUSIONES
CONCLUSIONS

Conclusiones

LasinvestigacionespresentadasenestaMemoriahancontribuidoa
expandir la aplicabilidad de la Nanotecnologa al anlisis de alimentos
mediante el desarrollo de diversos mtodos que presentan niveles
adecuados de sensibilidad y selectividad. El uso de microplacas ha
permitido la automatizacin de estos mtodos con un bajo consumo de
muestras y reactivos y una elevada velocidad de muestreo. A partir del
trabajorealizadoseextraenlassiguientesconclusiones:

Sehansintetizadonanopartculasdeslicedopadasconlosfluorforos
delargalongituddeondavioletadecresiloyazulnilo.Lautilizacin
de estos nanomateriales con fines analticos permite mejorar
simultneamentelaselectividadespectral,evitandolasealdefondo
de la matriz de la muestra, y la sensibilidad, consecuencia de su
elevada relacin superficie/volumen. Los principales resultados
obtenidosconestasnanopartculasseresumenacontinuacin:
o

La sntesis mediante el mtodo de formacin de

microemulsiones

en

micelas

inversas

proporciona

nanopartculas con un mayor porcentaje de fluorforo

encapsulado que con el mtodo Stber, que en general ha
sidoelmsutilizado.
o

Los nuevos nanomateriales desarrollados presentan mayor

estabilidad fotoluminiscente que los fluorforos orgnicos
endisolucin.

Lainteraccinentrelascargaspositivasdeestosfluorforos
y las cargas negativas de la matriz de slice favorece la

399

Conclusiones

estabilizacin de los fluorforos encapsulados, no siendo

necesariosuenlacecovalenteadichamatrizdeslice.
o

La

versatilidad

aplicabilidad

analtica

de

las

nanopartculasdeslicedopadasconazulnilosehapuesto
de manifiesto mediante su uso para formar marcadores en
fluorinmunoensayo heterogneo. Se ha demostrado su
utilidadparaladeterminacindemacromolculascomolas
protenasdesojaymolculaspequeascomoelantibitico
deusoveterinariomonensn.
o

Los dos mtodos de inmunoensayo desarrollados

constituyenunaalternativatilfrentealosmtodosELISA
normalmente usados para la determinacin de estas
especies, ya que alcanzan menores lmites de deteccin y
evitan la etapa adicional para la medida de la actividad
enzimtica.

La elevada sensibilidad obtenida con estos marcadores

permite realizar medidas de fluorescencia en superficie
slida utilizando pocillos de menor volumen que los
convencionales, lo que disminuye el consumo de reactivos
yelcostedelosensayos.

SehadesarrolladounnuevomtodoORACparaladeterminacinde
lacapacidadantioxidanteenzumosyvinosconelusodelfluorforo
de larga longitud de onda azul nilo como reactivo, mejorando la
selectividad espectral de las determinaciones convencionales basadas
enelusodefluorescenaodeficoeritrina.

400

Conclusiones

Se ha propuesto un nuevo mtodo enzimtico con deteccin

fluorimtricaparaladeterminacindepolifenolesenvinosbasadoen
el uso del fluorforo 8hidroxipireno 3sulfonato sdico como nuevo
sustrato de la laccasa y de nanopartculas de xido de terbio como
activadores de esta enzima. Se ha demostrado que el lmite de
deteccin del mtodo en presencia de estas nanopartculas es tres
vecesinferioralobtenidoensuausencia.

Se ha ampliado el campo de aplicacin de los procesos basados en

transferenciadeenergaderesonancialuminiscente(LRET)utilizando
nanocristales de iones lantnidos que presentan luminiscencia anti
Stokes, denominados upconverting phosphors, como dadores de
energa, y fluorforos orgnicos que emiten en el visible, como
aceptores. Las conclusiones ms destacables de este estudio son las
siguientes:
o

Se han obtenido seales LRET adecuadas utilizando

nanocristales dopados con Er(III), Ho(III) y Tm(III), con
dimetros medios entre 10 y 20 nm, como dadores y,
ficoeritrina, Rficoeritrina y Alexa Fluor 680 como
aceptores.

Mediante ensayos de afinidad homogneos, utilizando el

sistema biotinaestreptavidina, se ha obtenido la mayor
luminiscencia con el par formado por nanocristales
dopados con Er(III) y ficoeritrina. No obstante, se ha
demostrado que los pares formados por nanocristales
dopadosconHo(III)yTm(III)comodadoresyficoeritrina

401

Conclusiones

y Rficoeritrina como aceptores tambin originan seales

luminiscentesadecuadas,permitiendoentodosloscasosla
determinacindebiotinaanivelnM.

Se ha demostrado la utilidad analtica de la mayora de los mtodos

desarrolladosmediantesuaplicacinalanlisisdediversosalimentos,
tales como vinos, zumos y leche. Las propiedades analticas que
presentan estos mtodos han permitido que las determinaciones
realizadasnorequierantratamientosdemuestracomplejos,siendoslo
necesaria la dilucin de las muestras de vinos y zumos y la
desproteinizacindelasmuestrasdeleche.

402

Conclusions

TheinvestigationsincludedinthisDissertationhavecontributedto
expand the application of Nanotechnology to food analysis by the
developmentofseveralmethodsthatpresentadequatelevelsofsensitivity
and selectivity. The use of the microplate format has allowed the
automation of these methods, also featuring low sample and reagent
consumptionandarelativelyhighsamplethroughput.Bearinginmindthe
resultsobtained,thefollowingconclusionscanbedrawn:

Silica nanoparticles doped with the longwavelength fluorophores

cresyl violet and nile blue have been synthesized. The use of these
nanomaterials for analytical purposes allows the simultaneous
improvement of the spectral selectivity, by avoiding the background
signalofsimplematrix,andthesensitivity,thislaterasaconsequence
of their high surface/volume ratio. The main results obtained with
thesenanoparticlescanbesummarizedasfollows:
o

The reversemicelle microemulsion synthesis procedure

provides nanoparticles with a higher percentage of
encapsulated fluorophore than the Stber method, which
hasbeengenerallyused.

The newly developed nanomaterials feature better

photoluminescentstabilitythantheorganicfluorophoresin
solution.

The interaction between the positive charges of these

fluorophores and the negative charges of silica matrix
favours the stabilization of the encapsulated fluorophores,
whichavoidstheircovalentlinkingtothesilicamatrix.

403

Conclusions

Theversatilityandanalyticalusefulnessofnilebluedoped
silica nanoparticles hasbeen shownby their application to
the synthesis of tracers to be used in heterogeneous
fluoroimmunoassays. Their analytical potential has been
demonstratedbydevelopingmethodsforthedetermination
of macromolecules, such as soy proteins, and for small
molecules (haptens), namely the veterinary antibiotic
monensin.

Thetwoimmunoassaymethodsdevelopedcanberegarded
as useful alternatives to the ELISA methods commonly
used for the determination of these species, since lower
detectionlimitsareachievedandtheadditionofasubstrate
tomeasuretheenzymaticactivityofthetracerisavoided.

The high sensitivity obtained with these tracers allows the

performance of frontsurface fluorescence measurements
using wells with a lower volume than the conventional
ones,whichlowersreagentconsumptionanddecreasesthe
costofassays.

A new ORAC method to determine the antioxidant capacity in juice

andwinesampleshasbeendevelopedbyusingthelongwavelength
fluorophore nile blue as reagent. This approach has improved the
spectral selectivity of the conventional determinations based on the
useoffluoresceinorphycoerythrin.

404

Conclusions

Anewenzymaticmethodforpolyphenoldeterminationinwineswith
fluorometric detection has been developed using the fluorophore 8
hydroxypyrene3sulfonatetrisodiumandterbiumoxidenanoparticles
as new laccase substrate and activator, respectively. This study has
shown that the detection limit obtained using nanoparticles is about
threetimeslowerthanthatobtainedintheirabsence.

The application field of luminescence resonance energy transfer

(LRET)processeshasbeenexpandedusingnanocrystalsoflanthanide
ions capable of providing antiStokes luminescence, so called
upconverting phosphors. These nanocrystals have been used as
energydonorsandorganicfluorophoresemittinginthevisibleregion
asenergyacceptors.Themainconclusionsofthisstudyare:
o

Suitable LRET signals have been obtained using

nanocrystals doped with Er(III), Ho(III) y Tm(III), which
feature mean diameters between 10 and 20 nm, as donors,
andphycoerythrin,RphycoerythrinandAlexaFluor680
asacceptors.

The performance of homogeneous affinity assays for the

biotinstreptavidin system has shown that the highest
luminescenceisobtainedwiththepairintegratedbyEr(III)
doped nanocrystals and phycoerythrin. Nevertheless, it
has been shown that the pairs integrated by Ho(III) and
Tm(III)dopednanocrystalsasdonorsandphycoerythrin
and Rphycoerythrin as acceptors also provide adequate

405

Conclusions

luminescent signals, allowing the biotin determination at

nMlevelinallinstances.

The analytical usefulness of most developed methods has been

demonstratedbytheirapplicationtotheanalysisofseveralfoods,such
aswine,juiceandmilk.Theanalyticalfeaturesofthesemethodshave
allowedtheuseofverysimplesampletreatments,suchasthedilution
ofwineandjuicesamplesortheproteinremovalfrommilksamples.

406

ANEXO
ANNEX

Produccin Cientfica

Scientific Production

ProduccinCientfica

ARTCULOSCIENTFICOS

Tipodepublicacin
Autores
Ttulo

Revista
ISSN
ndicedeimpacto

Tipodepublicacin
Autores
Ttulo

Revista
ISSN
ndicedeimpacto

Tipodepublicacin
Autores
Ttulo

Revista
ISSN
ndicedeimpacto

Artculoscientfico
Juan GodoyNavajas, Mara Paz AguilarCaballos,
AgustinaGmezHens
Synthesis and caracterization of oxazinedoped
silicananoparticlesfortheirpotentialuseasstable
fluorescentreagents
JournalofFluorescence
Volumen20(2010),Pginas171180
10530509(Print)15734994(Online)
1.966 (31 posicin en la seccin de Qumica
AnalticadelJournalofCitationReportde2010)
Artculoscientfico
Juan GodoyNavajas, Mara Paz AguilarCaballos,
AgustinaGmezHens
Longwavelength fluorimetric determination of
food antioxidant capacity using nile blue as
reagent
JournalofAgriculturalandFoodChemistry
Volumen59(2011),Pginas22352240
00218561(Print)15205118(Online)
2.816 (12 posicin en la seccin de Qumica
AplicadadelJournalofCitationReportde2011)
Artculoscientfico
Juan GodoyNavajas, Mara Paz AguilarCaballos,
AgustinaGmezHens
Heterogeneous immunoassay for soy protein
determination using nile bluedoped silica
nanoparticles as labels and frontsurface long
wavelengthfluorimetry
AnalyticaChimicaActa
Volumen701(2011),Pginas194199
00032670
4.555(5posicinenlaseccindeQumicaAnaltica
delJournalofCitationReportde2011)

409

Anexo

Tipodepublicacin
Autores
Ttulo

Revista
ISSN
ndicedeimpacto

Tipodepublicacin
Autores
Ttulo
Revista
ISSN

Tipodepublicacin
Autores
Ttulo

Revista

Artculoscientfico
JuanGodoyNavajas,MaraPazAguilarCaballos,
AgustinaGmezHens
Determinationofmonensininmilksamplesby
frontsurfacelongwavelengthfluoroimmunoassay
usingnilebluedopedsilicananoparticlesaslabels
Talanta
Volumen94(2012),Pginas195200
00399140
3.498(12posicinenlaseccindeQumica
AnalticadelJournalofCitationReportde2012)

Artculoscientfico
JuanGodoyNavajas,MaraPazAguilarCaballos,
AgustinaGmezHens
Automaticdeterminationofpolyphenolsinwines
usinglaccaseandterbiumoxidenanoparticles
EnviadoaFoodChemistry
03088146

Artculoscientfico
JuanGodoyNavajas,TerhiRiuttamki,Tero
Soukka
Evaluationofdifferentdonoracceptorpairsforthe
developmentofhomogeneousbioaffinityassays
usingupconversionluminescenceresonance
energytransfer
Enredaccin

410

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CONTRIBUCIONESACONGRESOS

Autores
Ttulo
Tipodeparticipacin
Congreso
Lugaryfecha
decelebracin

Autores
Ttulo

Tipodeparticipacin
Congreso
Lugaryfecha
decelebracin

Autores
Ttulo
Tipodeparticipacin
Congreso

Lugaryfecha
decelebracin

Juan GodoyNavajas, Mara Paz AguilarCaballos,

AgustinaGmezHens
Synthesisofnileblue andcresylvioletdopedsilica
nanoparticlesfortheirpotentialuseaslabels
Comunicacinoral
XIII International Symposium of Luminiscence
Spectrometry
Bologna(Italy),del7al11deSeptiembrede2008

Juan GodoyNavajas, Mara Paz AguilarCaballos,

AgustinaGmezHens
Development and assessment of new luminescent
silica nanoparticles as labels in longwavelength
fluoroimmunoassays
Pster
12JornadasdeAnlisisInstrumental
Barcelona(Espaa),del21al23deOctubrede2008

Juan GodoyNavajas, Mara Paz AguilarCaballos,

AgustinaGmezHens
Long wavelength dyedoped silica nanoparticles as
potentiallabels
Pster
I Encuentro sobre Nanociencia y Nanotecnologa de
investigadores y tecnlogos de la Universidad de
Crdoba(INanoUCO)
Crdoba(Espaa),12deDiciembrede2008

411

Anexo

Autores
Ttulo

JuanGodoyNavajas
Investigacin del potencial analtico de nuevos
nanomateriales luminiscentes a larga longitud de
onda
Tipodeparticipacin Pster
Congreso
VIIJornadasDoctoralesAndaluzas
Lugaryfecha
Carmona (Sevilla, Espaa), del 28 de Junio al 3 de
decelebracin
Julio(2009)

Autores
JuanGodoyNavajas
Nuevas metodologas en anlisis de alimentos y
Ttulo
ambientalconelusodenanopartculas
Tipodeparticipacin ComunicacinOral
Congreso
I Congreso Cientfico de Investigadores en
Formacin
Lugaryfecha
Crdoba(Espaa),15y16deOctubrede2009
decelebracin

Autores
Juan GodoyNavajas, Mara Paz AguilarCaballos,
AgustinaGmezHens
Longwavelength
homogeneous
Ttulo
fluoroimmunoassay for the veterinary antibiotic
monensinusingdopedsilicananoparticles
Tipodeparticipacin Pster
Congreso
IIEncuentrosobreNanocienciayNanotecnologade
Investigadores y Tecnlogos de la Unversidad de
Crdoba(IINanoUCO)
Lugaryfecha
Crdoba(Espaa),15deEnerode2010
decelebracin

412

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Autores

Juan GodoyNavajas, Mara Paz AguilarCaballos,

AgustinaGmezHens
Determinacin de protenas de soja mediante
fluoroinmunoensayo a larga longitude de onda
utilizando nanopartculas de slice dopadas con
azulnilocomomarcador
ComunicacinOral
XII Reunin del grupo regional andaluz de la
SociedadEspaoladeQumicaAnaltica
Crdoba(Espaa),10y11deJuniode2010

Ttulo

Tipodeparticipacin
Congreso
Lugaryfecha
decelebracin

Autores

Juan GodoyNavajas, Mara Paz AguilarCaballos,

AgustinaGmezHens
Determinacin fluorimtrica a larga longitud de
ondadelaactividadantioxidantedemuestrasde
alimentosconazulnilocomoreactivo
Pster
XII Reunin del grupo regional andaluz de la
SociedadEspaoladeQumicaAnaltica
Crdoba(Espaa),10y11deJuniode2010

Ttulo

Tipodeparticipacin
Congreso
Lugaryfecha
decelebracin

Autores

T.Riuttamki(neRantanen),J.GodoyNavajas,E.
Harju,J.Hlsa,T.Soukka
Resonance energy transfer from upconverting
phosphorsdopedwithdifferentlanthanideions
Pster
International Conference on Luminiscence of
Lanthanides(ICLL)
Odessa(Ukraine),del5al9deSeptiembrede2010

Ttulo
Tipodeparticipacin
Congreso
Lugaryfecha
decelebracin

413

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Autores
Ttulo

Tipodeparticipacin
Congreso
Lugaryfecha
decelebracin

Autores
Ttulo
Tipodeparticipacin
Congreso
Lugaryfecha
decelebracin

Autores
Ttulo
Tipodeparticipacin
Congreso

Lugaryfecha
decelebracin

Juan GodoyNavajas, Mara Paz AguilarCaballos,

AgustinaGmezHens
Useofnilebluedopedsilicananoparticlesaslabels
in heterogeneous immunoassay for antibiotic
determination
Pster
IIIEncuentrosobreNanocienciayNanotecnologade
InvestigadoresAndaluces(IIINanoUCO)
Crdoba(Espaa),10y11defebrerode2011

Juan GodoyNavajas, Mara Paz AguilarCaballos,

AgustinaGmezHens
Frontsurface
longwavelength
fluorescence
immunoassayformonensindetermination
Pster
12th International Conference on Methods and
ApplicationsofFluorescence
Strasbourg (France), del 11 al 14 de Septiembre de
2011

JuanGodoyNavajas
Nuevas metodologas analticas en anlisis de
alimentosyambientalconelusodenanopartculas
ComunicacinOral
I Congreso Cientfico de Investigadores en
Formacin en Agroalimentacin de la eidA3 y II
Congreso Cientfico de Investigadores en Formacin
delaUniversidaddeCrdoba
Crdoba(Espaa),8y9demayode2012

414

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Autores
Ttulo

Tipodeparticipacin
Congreso

Lugaryfecha
decelebracin

Autores
Ttulo

Tipodeparticipacin
Congreso
Lugaryfecha
decelebracin

Juan GodoyNavajas, Mara Paz AguilarCaballos,

AgustinaGmezHens
Utilidad Analtica del uso combinado de
nanopartculasdeTb4O7paraladeterminacinde
antioxidantesenalimentos
Pster
IV Encuentro sobre Nanociencia yNanotecnologa
de Investigadores y Tecnlogos Andaluces (IV
NanoUCO)
Crdoba(Espaa),7y8defebrerode2013

JuanGodoyNavajas,MaraPazAguilarCaballos,
AgustinaGmezHens
Analyticalusefulnessofthecombineduseof
Tb4O7nanoparticlesandlaccaseenzymeforthe
determinationofantioxidantsinfoodsamples
Pster
TrendsinNanotechnology
Sevilla(Espaa),del9al13deseptiembrede2013

415

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