Sensors and Actuators B 69 2000.

153163
www.elsevier.nlrlocatersensorb

Configurations used in the design of screen-printed enzymatic

biosensors. A review
Miquel Albareda-Sirvent, Arben Merkoci, Salvador Alegret )
Grup de Sensors i Biosensors, Departament de Qumica,
Uniersitat Autonoma
de Barcelona, 08193 Bellaterra, Catalonia, Spain

`
Received 7 May 2000; accepted 19 May 2000

Abstract
Different thick-film biosensor configurations designed during the last decade are revised. These planar configurations are classified in
three groups: a. multiple-layer deposition biological deposition by hand or electrochemically., b. screen-printing of composite inks or
pastes using two or more steps biological deposition by screen-printing., c. one-step deposition layer or biocomposite strategy.
Different enzyme immobilisation procedures corresponding to these configurations are also revised. These procedures consist mainly
of enzyme adsorption onto transducer surface, biological immobilisation by cross-linking in a glutaraldehyde layer, and physical, electroor UV-polymerisation entrapment. Immobilisation into the bulk of different carbon-based inks or pastes is also presented. q 2000 Elsevier
Science S.A. All rights reserved.
Keywords: Enzyme immobilisation; Thick-film biosensors; Screen-printed biosensors; Screen-printing ink; Screen-printing paste

1. Introduction
Over the past few years interest has been increasing in
the application of simple, rapid, inexpensive and disposable biosensors in fields such as clinical, environmental or
industrial analysis. The most common disposable biosensors are those produced by thick-film technology.
A thick-film biosensor configuration is normally considered to be one which comprises layers of special inks
or pastes. deposited sequentially onto an insulating support or substrate. One of the key factors which distinguishes a thick-film technique is the method of film deposition, namely screen printing, which is possibly one of the
oldest forms of graphic art reproduction.
Screen printing seems to be one of the most promising
technologies allowing biosensors to be placed large-scale
on the market in the near future because of advantages
such as miniaturisation, versatility and low cost and particularly the possibility of mass production.
The use of thick-film technology for the production of
sensors and biosensors is an emerging field. The most
critical point in manufacturing thick-film biosensors is the
)
Corresponding author. Tel.: q34-93-581-1017; fax: q34-93-5812477.
E-mail address: salegret@gsb.uab.es S. Alegret..

sensing or active membrane and its adhesion to the transducer layer. Technical details on these planar techniques
for sensor and biosensor development have been reviewed
in previous works w1,2x
The aim of the present work is to review various
challenges reported in the literature in the last decade
related with the thick-film biosensors configurations as
well as the different enzyme immobilisation techniques
used. The revised configurations are classified in three
groups see Fig. 1.. The first one includes configurations
based on multiple layer deposition and enzyme immobilisation by hand or electropolymerisation, the second one
presents combined configurations based on composite inks
or pastes with printed enzyme immobilisation, and the
third one is a configuration based on a one-step deposition
layer of biocomposite ink or paste.
Ideally a thick-film biosensor configuration can be developed beginning with a conducting pad T which consists of a carbon ink or paste C., platinum Pt. or other
metal pastes. mixed or not with a mediator or catalyst M.
or any other components applied on a substrate S.. After
that, the free enzyme E. or mixed with an entrapment
agent m. or cross-linker c. glutaraldehyde mainly. g.,
and a stabiliser or additive s., etc. is applied in one or
more layers. In some configurations, the enzyme is mixed
with the cofactor F. and in others an outer selector layer

0925-4005r00r$ - see front matter q 2000 Elsevier Science S.A. All rights reserved.
PII: S 0 9 2 5 - 4 0 0 5 0 0 . 0 0 5 3 6 - 0

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M. Albareda-Sirent et al.r Sensors and Actuators B 69 (2000) 153163

In this review we will refer to this kind of abbreviated

notation that helps to see at a glance the thick-film biosensor immobilisation and fabrication procedures. In Fig. 2
we observe the summary of the abbreviated notation of all
revised configuration devices.
Some technical characteristics and analytical parameters
related to different configurations are given in Tables 1
and 2.

2. Configurations based on multiple-layer deposition

Fig. 1. Typical configurations used in designing screen-printed biosensors. i. Multiple layers by manual deposition, ii. screen printing of
enzymatic inks or pastes using two or more steps, iii. one-step configurations based on biocomposites ink or pastes. In all of these configurations,
layers are applied on a substrate with conducting tracks previously
formed by planar technologies. BI: biocomposite ink printed; c: layer of
material used for immobilisation glutaraldehyde, etc. deposited by manual deposition; E: enzyme layer manual deposition.; EI: enzymatic ink or
paste deposited by screen printing; L: outer protective layer CA, Nafion,
etc.. by manual deposition; S: substrate PVC, ceramic, etc.. with conducting tracks formed previously; T: conductive paste deposited by
screen printing. The mediators, stabilisers and additives, depending on the
device, can be included in any ink or paste.

L. like cellulose acetate CA. or Nafion, etc. is applied.

This layer is used to avoid interference, improve linear
range, etc. When the above mentioned layers are deposited
separately, a multiple-layer thick-film biosensor is obtained. A possible example of a multiple layer configuration can be abbreviated in the form: S-1TM-2Ecs-3L. In
this notation the individual fabrication steps or layers. are
numbered in Arabic numerals 1, 2, 3 . . . . and for each
step a capital letter refers to the principal components
enzyme, mediator, transducer, selector layer, etc.. and
with small letter any specific components cross-linker,
immobilisation matrix, stabiliser etc.. All components applied as a unique phase to form a layer are delimited by
hyphens e.g. -1TM-.. Explicit information between brackets can be added after a letter designating a component
i.e. cg., although frequently this is simplified to g.., so
the previous abbreviated notation can also be written as:
S-1TM-2Ecg.s-3L. This means that on the substrate S the
first layer 1TM. of the transducer mixed with mediator is
deposited and then a second layer is deposited over it
2Ecg.s. in which the enzyme E. is mixed with the
glutaraldehyde cross-linker cg. and a stabiliser or additive
s. Finally, a selector layer 3L. is applied. All designs have
previously incorporated conductive trucks, which are not
included on the notation configuration, except those used
as transducer materials.

All these configurations are based on sequential layer

deposition onto previously formed conducting tracks. The
multiple-layer configurations reported can be classified on
the base of biological immobilisation techniques adsorption, cross-linking, entrapment, and electropolymerisation.
which are carried out manually or electrochemically but
are not printed.
2.1. Enzyme immobilisation by adsorption.
Adsorption is used as a rapid and simple procedure,
especially for single-use biosensors. Physical adsorption
based on van der Waals attraction forces between enzyme
and a solid support surface mainly graphite. is the most
used method due to it simplicity. For this reason, the
method is used as a preliminary test before undertaking
further improved or complex. design. However, the most
important drawback is that bonding forces between enzyme and the support cannot be easily controlled. These
are weak forces and as a consequence, depending on
experimental conditions such as pH, ionic strength, temperature and solvent, the enzyme can be leached during the
assay.
The section describes different configurations see Fig.
2., which can be classified, in the following classes: I
S-1TM-2E., IIX S-1TM-2EF-3L. and IIIX S-1T-2EM-3L..
The prime symbol X . means a configuration with a third
layer added -3L.. This will simplify device classification
because there are identical configurations whose only difference is an added layer.
2.1.1. (I) S-1TM-2E
Cagnini et al. w3x and Palchetti et al. w4x used a S1TC.MRu.-2EChE. configuration to immobilise choline
oxidase onto a screen-printed electrode graphite-based ink
manually mixed with ruthenised activated carbon and applied over a polyester flexible film.. This biosensor was
used for monitoring pesticides.
2.1.2. (II X ) S-1TM-2EF-3L
A reagentless disposable biosensor for lactic acid based
on IIX class was developed by Sprules et al. w5x: S1TC.MMB.-2ELDH.FNAD.-3 L. The screen-printing
inks for the first layer were carbon-based and contained

M. Albareda-Sirent et al.r Sensors and Actuators B 69 (2000) 153163

Fig. 2. Different configuration classes employed in designing screen-printed biosensors. c: cross-linker; E: enzyme; F: cofactor; L: outer protective layer; m: polymer or gel matrix; M: mediator or catalyst; p:
electropolymer; p M : electropolymer acting as a mediator; S: substrate; s: stabilisers; T: transducer.

155

156

Table 1
Some details of most representative devices designed by manual or electro deposition of biological material multiple layer configuration.
Biological
moleculercofactor

Modifierr
applied potential
vs. AgrAgCl.

Immobilization methodr
material usedrmembrane

Printing materialr
components

Analyterdetection limit

Polyester
Polyester
PVC
PVC
PVC
Ceramic
support
Alumina
ceramic
PVC

COD
CODrAChE
LDHrNADq
LDHrNADq
ADH
AChE,
BChE
AChE,
BChE
AChE

Rur0.7 V SCE.
Rur0.7 V SCE.
MBry005 V
MBr0 V
MBr0 V
CoPCr0.350 V

Ad by epolr polyphenolr
Ad by epolrpolyphenolr
Ad by enrrCA
Ad by enrrWhatman Tissue
Ad by enrrCA
CLrglutaraldehyde

Co PCr0.4 V

CLrglutaraldehyde, BSA

CIrRu
CIrRu
CIrMB, C
CIrMB, C
CIrMB, C
Paste PtrCoPC,
AC.
PasterG, AC

0V

CLrglutaraldehyde

CI CoPC.

PVC

BChE

TCNQr0.1 V

CLrglutaraldehyde

PVC

AChE

CLrglutaraldehyde

Alumina
ceramics
Alumina

1. GOD
2. PuOD
1. GOD or AO

CoPCr0.1 V
SCE.
r0.520.6 V
r0.140.22 V
ry0.4 V

CI TCNQ, G,
HEC, To, EG.
CI CoPC.

Green tape
ceramics
Alumina
ceramics

2. GDH, ADHr
NADH
1. GOD
2. GOD, b-G
3. GOD, INV, MUT
GOD

Alumina
substrate
Au
Au

LDH, MDHr
NADH
GDHrNADH

CLrglutaraldehyde onto a
silaneted Pt
1. CLrglutaraldehyderCA

1. Paste PtrAg.

2. Adrr

2. Paste Ag.

1. 0.6 V Pt.,
0.35 V Pt-G.

-CLrglutaraldehyde onto
silaneted Ptr

Paste Pt.

r0.7 V

Paste RuO 2 .

r0.6 V SCE.

CLrglutaraldehyde, BSAr
PVB
EnrPVAPEr

Pesticidesr10y9 M
Pesticidesr2 mg ly1
Lactic acidr10y3 M
Lactic acidr10y3 M
Ethanolr10y3 M
Carbofuran or propoxurr
10 mgrl
Pesticides or heavy metalsr
- 0.3 nM
Dichlorvos or paraoxonr
6.0=10y9 or 7.0=10y9 M FIA.
PMSF or DIFPr
- 0.06
Paraoxan or dichlorvosr
6.5=10y7 or 1.7=10y6 M
1. Glucoser1 mM
2. Aminer0.06 mmolrl
Glucoser -10y3 mmolrl,
ethanolr - 0.05 mmolrl
Glucoser - 3=10y4 mmolrl,
ethanolr0.005 mmolrl
1. Glucoser - 0.001 mM
2. Lactoser - 0.5 mM
3. Sucroser
GlucosarFIA.

r0.2 V SCE.

-En by epolrMBrPVAc

Au thick film

Lactater2.8=10y5 M FIA.,
malater2.4=10y4 M FIA.
Glucoser1 mM FIA.

Pt conductor paste

Lifetime

37 weeks

340 days

) 2 weeks

Ref.

Configuration

w3x
w4x
w5x
w6x
w7x
w10x

S-1TM-2E
S-1TM-2E
S-1TM-2E-3L
S-1TM-2E-3L
S-1TM-2E-3L
S-1T-2M 3Ecs

w11x

S-1TM-2Ecs-3L

w12x

S-1TM-2Ecs

w13x

S-1TM-2Ec-3L

w14x

S-1TM-2E-3cs

w17x

S-1T-2Ecs

w18x

S-1T-2Ecs-3L
S-1T-2EF

1. 1 month
3. 14 days
Several
weeks
2 months

w19x

S-1T-2Ecs-3L

w20x

S-1T-2E-3L

w25x

S-1T-2Em

w27x

S-1TM-2Ep

Biological molecule: GOD, glucose oxidase; AChE, acetylcholinesterase; LDH, lactate dehydrogenase; COD, choline oxidase; ADH, alcohol dehydrogenase; INV, invertase; AAO, ascorbic acid oxidase;
MDH, malate dehydrogenase; GDH, glucose dehydrogenase; AO, alcohol oxidase; PuOD, putrescine oxidase; b-B, b-galactosidase; Inv, invertase; MUT, mutarotase; URE, urease.
Cofactor: NADH, nicotinamide adenine dinucleotide.
Mediatorrcatalyst: CoPC, cobaltphtalocyanine, TCNQ, tetracyanoquinodimethane; MB, methylene blue; FE, ferrocene, Ru, ruthenium.
Analyte: OP, organophosphorates; PMSF, phenylmethylsulphonylfluoride; DIFP, diisopropylfluorophosphate.
Immobilization method: Ad, adsorption; En, entrapment; Epol, electroplymerisation; cL, cross-linking.
Material components: Gl, glutaraldehyde; BSA, bovine serum albumin; CI, carbon ink; G, graphite; AC, acetone; TO, toluene; CH, cyclohexanone; CA, cellulose acetate; HEC, hydroxyethyl cellulose; PVB,
polyvinylbutyral; PVP, polyvinylpyrrolidine; PVAc, polyvinylacetate; PE, polyethylene; PU, polyurethane; PIB, polyisobutylene; UV paste, UV polymerizable paste; EG, ethylene glycol.

M. Albareda-Sirent et al.r Sensors and Actuators B 69 (2000) 153163

Substrate

Table 2
Some details of most representative devices designed by screen-printing of biocomposite pastes or biological inks
Biological
moleculercofactor

Modifierr
applied potential
vs. AgrAgCl.

Immobilization
methodrmembrane

Printing material

Analyterdetection limit

Lifetime

Ref.

Configuration

Polyester
PVC

LDHrNADq
AChE or BChE

ry0.35 V
CoPCr0.1 V

mM
Paraoxon or Dichlorvosr10y8 M

40 days

w29x
w30x

S-1T-2EFM
S-1TM-2Ecs-3L

AChE

CoPCr0.1 V

Pesticider

1 year

w31x

S-1TM-2Ecs-3L

Alumina
substrate
Polyamide
Alumina
ceramic

LDH

r0.35 V AgrPd.

70 h

w35x

S-1TEMs

GOD
Tyrosinase

FEr0.450.7 V
ry0.2V

Biocomposite or ink
Biocomposite or ink

LDH, NAD, PVP, silicone

AChE, BChE, HEC, BSA,
glutaraldehyde, graphite
AChE, HEC, PEI, lactitol,
glutaraldehyde.
DEAE, SiO 2 , graphite,
lactitol, UV paste
GOD-ferrocene, PVP, PVB
Tyrosinase, carbon ink

L-Lactater -9.1

PVC

Biocomposite or ink
Biocomposite or inkr
PU
Biocomposite or inkr
PU
Entrapment

30 days

w36x
w37x

S-1TEMs
S-1TEMs

PVC

GOD

CoPCr0.3 V

Biocomposite or ink

w38x

S-1TEMs

Fired
alumina
ceramics
Fired
Alumina
ceramics
Glass fiber

GOD

TTFr0.22 V

Biocomposite or ink

GOD

TTFr0.22 V

Biocomposite or inkr
PC

GOD

r1.150 V

Biocomposite or ink

AOT, TMOS, graphite,

ferrocene, GOD, CoPC, DPT
TTF, graphite, toluene,
GOD, PVP

y7

L-Lactater2=10

M FIA.

Glucosar2 mgrml
Pesticidesrfrom 2 to 9 mM e.g.
diethyldithiocarbamate 2 mM; 2,4dinitro-o-cresol 9 mM. FIA
Glucosar0.4 mM
Glucosar -1 mM

20 days

w17x

S-1TEMs

TTF or Pt, HEC, graphite,

GOD, PVP, EG

Glucosar - 0.01 mM Pt-G.;

-1 mM TTF-G.

)6 months

w19x

S-1TEMs

GOD, graphite, epotek, CH

Glucosar - 0.05 mM

60 days

w41x

S-1TEMs

Biological molecule: GOD, glucose oxidase; AChE, acetylcholinesterase; LDH, lactate dehydrogenase.
Cofactor: NAD, nicotinamide adenine dinucleotide.
Mediatorrcatalyst: CoPC, cobaltphtalocyanine; FE, ferrocene; TTF, tetrathiafulvalene.
Material components: CH, cyclohexanone; HEC, hydroxyethyl cellulose; UV paste, UV polymerizable paste; PVP, polyvinylpyrrolidine; EG, ethylene glycol; PEI, polyethyleneimine; DEAE,
diethalaminoethyl; PVB, polyvinylbutyral.

M. Albareda-Sirent et al.r Sensors and Actuators B 69 (2000) 153163

Substrate

157

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M. Albareda-Sirent et al.r Sensors and Actuators B 69 (2000) 153163

Meldola Blue MB. 2% wrw.. Lactate dehydrogenase

LDH. and cofactor NADq F. were dissolved in a buffer
and the solution was dropped onto the working area of the
electrodes. After drying at room temperature a cellulose
acetate solution was used to cover the working area of the
electrode. Using the same configuration, the same authors
developed another disposable screen-printed sensor strip
for the measurement of NADH and lactate w6x as well as
for alcohol in beverages w7x.
2.1.3. (III X ) S-1T-2EM-3L
Enzyme adsorption and casting an outer membrane to
protect the biocatalyst was applied by Zhang et al. w8x. This
system was used for simultaneous determination of maltose and glucose. The base electrodes were produced by
screen-printing a planar two-electrode design. The conducting tracks consisted of silver ink. A graphite pad was
deposited onto one of conducting tracks. A water solution
of enzymes glucose oxidase GOD. and amyloglucosidase
AG. and 1,1X-ferrocenedimethanol ferr. was deposited
onto the working area of the electrode. An outer membrane
3L., prepared by mixing hydroxyethyl cellulose HEC.,
Triton X100 and polyethylene glycol in distilled water,
was printed. The notation of this design is S-1TC.2EGOD, AG.Mferr.-3LHEC..
2.2. Enzyme immobilisation by cross-linking in a glutaraldehyde layer
It is possible to immobilise enzyme using intra- or
intermolecular cross-linking of enzyme molecules. This
method is based on the formation of three-dimensional
linkings between the biological material and bi- or multifunctional reagents. The resulting modified biological material is completely insoluble in water and can be adsorbed
onto a solid surface. Glutaraldehyde is one of the cross-linking reagents most used amongst other multifunctional
reagents w9x.
Configurations based on multiple -layer deposition using glutaraldehyde as a cross-linking agent for enzyme
immobilisation is one of the most reported methods for
enzyme immobilisation in planar biosensor devices. The
resulting 3D network affects the enzymatic kinetic and
diffusion characteristics. It should be optimised for each
enzyme trying to enhance its immobilisation and at the
same time to preserve its activity as well as the appropriate
diffusion characteristics.
In this type of immobilisation most authors use similar
configurations to those mentioned in the previous Section
2.1, but using a cross-linker and changing some chemical
components. Besides class IV S-1T-2TM-3Ecs., class V
S-1TM-2Ecs., class VI S-1TM-2E-3cs., class VII S-1T2Ecs. and class VIII S-1T-2EM-3s-4c., we find a small
variation wVX . S-1TM-2Ecs-3L and VIIX . S-1T-2Ec-3Lx
of the previous notations, being V and VII configuration
with a third protective layer 3L..

2.2.1. (IV) S-1T-2TM-3Ecs

For producing amperometric biosensors for pesticides
based on class IV configuration Skladal et al. w10,11x
prepared screen-printed electrode on a ceramic support,
using silver- and platinum-based pastes for reference and
working electrodes. The working electrode was modified
by depositing a graphite-based composite layer C. containing cobaltphtalocyanine CoPC. as a modifier and
acetylcellulose AC. as a binder. A mixture of choline
esterase ChE. solution, bovine serum albumin BSA.,
phosphate buffer and glutaraldehyde g. was applied to the
surface of the graphite composite electrode resulting in a
S-1TPt.-2TC.MCoPC.mAC.-3EChE.cg.sBSA. configuration.
2.2.2. (V) S-1TM-2Ecs
Screen-printed biosensors for use as detectors in a
flow-injection system for monitoring organophosphate pesticides were designed in this configuration w12x. Differently
from the previous configuration, a mediator CoPC was
mixed with the transducer in the first layer 1TM., followed by a unique second layer that included AChE,
glutaraldehyde and stabiliser 2 Egs.. The resulting profile
was SPVC.-1TC.MCoPC.-2EChE.cg.s.
2.2.3. (V X ) S-1TM-2Ecs-3L
For producing amperometric biosensors based on
cholinesterase, Kulys and DCosta w13x used a printing ink
prepared with tetracyanoquinodimethane TCNQ. dissolved in toluene and then mixed with graphite powder.
This conducting ink was applied over a PVC substrate.
The biocatalytic layer was then applied by covering each
modified electrode pad with a buffered cholinesterase solution containing glutaraldehyde. An outer protective layer is
applied, giving the final class VX configuration: SPVC.1TC.MTCNQ.-2EChE.cg.s-3L.
2.2.4. (VI) S-1TM-2E-3cs
A similar configuration was used by Hartley and Hart
w14x for amperometric measurement of organophosphate
pesticides previous to Rippeth et al. w12x. A gluteraldehyde
solution was dropped and spread over the AChE acetylcholinesterase. buffered solution layer previously deposited onto a SPCE screen-printed carbon electrode..
The configuration of a multiple-layer screen-printed
biosensor was the V class configuration, but the enzyme
and gluteraldehyde were added in different steps. A decrease of layer numbers can be observed in further works
w12x.
2.2.5. (VII) S-1T-2Ecs
Carsol et al. w15x developed a three-electrode configuration for uric acid and hypoxanthine detection for fish
freshness determination. AgrAgCl and silver tracks were
used as reference and counter electrodes respectively. The
working electrode was a carbon pad positioned over part
of the silver track. Before enzyme immobilisation, the

M. Albareda-Sirent et al.r Sensors and Actuators B 69 (2000) 153163

electrode was poised at 1.7 V vs. AgrAgCl for 20 s in

order to oxidise the carbon surface. This treatment was
found to be very important for the reproducibility of
biosensor behaviour. Then a solution was prepared by
mixing a phosphate buffer containing xanthine oxidase
XO., BSA and glutaraldehyde, and placed onto the carbon
surface. The configuration for this device is: S-1TC.2EXO.cg.sBSA.. The same authors w16x using the same
configuration immobilised enzyme on controlled pore glass
beads activated by glutaraldehyde.
Bilitewski et al. w17x use a similar approach in designing
biosensors to detect glucose in fruit juice and wine and for
determining the freshness of fish. They mixed GOD and
gluteraldehyde in phosphate buffer and applied it over the
working electrode previously printed with platinum paste,
giving a simple configuration class VII, S-1TPt.2EGOD.cg. without a mediator. In addition, they modified some platinum electrodes with 3-aminopropyltriethoxysilane.
2.2.6. (VII X ) S-1T-2Ec-3L
Glutaraldehyde immobilization on a transducer pad was
also chosen by Ruger
et al. w18x in the design of glucose

and ethanol biosensors using commercial thick-film electrodes. To avoid interference from oxidisable substrates
such as ascorbic acid, the biosensor was covered with a
membrane by dipping in a solution of cellulose acetate
CA. with acetone and cyclohexanone. A three-layer configuration sensor, S-1TPt.-2EGOD or AO.cg.-3LCA.
class VIIX was the result.
On a layer of enzyme immobilised by glutaraldehyde
on a platinum electrode previously modified with 3aminopropyltriethoxysilane, APTS., the same authors w19x
studied the effect of additional polycarbonate membranes
with different thickness, pore size and pore densities. It is
used for the determination of carbohydrates in food glucose, lactose and sucrose detection.. These sensors are
based on GOD, co-immobilized b-galactosidase and GOD,
and invertase, mutarotase and GOD, respectively.
The increase of the linear range when a membrane is
added can be observed from the reported works of
Bilitewski et al. w17,19x and Ruger
et al. w18x, but at the

same time we can notice the decrease of sensitivity. The

same authors show a higher reproducibility in cross-linked
GOD biosensors vs. the developed devices using biocomposite configuration see Section 4..
In the same way, Deman et al. w20x produced a glucose
sensor with a commercially available RuO 2 paste They
mixed the GOD enzyme, BSA and glutaraldehyde and then
dispensed it on the silanised electrode surface. After 24 h
the enzyme membrane is cross-linked and dip-coated with
a polyvinylbutyral membrane, resulting in a S-1TRuO 2 .2EGOD.cg.sBSA.-3L VIIX configuration.
2.2.7. (VIII) S-1T-2EM-3m-4c
Khan w21x designed a biosensor on a ceramic chip. The
chip surface was coated with an insulating glass layer

159

except for the Pt-coated working part of the chip. A paste

of GOD enzyme was previously adsorbed onto TTF tetrathiafulvalene. TCNQ tetracyanoquinodimethane. crystals. was deposited manually. Then a gelatin matrix wmgel.x
was spread over the sensor, and finally the dried gelatin-cast
electrode was dipped into a glutaraldehyde solution, giving
a final S-1TPt.-2EGOD.MTTF-TCNQ.-3mgel.-4cg.
configuration.
2.3. Enzyme immobilisation by entrapment
Entrapment within an adequate matrix m. can improve
enzyme stability. Moreover, immobilisation in matrices
like gels, polymers, pastes or inks can be as simple as
physical adsorption. Generally, the biological material is
mixed and well homogenised with the supporting material
and then applied over the electrode pad as an additional
membrane that has to be dried or polymerised.
Some of the matrices used are gelatines, polyurethanes,
polyvinyl alcohol w22x, carbon paste w23x or carbon ink
w24x. The principal advantage of this technique is its compatibility with mass fabrication techniques.
An S-1T-2Em class IX. configuration has been developed using enzyme immobilisation by entrapment.
2.3.1. (IX) S-1T-2Em
Silber et al. w25x manufactured biosensors for immobilising either enzymes lactate dehydrogenase LDH. or
malate dehydrogenase MDH. by entrapment in a polymer
matrix m. based on polyvinylacetatepolyethylene
copolymer dispersion PVPPE., giving a class IX configuration S-1TAu.-2ELDH or MDH.mPVAPE.. The
cofactor NAD is added in solution. The biosensors designed were used for determination of lactate and malate.
2.4. Enzyme immobilisation by electropolymerisation
Electropolymerisation is an efficient enzyme immobilisation method used in biosensor development. Conducting
polymers such as, polythiophene, polyaniline, polyindole
polypyrrole w26x, can be grown electrochemically on an
electrode surface, and it could be demonstrated that electrochemical polymerisation is an easy and attractive approach for the immobilisation of enzymes at electrode
surface. However, conducting polymers have additionally
been used in amperometric enzyme electrodes with the
intention of coupling the electron-transfer reaction between
enzyme and electrode via the ramified conducting network
of the polymer. Some of these electropolymers can also
serve as mediator, decreasing the work potential and
thereby avoiding interference from other species.
The polymer film is in general formed potentiostatically
in the presence of the enzyme at constant potentials between 650 and 800 mV vs. SCE. Often, the thickness of
the growing polymer film is controlled by measuring the

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M. Albareda-Sirent et al.r Sensors and Actuators B 69 (2000) 153163

charge transferred during the electrochemical polymerisation process. However, most authors have their individual
way of electrode surface pretreatment, choose specific
concentrations for the enzyme and the monomer, and use
different electrolyte salts and hence counter-anions incorporated in the growing polymer film.
The main advantages of entrapment of enzymes into
conducting polymer films during the electrochemical polymerisation process are the extremely simple one-step procedure and the control of the spatial distribution of the
immobilised enzyme.
However, poor adherence resulted in some electropolymer based membranes and incompatibilities with some
enzyme immobilisation methods have appeared, presenting
difficulties for it applications.
Although electropolymerisation is not a manual process,
biosensors based on this technique have been classified in
this section. This is due to the fact that this technique is
different from the screen-printed one and is related to an
entrapment polymer layer.
Usual configurations for devices based on electropolymers p. are: X. S-1T-2Ep M p M means an electropolymer acting as a mediator. and XI. S-1TM-2Ep.
2.4.1. (X) S-1T-2EpM
Silber and Hammp w27x immobilised the enzyme glucose dehydrogenase GDH. by electropolymerisation of
methylene blue MB. in the presence of GDH in a buffered
solution. MB acts at the same time as electropolymer
matrix and mediator p M .. The thick-film biosensors produced, S-1TAu.-2EGDH.p M MB., were used for
electro-catalytic oxidation of NADH in solution during
glucose determination.
2.4.2. (XI) S-1TM-2Ep
Wang et al. w28x used this kind of configuration for
screen-printed amperometric biosensors for glucose and
alcohol based on ruthenium-dispersed carbon inks. Firstly,
a ruthenium-containing carbon ink was printed on a rectangular alumina ceramics substrate. Then GODrpolyphenol
films pph. were grown over it electrochemically from a
solution containing phenol and GOD in phosphate buffer,
resulting in the configuration S-1TC.MRu.-2EGOD.ppph. with two applied layers yielded.
Cagnini et al. w3x used a similar procedure for immobilising AChE.

3. Screen printing of enzyme-containing inks or pastes

using two or more steps
This technology consists of printing enzyme-containing
inks over previous screen-printed electrodes; therefore the
main difference with the previous techniques is that the
biological material deposition is not manual but printed.

Four typical configurations have been reported: XII. S1T-2EFm, XIIIX . S-1TM-2Ecs-3L, XIVX . S-1T-2M-3E4L and XV. S-1T-2M-3E-4s-5c.
The abbreviated configuration presented as S-1T-2M3E-4s-5c, for example, means that a first layer of conducting paste e.g. graphite carbon 1TC.. is applied and then
the process is ended by a successive screen-printed layers
with mediator M., enzyme E., stabilisers s. and crosslinker c.. In other configurations we can observe other
components like polymer or binder m., cofactor F. and
selector layer L..
Some technical characteristics and analytical parameters
related to the usual device configuration are given in Table
2.
3.1. (XII) S-1T-2TEFm
Yoon and Kim w29x fabricated thick-film electrodes on a
polyester sheet using a conventional screen-printing process. Three silver conducting tracks were first applied to a
polyester sheet and then graphite ink was printed on the
end of two conducting tracks for the counter and the
working electrodes. The nonmodified part of the conducting tracks was then covered by UV-polymerisation insulation ink. Finally, ink containing activated carbon, enzyme
LDH, cofactor NADq, PVP and silicone oil was printed on
the working electrode pad. The resulting configuration was
S-1TC.-2TC.ELDH.FNADH. mPVP..
3.2. (XIII X ) S-1TM-2Ecs-3L
S-1TM-2Ecs-3L configuration has also been developed,
where the mediator is mixed with graphite and a protective
layer is added. All the layers are printed except for the last
one 3L.. Using this configuration, Hart et al. w30x screenprinted electrodes onto clear PVC. The conducting track
consisted of a graphite ink. A graphite ink pad was printed
over the nonterminal end of the track. Over this, a further
graphite pad consisting of graphite ink containing cobalt
phthalocyanine CoPC. was then printed. An insulation
shroud of vinyl ink was then screen-printed to the electrodes, apart from the terminals and the pads. The enzyme
ink was prepared by mixing in a proper buffer acetyl
cholinesterase, hydroxyethyl cellulose HEC., bovine
serum albumin BSA., glutaraldehyde g. and graphite
powder to improve the viscosity. A thin outer membrane
of polyurethane was then applied to prevent the enzyme
layer from being washed off in the stirred aqueous sample.
The prepared screen-printed biosensors S-1TC.MCoPC.2EAChE.cg.sBSA.mHEC.-3L were used to determine
pesticides through inhibition of cholinesterase.
Recently, Hart and Collier w31x designed thick-film
biosensors by printing enzyme acetyl cholinesterase in inks
similar to those used in a previously commented work w30x,
but adding a copolymer of vinyl pyrrolidone and dimethylaminoethyl methacrylate, and polyethyleneimine. The

M. Albareda-Sirent et al.r Sensors and Actuators B 69 (2000) 153163

long-term storage stability of electrodes was highest when

the enzyme matrix consisted of copolymer.
3.3. (XIV X ) S-1T-2M-3E-4L
In previous works w32,33x using the same technology,
Hart and Collier w31x developed an amperometric enzyme
electrode for lactate. In this case the mediator was not
CoPC but an electrocatalyst Pt electrodeposited separately
from a solution of hexachloroplatinic acid., and the enzyme lactate oxidase. is printed without BSA. Therefore,
if we compare these devices with those most recently
developed w31x fewer procedure steps can be observed: the
mediator is printed within the transducer and not separately, and BSA is added to increase enzyme stabilisation.
Finally a third layer of cellulose acetate AC. or nafion N.
is deposited by dip-coating or by ink-jet printing. The final
configuration was: S-1TC.-2MPt.-3ELDH.-4LAC or
N..
3.4. (XV) S-1T-2M-3E-4s-5c
In contrast to the above, Newman and Turner. w34x have
prepared a glucose sensor using a hybrid technology. First,
they used a screen-printing technology to draw the tracks,
and then ink-jet printing for the enzyme deposition. This
ink is based on TTF dissolved in ethanol. Tetrabutylammonium perchlorate was added to increase the conductivity of
the mediator solution. GOD was dissolved in phosphate
buffer pH 7. and deposited using the same print pattern. A
layer of BSA followed by a layer of glutaraldehyde were
deposited on some sensors to aid the retention of the
enzyme. This technique implies more steps, consequently
the final configurations results as follows: S-1TAg, C.2MTTF, TBA.-3EGOD.-4sBSA.-5cg..

4. Configurations based on one-step deposition layer.

Biocomposite strategy
In this configuration, enzymes and other materials such
as graphite, mediators, catalysts, stabilisers, polymers etc.
are mixed together forming the so-called biocomposite ink
or paste which is screen-printed onto the substrate producing biosensors in a single, one-step procedure.
Of the existing techniques, it is this one that offers
greater rapidity and simplicity in the manufacturing process. Nevertheless, it is also the technique that requires the
most complex optimisation process, given that there is the
need to bear in mind the materials of varying nature and
characteristics., maintaining their initial properties intact.
This configuration would be denoted as S-1TEMs and
represents the most simple configuration. The main device
configurations are summarised in Table 2. If an additional
conducting layer is applied onto these conducting tracks

161

we consider that more than one step has been added, and
this will be included in Section 3.
Khan w21x designed three printed electrodes on a ceramic chip based on this configuration. The chip surface
was coated with an insulating glass layer except for the Pt
coated working part of the chip. A printable pasterink of
GOD previously enzyme was adsorbed on to TTFTCNQ
crystals. was prepared by thorough mixing with a polyester
binder poly isobutylene PIB. and N-butyl carbital acetate
NBC.. in adequate solvent heptane. and printed over the
working electrode.
Rohm et al. w35x developed a UV-polymerizable enzyme paste to be used in screen-printed L-lactate sensors
based on this configuration. Firstly, thick-film transducers
printed with platinum conducting paste on fired aluminium oxide ceramics. were produced. After that a UVpolymerizable and water-soluble paste p UV . was mixed
with a proper buffer, different amounts of a porous SiO 2
material, graphite andror solutions of diethanolaminoethyl
DEAE.-dextran and lactitol in potassium phosphate buffer
were added, and the resulting enzyme paste was screen
printed on the surface of a platinum working electrode of
each electrode system. Polymerisation was performed by
UV irradiation.
The application of this kind of configuration by Nagata
et al. w36x in the fabrication of a glucose sensor by the
screen printing technique was assayed in three types of
ferrocene-modified GOD. The ink used consisted of only
four components: modified GOD, two kinds of polymer
resin as binder, and organic solvent. Polyvinylpyrrolidine
PVP. and polyvinylbutyral PVB. were used. Therefore a
mixture of water-soluble and nonsoluble polymers was
used to provide a proper environment for ferrocene-GOD.
The polymers were dissolved in n-pentanol for printing.
The ink was printed onto the polymide film previous
patterned with thin cooper foil and dried immediately.
Wang et al. w37x modified inks by thoroughly mixing a
commercial carbon ink with the enzyme tyrosinase. Typical preparation was 0.6% enzymerink wrw.. The prepared amperometric biosensors were used to test various
pesticides and herbicides through the decrease in the substrate catechol. steady-state current, caused by the inhibition of enzyme.
Solgel graphite composites were also prepared for
developing screen-printable biosensors for surfactants w38x.
The solgel graphite mixture was prepared by mixing
sulfosuccinate AOT., tetramethylorthosilicate TMOS.
and water, with graphite powder mixed previously with
ferrocene, GOD, buffer solution and diphenylthiocarbazone DPT.. The paste prepared was printed onto the
pretreated PVC substrate.
Bilitewski et al. w17,19x prepared enzyme ink by mixing
graphite powder with saturated solution of TTF in toluene
until the latter was evaporated. Meanwhile they dissolved
GOD in polyvinylpyrrolidine solution and then added the
TTF-coated graphite powder. The same developed biosen-

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M. Albareda-Sirent et al.r Sensors and Actuators B 69 (2000) 153163

sors have been tested by changing TTF-graphite by Pt-graphite. After thorough mixing, the ink prepared was applied
to the working electrodes by screen-printing. The devices
prepared were applied to carbohydrate determination.
Not only can enzymatic pastes be produced, we have
also encountered inmunosensors made by screen printing.
An example is a solgel derived thick-film amperometric
immunosensor by Wang and Pamidi w39x. The solgel
solution was prepared by mixing tetraethoxysilane TEOS.,
ethanol, water and HCl. Hydroxyproyl cellulose and RIgG
rabbit IgG. were added to the solgel solution and mixed
thoroughly. Subsequently, a mixture of the above solgel
solution was mixed with graphite and screen-printed onto
ceramic substrates.
A screen-printed glucose biosensor was reported recently by Wang and Zhang w40x. An ink with enzyme GOD
as well as mediator cupric hexacyanoferrate. was printed.
Finally, Galan-Vidal
et al. w41x designed a glucose

biosensor based on a graphiteepoxy screen-printable biocomposite. The GOD graphiteepoxy pastes were prepared
by dispersing GOD and graphite powder in an epoxy resin.
The composite viscosity was adjusted with cyclohexanone.
The paste prepared was printed onto a glass fibre circuit
board patterned with copper tracks by a conventional
photolithography process. The same procedure was implemented in a three-electrode configuration w42x.
From observations of different configurations that used
biocomposite ink or paste strategies, it can be concluded
that the technology used was approximately the same, the
only difference being the reagents employed.

5. Final remarks
The major advantages of the thick-film process are the
versatility in material preparation and the simplicity in film
formation, which generally results in lowering manufacturing cost. Research in the field of thick-film biosensors is
continually improving and its evolution is focused on ways
of producing biosensors strips for small portable devices
by allowing the monitoring of different species of interest
such as glucose and other clinical parameters or pollutants
pesticides etc..
The limiting step for commercialisation is mainly due to
the immobilisation procedure of the biological molecule,
mediators and other additives that are not suitable, in most
cases, to mass production, low manufacturing costs or
reproducibility.
The developed planar biosensors described in this review can be used in a wide range of analytical procedures
from Adrop on assayB, which can be useful for simple field
analysis, to automated systems such as flow injection
analysis.
The modification of commercially available or homemade inks for electrode printing, and the wide range of
possibilities with respect to the substrate material, allow

the development of low-cost and high-performance biosensors, which can be applied large-scale in any field.
Currently there is a tendency towards Aone step depositionB configuration due to its simplicity and applicability
in mass biosensor production. The use of biocomposite
inks and the absence of outer coatings greatly simplify the
fabrication of screen-printed biosensors Nevertheless multiple-layer technology is still used with the aim of improving the analytical characteristics of the biosensor devices.
Some authors began firstly with a simple configuration and
then continued their work by adding additional membranes, which means increasing fabrication steps. Both
trends make the tendency of two important issues clear:
simple configuration and good analytical performance.
Maybe at the present state of knowledge, these two factors
seem to be practically contradictory, and as a result, some
authors are directing their research towards one-step configuration, with others moving towards more-than-one-step,
in the search for higher analytical performance. In addition, we can observe better results in manual enzymatic
immobilisation biosensors than in devices with a screenprinted biological layer. At the present stage of this technology, manual cross-linking immobilisation using glutaraldehyde is the technique most used, and shows the best
results.
Whatever the case may be, the development of new
pastes and inks that incorporate as much bioelectrocomponents and as few steps as possible is clearly the most
adequate strategy in the design of new screen-printed
biosensor devices. The so-called biological inks, or
biofilms, compatible with automatic process are a new and
interesting field of research, directly related with the improvement of biosensor thick-film technology.
Acknowledgements
A. Merkoci and M. Albareda-Sirvent acknowledge
grants from Direccio General de Recerca Generalitat de
Catalunya., Barcelona DOGC no 3050, 5r1r00. and
Comision Interministerial de Ciencia y Tecnologia
CICYT., Madrid PN 9634750905., respectively. The authors also acknowledge CICYT for its financial support.
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Biographies
Miquel Albareda-Sirent graduated in 1996 from the Autonomous University of Barcelona. He is a PhD student at the Sensor and Biosensor
Group of the Department of Chemistry of this university and is currently
developing analytical instrumentation such as enzyme-based electrochemical biosensors.
Arben Merkoci graduated in 1986 from the University of Tirana, Albania,
and obtained his PhD in chemistry from the same university in 1991.
Since 1997 he has been working as invited professor at the Sensor and
Biosensor Group of Autonomous University of Barcelona. His main
research interests concern the design of composite and biocomposite
materials for chemo- and biosensors using amperometric, potentiometric
and stripping techniques.
Salador Alegret became professor of analytical chemistry at the Autonomous University of Barcelona in 1991. He is the head of the Sensor
and Biosensor Group of the Department of Chemistry of the UAB. He is
currently devoted to the development of electrochemical chemo- and
biosensors based on amperometric, potentiometric and ISFET transducers
in chemical, enzymatic, immunological and DNA systems. The resulting
sensor devices have been applied in automated analytical systems based
on bioinstrumentations and biomimetic instrumentation concepts for monitoring and process control in different fields, such as biomedicine,
environment and chemical industry.