Silicon (2010) 2:33–39
DOI 10.1007/s12633-010-9038-7
ORIGINAL PAPER
Synthesis of Enzyme and Quantum Dot in Silica
by Combining Continuous Flow and Bioinspired Routes
Siddharth V. Patwardhan & Carole C. Perry
Received: 4 August 2009 / Accepted: 11 March 2010 / Published online: 30 April 2010
# Springer Science+Business Media BV 2010
Abstract In this contribution, we demonstrate the potential
of combining bioinspired synthesis and continuous flow
processing to generate functional materials with possible
applications in catalysis, biocatalysis and photonic devices.
Specifically, we have prepared invertase immobilized on
silica while preserving its enzymatic activity. Furthermore,
we present routes to synthesize silica and gold colloid
composite materials (Au@SiO2) and demonstrate that the
colloids retain their optical activity.
Keywords Biomimetic . Flow chemistry . Enzymes .
Green chemistry
1 Introduction
In this article, we provide results from a proof-of-concept
study of the synthesis of silica based hybrid functional
materials. We achieve this by using a unique combination
of bioinspired materials technology and flow chemistry
(Scheme 1); this, to our knowledge, has not been reported
before. The key advantages that this method offers are: (a)
Electronic supplementary material The online version of this article
(doi:10.1007/s12633-010-9038-7) contains supplementary material,
which is available to authorized users.
S. V. Patwardhan (*)
Department of Chemical and Process Engineering,
University of Strathclyde,
James Weir Building, 75 Montrose Street,
Glasgow G1 1XJ, UK
e-mail: Siddharth.Patwardhan@strath.ac.uk
S. V. Patwardhan : C. C. Perry
School of Science and Technology, Nottingham Trent University,
Clifton Lane,
Nottingham NG11 8NS, UK
control over the physical properties of the materials (e.g.
porosity or morphology) achieved by using bioinspired
additives; (b) rapid synthesis (requires 1–3 min); (c) use of
environmentally benign or mild conditions (compatible with
sensitive materials including enzymes); (d) potential for high
throughput / screening; and (e) versatility of the method that
can be applied to a range of materials.
Flow chemistry has the potential for high throughput and
continuous processing, thereby reducing the time for
materials synthesis and process optimisation [1]. The
continuous flow synthesis of nanomaterials using micro-
tubes or microfluidic devices has recently been shown to
produce monodisperse inorganic nano–particles and micro–
particles by precise control of reactant mixing and
nucleation [2-6]. However, only rarely has any demonstra-
ble control over complex chemistries (biomolecule-inorganic
particle hybrids) [7, 8] or materials properties, such as shape
or morphology, been achieved [9, 10]. This is primarily due
to the use of tedious or time consuming synthesis methods,
use of non-biologically friendly solvents and high temper-
atures. However, control over the preparation of inorganic
materials can be achieved using bioinspired approaches
operating under environmentally benign conditions (i.e.
“green” or “soft” routes), but to date only for batch
processing [11-15]. The soft routes are compatible with
biological materials, proteins and enzymes and a range of
nanostructured and functional materials can be produced
including oxides of Ge, Zn, Zr, Ga and Ti and metallic and
bimetallic particles of elements such as Au, Ag, FePt, CoPt
and Pd [16].
The first aim of this study was to test the feasibility of
the hypothesis that bioinspired materials technology and
flow chemistry can be combined to produce materials
rapidly and under mild conditions. Furthermore, we sought
to demonstrate the versatility of this method for the
34
Scheme 1 Schematic represen-
tation of the unique combination
of bioinspired materials
synthesis and continuous flow
for generating functional
hybrid nano/bio-materials
preparation of hybrid materials in the two examples
described below.
Enzyme stability is one of the bottlenecks of enzyme
implementation in an industrial biotechnology context [16].
A solution is to immobilise the enzymes such that they
retain their activity under process conditions [16]. Current
technologies for the protection of enzymes to improve
stability consist of the encapsulation of enzymes into
polymer based matrices or adsorption on inorganic porous
materials; some of which have been produced by conven-
tional sol-gel approaches [17]. In both cases, the surround-
ing matrix can sometimes cause loss of catalytic activity
due to reduced diffusion and low accessibility of the
catalytic sites. These drawbacks should be overcome by
immobilization of the enzymes [18, 19]. One of the
promising candidates for enzyme supports is amorphous
silica due to its proven biocompatibility in the amorphous
state [20]. There are numerous reports of laboratory based
experiments indicating the successful application of silica,
as monoliths and particles, for the controlled encapsulation,
stabilisation and/or release of a variety of biomolecules
such as vitamins, enzymes, drugs and cells [21, 22].
Immobilisation of enzymes in silica in a batch mode has
been previously reported using traditional or bioinspired
approaches for a range of biomolecules and has been
detailed in recent reviews [23-26]. We have therefore
applied flow chemistry and bioinspired technology to
prepare supported enzymes.
Quantum dots (QD) such as gold nanoparticles, exhibit
unique chemical and physical properties, and have been
successfully used in several applications including biosens-
ing [27, 28], optoelectronics [29, 30] and catalysis [31-34].
However, it is difficult to avoid the aggregation of nano-
particles (NP), especially when they are transferred from
one solution to another, thus significantly limiting their
current potential. In order to overcome such limitations, QD
have been typically encapsulated in silica particles or glass,
however, their properties depend on the processing con-
ditions used to prepare the silica or glass and include
solvents, pH, temperature and the use of stabilising agents.
For example, gold colloids show prominent shifts in surface
Silicon (2010) 2:33–39
plasmon bands even upon small changes in the refractive
index (RI) of the solvent used [29]. These processing
conditions in turn have been shown to affect the state of
aggregation and stability of the QD, silica shell thickness
as well as shell porosity, thereby consequently controlling
the optical properties of QD. Supporting functional nano-
particles such as gold colloids onto silica using our method
(i.e. a combination of bioinspired materials technology and
flow processing) is explored in this work.
2 Experimental Section
2.1 Chemical Reagents
Sodium metasilicate nonahydrate (the silica precursor),
pentaethylenehexamine (PEHA), used as the bioinspired
catalyst for silica preparation, gold colloid solution (20 nm),
2,3,5-triphenyl 2H-tetrazolium chloride (TPTZ), sucrose,
sodium borohydride, gold(III) chloride solution and invertase
were purchased from Sigma-Aldrich while sodium hydroxide
was obtained from Fisher Scientific. All reagents were used
without further purification. Distilled and deionised water
(conductivity <1 μS) was used as a solvent, for the preparation
of buffers and washing precipitated samples.
2.2 Silica Synthesis in Continuous Flow
Two types of 5 mL coil reactors—a FlowSyn™ (supplied
by Uniqsis; PEEK tubing) and a PVC tube reactor run by
syringe pumps—were employed. The latter provides a
simple set-up for preliminary studies and flexibility for
reactant injection/mixing. The FlowSyn™ is programmable
and allows precise control of operational parameters with a
wide range of flow rates. The tube internal diameters were
1 mm for FlowSyn™ and 1.5 mm for the syringe pump
(typical length of ∼6.3 m). In all cases, two streams of stock
solutions of reactants were used and a T-mixer fitted prior
to the coil reactors. The procedures for silica synthesis
using a bioinspired catalyst, such as PEHA, were similar to
those reported previously [15, 35]. The concentrations of
Silicon (2010) 2:33–39
sodium silicate and the catalysts were varied from 20 mM
to 40 mM in order to vary the ratio of silicon from the silica
precursor and nitrogen from PEHA (defined as Si:N) from
0.5 to 2 (see Table S1). Residence times between 20 s and
5 min were explored (corresponding flow rates were
15 mL min-1 to 1 mL min-1). Reactions at higher concen-
trations of the silica precursor or at longer residence times
were not possible due to blockage issues caused by rapidly
precipitating particles. Products obtained from the coil
reactors were centrifuged and washed thoroughly with
distilled and deionised water, collected as powders after
lyophilisation and weighed. For statistical analysis, selected
samples were prepared in triplicate and analysed as
described below.
2.3 Sample Characterisation
Thermal Gravimetric Analysis (TGA) was performed
using a Mettler Toledo TGA/SDTA851 e instrument
equipped with a TSO 801Ro autosampler. Samples
underwent heating from 20o to 900°C at a rate of 10°C/
min. Any loss in weight between 250o and 600°C was
recorded as the amount of organics present. For samples
containing enzyme, the weight of enzyme was calculated
by subtracting the weight of organics present in the
sample, prepared in the absence of enzyme under identical
conditions, from the total weight loss between 250o and
600°C. Attenuated Total Reflection Fourier Transform
Infrared spectroscopy (ATR-FTIR) was used to confirm
silica formation and to detect the bioinspired catalyst and
enzyme. Spectra were collected using a Magna IR-750
spectrometer with 512 scans at a resolution on 4 cm-1.
Samples for Scanning Electron Microscopy (SEM) were
prepared by placing powders on double-sided sticky
carbon tape on an aluminium sample holder. The samples
were then plasma coated with gold in order to minimise
any noise caused by sample charging and analysed at
20 kV using a JEOL JSM 840A equipped with Energy
Dispersive X-ray Analysis (EDXA) for elemental analysis.
Transmission Electron Microscopy (TEM) was performed
using a JEOL 2010. The samples were suspended in ethanol
and drop coated onto carbon coated copper or nickel grids for
TEM analysis.
Gold colloid solutions, before occlusion in silica, were
analyzed by scanning from 400–900 nm using a Unicam
UV2 UV-VIS spectrometer. The optical activity of the gold
colloids in the solid state (when entrapped in silica) was
measured using a Jasco V-670 spectrophotometer by
scanning from 200–2000 nm. The activity of free and
immobilised invertase was monitored by the reaction
producing glucose and fructose from sucrose using 2,3,5-
triphenyl 2H-tetrazolium chloride (TPTZ). TPTZ forms a
water-insoluble deep red pigment with an absorbance
35
maximum at ca. 535 nm with glucose [36]. Upon
incubation of samples with TPTZ, solutions were scanned
from 400–900 nm using a Unicam UV2 UV-VIS spec-
trometer.
3 Results and Discussion
3.1 Optimisation of Conditions for the Synthesis
of the Silica Support by Flow Processing
Since the combination of bioinspired synthesis and contin-
uous flow has not been investigated before, the first stage
of this study was focused on optimising the conditions
for the preparation of the silica support in the absence of
the enzyme or QD. It is noted that the continuous flow
synthesis of silica particles using the Stöber route has
been reported previously [37], however, this method
requires high pH, uses hazardous alkoxysilanes and
therefore is not suitable for the in situ immobilization of
sensitive materials such as enzymes. In this study, pentae-
thylenehexamine (PEHA) was chosen as the bioinspired
catalyst based on its ability to control silica particle size and
materials properties such as surface area [15, 35], however,
a range of other catalysts developed by us and others could
be explored for use in silica synthesis [15, 35, 38]. The
industrial production of precipitated silica commonly
utilizes sodium silicate and therefore this precursor was
employed in this study.
The yield and particle size of silica particles increased
with the residence time, as expected. The particle size
increased from <100 nm for a residence time of 20 s to
>200 nm for a residence time higher than 60 s (Fig. 1a). In
terms of precipitate collected, very little material was
obtained for the 20 s residence time (Fig. 1b). A residence
time as short as 60 seconds was sufficient to produce silica
particles (see Fig. 1b for Si:N=1 and 2), while particle
growth was complete in 3–5 min. This rate of silica
preparation is very quick compared to silica formation
using the Stöber method which takes several hours in batch
synthesis and at least 15 min in continuous mode [37, 39].
Additionally, as an example, for Si:N=1, the amount of
PEHA incorporated in silica increased up to ∼20% as
residence time increased (Fig. 1c). The chemical composi-
tion of the samples was studied using Energy Dispersive
X-ray analysis (EDXA) (Fig. 1d) and Fourier Transform
Infrared Spectroscopy (FTIR) (Fig. 1e). EDXA data
shows the presence of silicon in the products prepared
for residence times higher than 20 s. The FTIR spectra of
samples collected after residence times of 20 s and 60 s at
Si:N=0.5 were identical to PEHA (amine and CH related
peaks highlighted in Fig. 1e) and lacked characteristic
silica peaks between 1100-800 cm-1. For residence times
36
Fig. 1 a Particle size as a function of residence time monitored using
TEM. Scale bars=100 nm. The yield of silica precipitated b and
occluded PEHA c as a function of residence time and concentration of
above 60 s, the formation of silica was evident even for
the lowest precursor to catalyst ratio studied, while for
higher catalyst concentrations, silica formation was possi-
ble even at a residence time of 20 s (spectrum 5 in
Fig. 1e).
Sample morphologies were investigated using Scanning
Electron Microscopy (SEM). For Si:N=0.5 at a residence
time of 20 s, where FTIR (Fig. 1e) and EDXA (Fig. 1d)
indicated an absence of silica, the material was featureless
and contained large (>10 μm) granules, presumably of
PEHA alone (Figure S1). For residence times >60 s,
particles of ∼300 nm were formed for Si:N=1 (Fig. 2a, b)
which were confirmed to be silica by FTIR and EDXA
analysis (Fig. 1d, e), and are consistent with the literature
[15, 35]. We have reported previously that with PEHA,
particle sizes and morphologies can be easily tuned by
Fig. 2 SEM images of samples collected using PEHA and Si:N=1 at a, b pH 7 and c pH 6.5. Scale bars=1 μm for a and c, 5 μm for b
Silicon (2010) 2:33–39
reactants. Representative d EDXA and e FTIR spectra of selected
silica samples prepared using PEHA
changing the solution pH [15]. In this study, when the pH
in the tube reactor was reduced to 6.5, a continuous
network of smaller (<100 nm size) particles was produced
(Fig. 2c). From the results presented, it is clear that
bioinspired routes can be combined with flow approaches
for the continuous production of silica particles. Further-
more, the operating parameters for the materials synthesis
can be readily controlled to tune materials properties such
as morphology and composition.
3.2 Silica Supported Enzymes
In order to apply this technology and to check the
suitability of this approach for the generation of functional
materials, we synthesised hybrid materials by encapsulating
invertase. Invertase is a hydrolytic enzyme also called beta
Silicon (2010) 2:33–39
fructofuranoside, E.C. 3.2.1.26, pI 3.8, which catalyses the
hydrolysis of sucrose to give glucose and fructose. In our
study, the encapsulation of invertase was achieved by
introducing it in solutions of the bioinspired catalyst
(PEHA in this case), and allowing it to mix with the silica
precursor only immediately prior to entering the coil reactor.
The concentration of invertase in the reaction mixture and the
residence time were varied from 0.5–2 mg mL-1 and 20–180 s
respectively for a fixed concentration (30 mM) of the silica
precursor and Si:N=1. The characterisation of collected
samples using FTIR (Fig. 3a) and EDXA (Figure S2)
established the formation of silica and occlusion of the
enzyme for residence times as short as 20 s. TGA analysis
revealed that up to 18% by weight of the material was
enzyme (Figure S3), thereby confirming the successful
immobilisation of invertase in silica.
Enzyme activity was monitored using 2,3,5-triphenyl
2H-tetrazolium chloride (TPTZ) which forms a water-
insoluble deep red pigment (an absorbance maximum at
ca. 535 nm) with glucose—one of the products of the
enzymatic reaction (Fig. 3b, 1) [36]. The free and supported
enzyme gave a similar colour and spectra (Fig. 3b, 2 and 3),
while silica lacking in loaded invertase did not show any
colour (Fig. 3b, curve 4). This data demonstrates that
invertase supported on silica is active and we believe that
the results are of importance from an application point of
Fig. 3 a FTIR spectrum of
invertase encapsulated in silica.
b UV-Vis. spectra and photos
(insets) of samples of glucose
(1), free (2) or supported
invertase (3) and silica without
invertase (4) in the presence of
sucrose and TPTZ. c TEM
image of Au@SiO2; insets show
photos of silica (i) and
Au@SiO2 prepared with method
1 and 2 (ii, iii respectively).
d Absorption spectra of
Au@SiO2
37
view. Although the enzyme activity was studied only
qualitatively, one could speculate that the success of
invertase encapsulation could be due to the application of
bioinspired synthesis of silica performed under neutral
pH and aqueous conditions thereby retaining the enzyme
activity. However, it is noted that there are reported examples
of the preparation of enzymes encapsulated in silica under a
wider pH range where enzymes have retained their activities
[26]. Further studies are being directed towards understand-
ing the chemical and thermal stability of immobilised
invertase and the effect of materials properties on enzyme
performance.
3.3 Synthesis and Characterization of Au@SiO2
Quantum dots, which have found applications in areas as
diverse as biosensing to catalysis, typically suffer due to
aggregation and sensitivity towards solvents used and are
often occluded in silica particles or glass. However,
encapsulation protocols used could affect the properties of
QDs. We therefore sought to apply our mild synthesis of
silica to occlude gold nanoparticles and studied their optical
properties before and after encapsulation. The preparation
of Au@SiO2 was achieved by two methods—(1) pre-
formed gold nanoparticles (NPs) were included in one of
the reactant streams prior to silica formation; (2) the gold
38
NP precursor (AuCl4-) was introduced into one of the
reactants in the flow synthesis. The precursor was success-
fully reduced off-line post-silica synthesis using NaBH4 to
form occluded gold NPs. Figure 3c shows images of typical
white silica particles which did not contain any gold NPs
(i), while the formation of Au@SiO2 using method 1 and 2
can be seen from inset (ii) and (iii) respectively. The pink
colour indicates the successful synthesis of Au@SiO2. The
formation of 7±3 nm gold NPs in silica upon post-
reduction was observed (Fig. 3c). Silica occluded gold
nanoparticles and other quantum dots have been known to
be suitable for applications in optical devices [29]. We
therefore analysed the optical properties of these materials
in the solid state (Fig. 3d). The Au@silica samples exhibited
a broad band centred at ∼550 nm (Fig. 3d curve 2, 3). This
absorption was absent in the silica sample without the gold
colloids (Fig. 3d curve 1) but is similar to that observed for
the gold colloid in solution (Fig. 3d, curve 4). The results
demonstrate that the new method described herein is capable
of encapsulating QDs and further work is directed towards
optimisation of processing conditions.
4 Conclusions
We have demonstrated the potential of combining bioinspired
nanomaterials synthesis and continuous flow processing—
two rapidly developing fields—in generating nanostructured
materials with potential applications in biochemistry, catalysis
and photonic devices. Key features of this method include (a)
a substantial reduction in preparation time; (b) the room
temperature and neutral pH processing using non-hazardous
chemicals (removing the need for specialised heat/pressure/
pH resistant materials thereby reducing operational costs); (c)
better control of product properties (e.g. control of morphol-
ogies); and (d) ease of incorporation of complex chemistries—
all features suitable for straightforward scale-up for materials
production. Optimisation and technological improvements
in the design of the continuous flow syntheses presented
herein are being undertaken and include close consideration to
aspects such as mixing, online monitoring and flow geome-
tries, dimensions and rates which could affect properties such
as morphology and growth rates of synthetic and biological
minerals [40, 41].
Acknowledgment We thank the University of Strathclyde for
Faculty of Engineering Scholarship, Research Support Fund and the
US AFOSR (grant code FA9500-06-1-0154) for financial support. We
also thank Dr. D. Belton, Dr. A. Rai, and Dr. A. Prabhune (National
Chemical Laboratory, Pune) for their help in the flow apparatus set-up,
experiments with Au NPs and invertase assay respectively.
Supporting Information Available SEM of materials produced
using PEHA at short residence time; EDXA and TGA data for
invertase-silica composites.
Silicon (2010) 2:33–39
References
1. de Mello AJ (2006) Control and detection of chemical reactions in
microfluidic systems. Nature 442:394–402
2. Song Y, Hormes J, Kumar CSSR (2008) Microfluidic synthesis of
nanomaterials. Small 4:698–711
3. Jahn A, Reiner JE, Vreeland WN, DeVoe DL, Locascio LE,
Gaitan M (2008) Preparation of nanoparticles by continuous flow
microfluidics. J Nanopart Res 10:925–934
4. Kawase M, Miura K (2007) Fine particle synthesis by continuous
precipitation usinf a tubular reactor. Adv Powder Technol 18:725–
738
5. Edel JB, Fortt R, de Mello JC, de Mello AJ (2002) Microfluidic
routes to the controlled production of nanoparticles. Chem
Commun 10:1136–1137
6. Nakamura H, Yamaguchi Y, Miyazaki M, Maeda H, Uehara M,
Mulvaney P (2002) Preparation of CdSe nanocrystals in a micro-
flow-reactor. Chem Commun 23:2844–2845
7. Khan SA, Jensen KF (2007) Microfluidic synthesis of titania
shells on colloidal silica. Adv Mater 19:2556–2560
8. Ju JX, Zeng CF, Zhang LX, Xu NP (2006) Continuous synthesis
of zeolite NaA in a microchannel reactor. Chem Eng J 116:115–
121
9. Xu S, Nie Z, Seo M, Lewis P, Kumacheva E, Stone HA, Garstecki
P, Weibel DB, Gitlin I, Whitesides GM (2005) Generation of
monodisperse particles by using microfluidics. Angew Chem Int
Ed 44:724–728
10. Boleininger J, Kurz A, Reuss V, Sönnichsen C (2006) Micro-
fluidic continuous flow synthesis of rod-shaped gold and silver
nanocrystals. Phys Chem Chem Phys 8:3824–3827
11. Patwardhan SV, Clarson SJ, Perry CC (2005) On the role(s) of
additives in bioinspired silicification. Chem Commun 9:1113–
1121
12. Meldrum FC, Cölfen H (2008) Controlling mineral morphologies
and structures in biological and synthetic systems. Chem Rev
108:4332–4432
13. Brutchey RL, Morse DE (2008) Silicatein and the translation of its
molecular mechanism of biosilicification into low temperature
nanomaterial synthesis. Chem Rev 108:4915–4934
14. Dickerson MB, Sandhage KH, Naik RR (2008) Protein–and
peptide-directed syntheses of inorganic materials. Chem Rev
108:4935–4978
15. Belton DJ, Patwardhan SV, Annenkov VV, Danilovtseva EN,
Perry CC (2008) From biosilicification to tailored materials:
Optimizing hydrophobic domains and resistance to protonation of
polyamines. Proc Natl Acad Sci USA 105:5963–5968
16. (2000). New Biocatalysts: Essential Tools for a Sustainable 21st
Century Chemical Industry, A report by Council for Chemical
Research.
17. El Rassy H, Maury S, Buisson P, Pierre AC (2004) Hydrophobic
silica aerogel-lipase biocatalysts: Possible interactions between
the enzyme and the gel. Journal of Non-Crystalline Solids
350:23
18. Kim J, Grate JW, Wang P (2006) Nanostructures for enzyme
stabilization. Chem Eng Sci 61:1017–1026
19. Mateo C, Palomo JM, Fernandez-Lorente G, Guisan JM,
Fernandez-Lafuente R (2007) Improvement of enzyme activity,
stability and selectivity via immobilization techniques. Enzym
Microb Tech 40:1451–1463
20. Barbe C, Bartlett J, Kong LG, Finnie K, Lin HQ, Larkin M,
Calleja S, Bush A, Calleja G (2004) Silica particles: A novel drug-
delivery system. Adv Mater 16:1959–1966
21. Gill I, Ballesteros A (1998) Encapsulation of Biologicals within
Silicate, Siloxane, and Hybrid Sol–Gel Polymers: An Efficient
and Generic Approach. J Am Chem Soc 120:8587
Silicon (2010) 2:33–39
22. Gill I, Ballesteros A (2000) Bioencapsulation within synthetic
polymers (Part 1): sol-gel encapsulated biologicals. Trends
Biotechnol 18:282–296
23. Livage J, Coradin T, Roux C (2001) Encapsulation of biomolecules
in silica gels. J Phys Condens Matter 13:R673–R691
24. Betancor L, Lucarift HR (2008) Bioinspired enzyme encapsula-
tion for biocatalysis. Trends Biotechnol 26:566–572
25. Szymanska K, Bryjak J, Mrowiec-Bialon J, Jarzebski AB (2007)
Application and properties of siliceous mesostructured cellular
foams as enzymes carriers to obtain efficient biocatalysts.
Microporous Mesoporous Mater 99:167–175
26. Lebert JM, Forsberg EM, Brennan JD (2008) Solid-phase assays
for small molecule screening using sol-gel entrapped proteins.
Biochem Cell Biol 86:100–110
27. Bruchez M, Moronne M, Gin P, Weiss S, Alivisatos AP (1998)
Semiconductor Nanocrystals as Fluorescent Biological Labels.
Science 281:2013
28. Chan WCW, Nie SM (1998) Quantum Dot Bioconjugates for
Ultrasensitive Nonisotopic Detection. Science 281:2016
29. Mulvaney P, Liz-Marzán LM, Giersig M, Ung T (2000) Silica
encapsulation of quantum dots and metal clusters. J Mater Chem
10:1259–1270
30. Nann T, Mulvaney P (2004) Single Quantum Dots in Spherical
Silica Particles. Angew Chem Int Ed 43:5393
31. Astruc D, Lu F, Aranzaes JR (2005) Nanoparticles as recyclable
catalysts: The frontier between homogeneous and heterogeneous
catalysis. Angew Chem Int Ed 44:7852–7872
32. Miller JT, Kropf AJ, Zha Y, Regalbuto JR, Delannoy L, Louis C,
Bus E, van Bokhoven JA (2006) The effect of gold particle size
39
on Au-Au bond length and reactivity toward oxygen in supported
catalysts. J Catalysis 240:222–234
33. Qian K, Huang W, Fang J, Lv S, He B, Jiang Z, Wei S (2008)
Low-temperature CO oxidation over Au/ZnO/SiO2 catalysts:
Some mechanism insights. J Catalysis 225:269–278
34. Wang L-C, Liu Y-M, Chen M, Cao Y, He H-Y, Fan K-N (2008)
MnO2 nanorod supported gold nanoparticles with enhanced
activity for solvent-free aerobic alcohol oxidation. J Phys Chem
C 112:6981–6987
35. Belton D, Patwardhan SV, Perry CC (2005) Spermine, spermidine
and their analogues generate tailored silicas. J Mater Chem
15:4629–4638
36. Phadtare S, D’Britto V, Pundle A, Prabhune A, Sastry M (2004)
Invertase-lipid biocomposite films: Preparation, characterization,
and enzymatic activity. Biotechnol Progr 20:156–161
37. Khan SA, Gunther A, Schmidt MA, Jensen KF (2004) Micro-
fluidic synthesis of colloidal silica. Langmuir 20:8604–8611
38. Belton D, Patwardhan SV, Perry CC (2005) Putrescine homo-
logues control silica morphogenesis by electrostatic interactions
and the hydrophobic effect. Chem Commun 27:3475–3477
39. Stöber W, Fink A, Bohn E (1968) Controlled growth of
monodisperse silica spheres in the micron size range. J Colloid
Interface Sci 26:62
40. Patwardhan SV, Mukherjee N, Clarson SJ (2001) Formation of
fiber-like amorphous silica structures by externally applied shear.
J Inorg Organomet Polymer 11:117–121
41. Clarson SJ, Steinitz-Kannan M, Patwardhan SV, Kannan R, Hartig
R, Schloesser L, Hamilton DW, Fusaro JKA, Beltz R (2009) Some
observations of diatoms under turbulence. SILICON 1:79–90