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Silicon (2010) 2:33–39

DOI 10.1007/s12633-010-9038-7


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 control over the physical properties of the materials (e.g.
of combining bioinspired synthesis and continuous flow porosity or morphology) achieved by using bioinspired
processing to generate functional materials with possible additives; (b) rapid synthesis (requires 1–3 min); (c) use of
applications in catalysis, biocatalysis and photonic devices. environmentally benign or mild conditions (compatible with
Specifically, we have prepared invertase immobilized on sensitive materials including enzymes); (d) potential for high
silica while preserving its enzymatic activity. Furthermore, throughput / screening; and (e) versatility of the method that
we present routes to synthesize silica and gold colloid can be applied to a range of materials.
composite materials (Au@SiO2) and demonstrate that the Flow chemistry has the potential for high throughput and
colloids retain their optical activity. continuous processing, thereby reducing the time for
materials synthesis and process optimisation [1]. The
Keywords Biomimetic . Flow chemistry . Enzymes . continuous flow synthesis of nanomaterials using micro-
Green chemistry tubes or microfluidic devices has recently been shown to
produce monodisperse inorganic nano–particles and micro–
particles by precise control of reactant mixing and
1 Introduction nucleation [2-6]. However, only rarely has any demonstra-
ble control over complex chemistries (biomolecule-inorganic
In this article, we provide results from a proof-of-concept particle hybrids) [7, 8] or materials properties, such as shape
study of the synthesis of silica based hybrid functional or morphology, been achieved [9, 10]. This is primarily due
materials. We achieve this by using a unique combination to the use of tedious or time consuming synthesis methods,
of bioinspired materials technology and flow chemistry use of non-biologically friendly solvents and high temper-
(Scheme 1); this, to our knowledge, has not been reported atures. However, control over the preparation of inorganic
before. The key advantages that this method offers are: (a) materials can be achieved using bioinspired approaches
operating under environmentally benign conditions (i.e.
Electronic supplementary material The online version of this article “green” or “soft” routes), but to date only for batch
(doi:10.1007/s12633-010-9038-7) contains supplementary material, processing [11-15]. The soft routes are compatible with
which is available to authorized users. biological materials, proteins and enzymes and a range of
S. V. Patwardhan (*) nanostructured and functional materials can be produced
Department of Chemical and Process Engineering, including oxides of Ge, Zn, Zr, Ga and Ti and metallic and
University of Strathclyde,
bimetallic particles of elements such as Au, Ag, FePt, CoPt
James Weir Building, 75 Montrose Street,
Glasgow G1 1XJ, UK and Pd [16].
e-mail: Siddharth.Patwardhan@strath.ac.uk The first aim of this study was to test the feasibility of
the hypothesis that bioinspired materials technology and
S. V. Patwardhan : C. C. Perry
flow chemistry can be combined to produce materials
School of Science and Technology, Nottingham Trent University,
Clifton Lane, rapidly and under mild conditions. Furthermore, we sought
Nottingham NG11 8NS, UK to demonstrate the versatility of this method for the
34 Silicon (2010) 2:33–39

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 plasmon bands even upon small changes in the refractive
described below. index (RI) of the solvent used [29]. These processing
Enzyme stability is one of the bottlenecks of enzyme conditions in turn have been shown to affect the state of
implementation in an industrial biotechnology context [16]. aggregation and stability of the QD, silica shell thickness
A solution is to immobilise the enzymes such that they as well as shell porosity, thereby consequently controlling
retain their activity under process conditions [16]. Current the optical properties of QD. Supporting functional nano-
technologies for the protection of enzymes to improve particles such as gold colloids onto silica using our method
stability consist of the encapsulation of enzymes into (i.e. a combination of bioinspired materials technology and
polymer based matrices or adsorption on inorganic porous flow processing) is explored in this work.
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 2 Experimental Section
due to reduced diffusion and low accessibility of the
catalytic sites. These drawbacks should be overcome by 2.1 Chemical Reagents
immobilization of the enzymes [18, 19]. One of the
promising candidates for enzyme supports is amorphous Sodium metasilicate nonahydrate (the silica precursor),
silica due to its proven biocompatibility in the amorphous pentaethylenehexamine (PEHA), used as the bioinspired
state [20]. There are numerous reports of laboratory based catalyst for silica preparation, gold colloid solution (20 nm),
experiments indicating the successful application of silica, 2,3,5-triphenyl 2H-tetrazolium chloride (TPTZ), sucrose,
as monoliths and particles, for the controlled encapsulation, sodium borohydride, gold(III) chloride solution and invertase
stabilisation and/or release of a variety of biomolecules were purchased from Sigma-Aldrich while sodium hydroxide
such as vitamins, enzymes, drugs and cells [21, 22]. was obtained from Fisher Scientific. All reagents were used
Immobilisation of enzymes in silica in a batch mode has without further purification. Distilled and deionised water
been previously reported using traditional or bioinspired (conductivity <1 μS) was used as a solvent, for the preparation
approaches for a range of biomolecules and has been of buffers and washing precipitated samples.
detailed in recent reviews [23-26]. We have therefore
applied flow chemistry and bioinspired technology to 2.2 Silica Synthesis in Continuous Flow
prepare supported enzymes.
Quantum dots (QD) such as gold nanoparticles, exhibit Two types of 5 mL coil reactors—a FlowSyn™ (supplied
unique chemical and physical properties, and have been by Uniqsis; PEEK tubing) and a PVC tube reactor run by
successfully used in several applications including biosens- syringe pumps—were employed. The latter provides a
ing [27, 28], optoelectronics [29, 30] and catalysis [31-34]. simple set-up for preliminary studies and flexibility for
However, it is difficult to avoid the aggregation of nano- reactant injection/mixing. The FlowSyn™ is programmable
particles (NP), especially when they are transferred from and allows precise control of operational parameters with a
one solution to another, thus significantly limiting their wide range of flow rates. The tube internal diameters were
current potential. In order to overcome such limitations, QD 1 mm for FlowSyn™ and 1.5 mm for the syringe pump
have been typically encapsulated in silica particles or glass, (typical length of ∼6.3 m). In all cases, two streams of stock
however, their properties depend on the processing con- solutions of reactants were used and a T-mixer fitted prior
ditions used to prepare the silica or glass and include to the coil reactors. The procedures for silica synthesis
solvents, pH, temperature and the use of stabilising agents. using a bioinspired catalyst, such as PEHA, were similar to
For example, gold colloids show prominent shifts in surface those reported previously [15, 35]. The concentrations of
Silicon (2010) 2:33–39 35

sodium silicate and the catalysts were varied from 20 mM maximum at ca. 535 nm with glucose [36]. Upon
to 40 mM in order to vary the ratio of silicon from the silica incubation of samples with TPTZ, solutions were scanned
precursor and nitrogen from PEHA (defined as Si:N) from from 400–900 nm using a Unicam UV2 UV-VIS spec-
0.5 to 2 (see Table S1). Residence times between 20 s and trometer.
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 3 Results and Discussion
were not possible due to blockage issues caused by rapidly
precipitating particles. Products obtained from the coil 3.1 Optimisation of Conditions for the Synthesis
reactors were centrifuged and washed thoroughly with of the Silica Support by Flow Processing
distilled and deionised water, collected as powders after
lyophilisation and weighed. For statistical analysis, selected Since the combination of bioinspired synthesis and contin-
samples were prepared in triplicate and analysed as uous flow has not been investigated before, the first stage
described below. of this study was focused on optimising the conditions
for the preparation of the silica support in the absence of
2.3 Sample Characterisation the enzyme or QD. It is noted that the continuous flow
synthesis of silica particles using the Stöber route has
Thermal Gravimetric Analysis (TGA) was performed been reported previously [37], however, this method
using a Mettler Toledo TGA/SDTA851 e instrument requires high pH, uses hazardous alkoxysilanes and
equipped with a TSO 801Ro autosampler. Samples therefore is not suitable for the in situ immobilization of
underwent heating from 20o to 900°C at a rate of 10°C/ sensitive materials such as enzymes. In this study, pentae-
min. Any loss in weight between 250o and 600°C was thylenehexamine (PEHA) was chosen as the bioinspired
recorded as the amount of organics present. For samples catalyst based on its ability to control silica particle size and
containing enzyme, the weight of enzyme was calculated materials properties such as surface area [15, 35], however,
by subtracting the weight of organics present in the a range of other catalysts developed by us and others could
sample, prepared in the absence of enzyme under identical be explored for use in silica synthesis [15, 35, 38]. The
conditions, from the total weight loss between 250o and industrial production of precipitated silica commonly
600°C. Attenuated Total Reflection Fourier Transform utilizes sodium silicate and therefore this precursor was
Infrared spectroscopy (ATR-FTIR) was used to confirm employed in this study.
silica formation and to detect the bioinspired catalyst and The yield and particle size of silica particles increased
enzyme. Spectra were collected using a Magna IR-750 with the residence time, as expected. The particle size
spectrometer with 512 scans at a resolution on 4 cm-1. increased from <100 nm for a residence time of 20 s to
Samples for Scanning Electron Microscopy (SEM) were >200 nm for a residence time higher than 60 s (Fig. 1a). In
prepared by placing powders on double-sided sticky terms of precipitate collected, very little material was
carbon tape on an aluminium sample holder. The samples obtained for the 20 s residence time (Fig. 1b). A residence
were then plasma coated with gold in order to minimise time as short as 60 seconds was sufficient to produce silica
any noise caused by sample charging and analysed at particles (see Fig. 1b for Si:N=1 and 2), while particle
20 kV using a JEOL JSM 840A equipped with Energy growth was complete in 3–5 min. This rate of silica
Dispersive X-ray Analysis (EDXA) for elemental analysis. preparation is very quick compared to silica formation
Transmission Electron Microscopy (TEM) was performed using the Stöber method which takes several hours in batch
using a JEOL 2010. The samples were suspended in ethanol synthesis and at least 15 min in continuous mode [37, 39].
and drop coated onto carbon coated copper or nickel grids for Additionally, as an example, for Si:N=1, the amount of
TEM analysis. PEHA incorporated in silica increased up to ∼20% as
Gold colloid solutions, before occlusion in silica, were residence time increased (Fig. 1c). The chemical composi-
analyzed by scanning from 400–900 nm using a Unicam tion of the samples was studied using Energy Dispersive
UV2 UV-VIS spectrometer. The optical activity of the gold X-ray analysis (EDXA) (Fig. 1d) and Fourier Transform
colloids in the solid state (when entrapped in silica) was Infrared Spectroscopy (FTIR) (Fig. 1e). EDXA data
measured using a Jasco V-670 spectrophotometer by shows the presence of silicon in the products prepared
scanning from 200–2000 nm. The activity of free and for residence times higher than 20 s. The FTIR spectra of
immobilised invertase was monitored by the reaction samples collected after residence times of 20 s and 60 s at
producing glucose and fructose from sucrose using 2,3,5- Si:N=0.5 were identical to PEHA (amine and CH related
triphenyl 2H-tetrazolium chloride (TPTZ). TPTZ forms a peaks highlighted in Fig. 1e) and lacked characteristic
water-insoluble deep red pigment with an absorbance silica peaks between 1100-800 cm-1. For residence times
36 Silicon (2010) 2:33–39

Fig. 1 a Particle size as a function of residence time monitored using reactants. Representative d EDXA and e FTIR spectra of selected
TEM. Scale bars=100 nm. The yield of silica precipitated b and silica samples prepared using PEHA
occluded PEHA c as a function of residence time and concentration of

above 60 s, the formation of silica was evident even for changing the solution pH [15]. In this study, when the pH
the lowest precursor to catalyst ratio studied, while for in the tube reactor was reduced to 6.5, a continuous
higher catalyst concentrations, silica formation was possi- network of smaller (<100 nm size) particles was produced
ble even at a residence time of 20 s (spectrum 5 in (Fig. 2c). From the results presented, it is clear that
Fig. 1e). bioinspired routes can be combined with flow approaches
Sample morphologies were investigated using Scanning for the continuous production of silica particles. Further-
Electron Microscopy (SEM). For Si:N=0.5 at a residence more, the operating parameters for the materials synthesis
time of 20 s, where FTIR (Fig. 1e) and EDXA (Fig. 1d) can be readily controlled to tune materials properties such
indicated an absence of silica, the material was featureless as morphology and composition.
and contained large (>10 μm) granules, presumably of
PEHA alone (Figure S1). For residence times >60 s, 3.2 Silica Supported Enzymes
particles of ∼300 nm were formed for Si:N=1 (Fig. 2a, b)
which were confirmed to be silica by FTIR and EDXA In order to apply this technology and to check the
analysis (Fig. 1d, e), and are consistent with the literature suitability of this approach for the generation of functional
[15, 35]. We have reported previously that with PEHA, materials, we synthesised hybrid materials by encapsulating
particle sizes and morphologies can be easily tuned by invertase. Invertase is a hydrolytic enzyme also called beta

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 37

fructofuranoside, E.C., pI 3.8, which catalyses the view. Although the enzyme activity was studied only
hydrolysis of sucrose to give glucose and fructose. In our qualitatively, one could speculate that the success of
study, the encapsulation of invertase was achieved by invertase encapsulation could be due to the application of
introducing it in solutions of the bioinspired catalyst bioinspired synthesis of silica performed under neutral
(PEHA in this case), and allowing it to mix with the silica pH and aqueous conditions thereby retaining the enzyme
precursor only immediately prior to entering the coil reactor. activity. However, it is noted that there are reported examples
The concentration of invertase in the reaction mixture and the of the preparation of enzymes encapsulated in silica under a
residence time were varied from 0.5–2 mg mL-1 and 20–180 s wider pH range where enzymes have retained their activities
respectively for a fixed concentration (30 mM) of the silica [26]. Further studies are being directed towards understand-
precursor and Si:N=1. The characterisation of collected ing the chemical and thermal stability of immobilised
samples using FTIR (Fig. 3a) and EDXA (Figure S2) invertase and the effect of materials properties on enzyme
established the formation of silica and occlusion of the performance.
enzyme for residence times as short as 20 s. TGA analysis
revealed that up to 18% by weight of the material was 3.3 Synthesis and Characterization of Au@SiO2
enzyme (Figure S3), thereby confirming the successful
immobilisation of invertase in silica. Quantum dots, which have found applications in areas as
Enzyme activity was monitored using 2,3,5-triphenyl diverse as biosensing to catalysis, typically suffer due to
2H-tetrazolium chloride (TPTZ) which forms a water- aggregation and sensitivity towards solvents used and are
insoluble deep red pigment (an absorbance maximum at often occluded in silica particles or glass. However,
ca. 535 nm) with glucose—one of the products of the encapsulation protocols used could affect the properties of
enzymatic reaction (Fig. 3b, 1) [36]. The free and supported QDs. We therefore sought to apply our mild synthesis of
enzyme gave a similar colour and spectra (Fig. 3b, 2 and 3), silica to occlude gold nanoparticles and studied their optical
while silica lacking in loaded invertase did not show any properties before and after encapsulation. The preparation
colour (Fig. 3b, curve 4). This data demonstrates that of Au@SiO2 was achieved by two methods—(1) pre-
invertase supported on silica is active and we believe that formed gold nanoparticles (NPs) were included in one of
the results are of importance from an application point of the reactant streams prior to silica formation; (2) the gold

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
38 Silicon (2010) 2:33–39

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