Glucose Oxidase Encapsulated Polyvinyl Alcohol–Silica Hybrid Films

May 19, 2013 - ... Department of Polymer Engineering, 8 Kl. Ohridski, 1756 Sofia, Bulgaria ..... Journal of The Electrochemical Society 2015 162 (1), ...
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Glucose Oxidase Encapsulated Polyvinyl Alcohol−Silica Hybrid Films for an Electrochemical Glucose Sensing Electrode Umesh Lad,† Girish M. Kale,*,† and Rayna Bryaskova‡ †

Institute for Materials Research, SPEME, University of Leeds, Leeds LS2 9JT, U.K. University of Chemical Technology and Metallurgy, Department of Polymer Engineering, 8 Kl. Ohridski, 1756 Sofia, Bulgaria

‡

S Supporting Information *

ABSTRACT: An amperometric glucose enzyme electrode was developed by the immobilization of glucose oxidase (GOD) in a composite material based on polyvinyl alcohol (PVA) and partially prehydrolyzed tetraethyl orthosilicate (pphTEOS) on the surface of “in-house” fabricated graphite electrodes. For comparison, silver and gold nanoparticles (Ag/AuNPs) embedded in the PVA-pphTEOS matrix was prepared through a novel method via sol−gel process based on the in situ chemical reduction of Ag or Au ions using PVA as a reducing agent and stabilizer. The successful incorporation of Ag and AuNPs ranging from 5 to 7.5 and 4.5−11 nm, respectively, in the PVA-pphTEOS matrix was confirmed by UV−vis spectroscopy, TEM, and EDX analysis. The PVA-TEOS matrix was also characterized by FTIR spectroscopy. The analytical performance of the enzyme electrodes were studied in terms of linear ranges, sensitivities, response times, limits of detection, reproducibility and stability.

S

PVA-based support materials for the immobilization of GOD.7 The PVA matrices were chemically cross-linked with glutaraldehyde, freeze−thawed, prepared with silicate sol−gel and alumina sol−gel. Both types of sol−gel methods were brittle but with the addition of PVA increased the mechanical strength. Because of the compaction and low-conductivity of polymer membranes, it can be difficult for the substrate to infiltrate into the enzyme membrane and for the electrons to effectively transfer between the enzyme membrane and the electrode.11 To improve on this, research groups have employed PVA and other polymers to form composite matrices with NPs to enhance the electron transport to the electrode surface while retaining their original beneficial physical properties. Nanoparticles possess unique chemical and physical properties, that have applications in surface Raman spectroscopy, display devices, catalysts, microelectronics, and have improved sensing devices, such as electrochemical sensors and biosensors.12−16 Different sizes and compositions of NPs provide different functions and advantages to biosensor devices, namely, enhanced electron transfer and immobilization of enzymes.17−22 Metal NPs, such as Au, Ag, Pt, Cu, etc., have excellent conductivity that have been described to act as “electronic wires” enhancing electronic communication between the enzyme and transducer surface giving improved sensitivity.21,22 In addition, metal NPs can provide suitable biocompatible microenvironment, that enables the biomolecules

ince the initial concepts of the glucose enzyme electrode was proposed,1 glucose biosensors have prompted a vast number of research groups to develop new biosensors and advanced materials for a wide range of applications. Even with the success story of the glucose sensor, many researchers are not deterred from developing new materials and innovative detection strategies to sense glucose as there is an abundant scope for improvement of accuracy and consistency of glucose measurements. Such huge market size and simplicity of sensing reaction chemistry makes diabetes a model disease for developing new biosensing concepts. Amperometric enzyme electrodes, based on glucose oxidase (GOD), have played a leading role in the move to simple easy-to-use blood sugar testing and point-ofcare diagnostics. Enzymes have been immobilized in many support materials, but polymers, such as PVA,2−7 offer unique characteristics such as excellent gel-forming properties, good film-forming ability, and notably good permeability. This hydrogel is frequently used in biomedical applications and is an ideal material for enzyme immobilization because of its nontoxic nature and good biocompatibility. Although PVA has a high swelling index it however, dissolves readily when not cross-linked8 or stabilized by other means. Zuo et al. demonstrated the entrapment of GOD on a screen-printed electrode with a silica sol−gel/PVA composite film.9 The silica sol−gel/PVA composite material is endowed with a great amount of hydroxyl groups, providing a biocompatible microenvironment for the encapsulation of GOD. Therefore, the loss of biological activity of GOD can be avoided efficiently.10 Wong and Aziz carried out a comparative study of © XXXX American Chemical Society

Received: March 8, 2013 Accepted: May 19, 2013

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to retain their activity once immobilized.18 It is well-known that the active sites of redox enzymes are buried and insulated deep within their protein shell and as a consequence cannot take part in direct electron exchange with standard electrodes.23 However, AuNPs have been found to shorten the electron transfer distances and mediate charge transport.24−27 Zhang et al. evaluated a silica network electrode with and without AuNPs in the presence of ferricyanide.28 Without AuNPs there was no response to ferricyanide due to a layer blocking the diffusion of ferricyanide toward the electrode surface. However, when the AuNPs were self-assembled on the electrode a quasi-reversible response was obtained. The continuous array of AuNPs within the silica network aided electron transfer. Only a few studies concerning the effect of AgNPs on the amperometric response of GOD electrodes are reported up to now and have mainly involved the entrapment of the AgNPs/ GOD within a polymer network4,29,30 or cross-linking within the a polymer network.30−33 However, AgNPs have been employed with other enzyme based biosensors. Gan et al.34 demonstrated the enhanced electron exchange of myoglobin using AgNPs to shuttle electrons to a pyrolytic graphite electrode. Liu and Hu et al.35 also fabricated a biosensor based on myoglobin immobilized on AgNPs doped carbon nanotubes film with composite sol−gel techniques. The direct electrochemistry of hemoglobin in hemoglobin−Ag sol films has also been described.27 Polymers used to stabilize metal NPs include, Nafion, chitosan, polyvinyl butyral and PVA. They serve a dual purpose by not only capping the NPs but entrapping GOD for biosensor applications. There are numerous ways of synthesizing colloidal Ag and AuNPs of varying size and shape, for example, using templates, photochemistry, seeds, electrochemistry, and radiolysis.36−42 In a study by Gautam et al.,43 PVA was used under hot conditions to synthesize AgNPs doped PVA to form a Ag-PVA composite structure. Filippo et al. also used a similar method in the development of a hydrogen peroxide sensor.44 To reinforce the PVA structure, Zuo et al.29 employed PVA/ silica sol−gel as a support for colloidal AgNPs and GOD. The AgNPs enhanced biosensor exhibited a good linear range, a rapid response and twice the sensitivity compared to those electrodes without AgNPs. In this study, we present the immobilization of GOD within PVA-pphTEOS matrix at an “in-house” fabricated graphite electrode for the determination of glucose. For comparison, we also prepared Ag and AuNPs embedded in the PVA-pphTEOS matrix prepared via sol−gel process based on the in situ reduction of Ag or Au ions by chemical reduction using PVA as a reducing agent and stabilizer. This work on composite films is in the interest of electrochemical biosensor field, but has also been an interest for application in the biomedical field as antibacterial coating materials.45,46

by the supplier without any further purification. Deionized water (Milli-Q) was used in all experiments. All glassware was cleaned in freshly prepared HNO3/HCl (1:3) solution, thoroughly rinsed with deionized water and dried before use. Preparation of PVA-Ag/AuNPs and pphTEOS. In a typical synthesis, PVA solution (5%) was prepared by dissolving 5.0 g of PVA in 95 mL of water under magnetic stirring and heating to 80 °C for 30 min in order to obtain a colorless PVA solution. To obtain AgNPs, different amounts of AgNO3 (50−1000 mg dissolved in 0.5 mL water) was added to 25 mL PVA (5%), stirred and heated for 1 h at 100 °C under reflux. The color of the solution in the resulting samples deepens gradually from an achromatic color in the beginning to a deep yellow-brownish equilibrium color. To retain a stable Ag colloid structure consisting of AgNPs capped in PVA molecules, the sample were cooled to 20−25 °C temperature just after the reaction and before employing for further experimentation. For PVAAuNPs, different amounts of HAuCl4 (1−10 mg dissolved in 0.5 mL of water) was added to 25 mL PVA (5%), stirred and heated for 1 h at 100 °C under reflux. The pale yellow color in the beginning slowly changes to purple before settling at a deep red color. All composite solutions were stored in dark bottles at 4 °C. A homogeneous standard of pphTEOS was prepared under magnetic stirring of 1 mL TEOS, 1 mL water and 0.1 mL of HNO3 in a small vial at room temperature for 60 min. Next, an aliquot of pphTEOS (0.3 mL) and 5% PVA (1 mL) solutions were mixed thoroughly under magnetic stirring. The solution mixture was freshly prepared prior to the fabrication of every enzyme electrode. Preparation of the Graphite Electrode and Immobilization of Glucose Oxidase. The in-house fabricated graphite rod electrode surfaces (area = 0.196 cm2) were treated on (1) p660 then (2) p1200 grade of emery paper, and brought to an almost mirror like finish by polishing on (3) 80 g m−2 paper. The graphite electrodes were reused after polishing through steps 1−3 as described. Prior to any modifications, the electrodes were cycled between −0.5 and +1.2 V (vs Ag/AgCl) at 50 mV s−1 in 0.1 M PBS (pH 7.0) until a stable profile was obtained. A 20 μL aliquot solution of PVA-pphTEOS (volume ratio 1:0.3 respectively) and a 5 μL aliquot of GOD (10 U μL−1) was mixed in an ultrasonic bath for 1 min to accelerate the dispersion of the enzyme in the pphTEOS and PVA mixture. Subsequently, a 5 μL sample of this mixture was spread over the surface of the graphite electrode followed by drying in air for 24 h. The resulting electrodes were denoted as PVApphTEOS-GOD. Alternatively, Ag or AuNPs electrodes (denoted as PVA-Ag/AuNPs-pphTEOS-GOD) were fabricated using a similar method but using PVA containing Ag or AuNPs. All the enzyme electrodes were stored at 4 °C when not in use. Apparatus and Electrochemical Measurements. UV− vis absorption spectra of the composite films and solution were recorded at room temperature in the wavelength range of 200− 800 nm using Perkin-Elmer Lambda UV−vis-NIR spectrometer. IR spectra of the films were recorded in transmittance mode in the range of 400−4000 cm−1 using Bruker Vertex FTIR. Transmission electron microscopy (TEM) images were recorded on a FEG-TEM (Phillips CM 200 field emission gun TEM). Samples were prepared by spin coating of the precursor solutions on carbon-coated grids using a microprocessor controlled spin coater (Model GP3-8 Spincoat, PI-KEM Ltd., U.K.) and dried under air at room temperature.

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MATERIALS AND METHODS Reagents and Materials. Polyvinyl alcohol (PVA, 87−89% hydrolyzed, average MW = 13 000−23 000, Sigma-Aldrich), tetraethyl orthosilicate (TEOS, ≥99.0%, Aldrich), nitric acid (HNO3, 2 M, Riedel-de Haën), silver nitrate (AgNO3, AcrosOrganics), auric chloride (HAuCl4, 99.99%, Aldrich), graphite rod (99.99%, Goodfellows), glucose oxidase (GOD, Type X-S (192 U mg−1, Sigma-Aldrich), D(+)-glucose (SigmaAldrich), ferrocene monocarboxylic acid (FMCA, ≥97.0%, Fluka), and phosphate buffer solution (PBS, 1.0 M, pH 7.0, Sigma-Aldrich) were used in respective experiments as supplied B

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by its ability to reduce and stabilize at such a high AgNO3 content. The absorption spectra of AgNPs in solution and in the form of a dried film prepared in the presence of PVA and added to pphTEOS show that the AgNPs do not agglomerate to form particles of a larger size or become destabilized. The same process was adopted for the formation of AuNPs within PVA and PVA-pphTEOS matrix. The formation of AuNPs is evidenced from UV−vis spectroscopy by the appearance of strong absorption bands at ∼520 nm53,54 in the form of solution (SI Figure S-3) and in the form of films (Figure 2). Again, the

All electrochemical experiments were performed with the SI1287 Electrochemical Interface (Solartron, U.K.) at 25 °C with a conventional 50 mL three-electrode system compromising of a platinum wire auxiliary (Model XM110, Radiometer Analytical), Ag/AgCl (3 M KCl) reference (Model REF321, Radiometer Analytical) and “in-house” fabricated graphite rod as the working or sensing electrodes. The amperometric and cyclic voltammetric detection of glucose were performed in nitrogen purged solutions of PBS (0.2 M) in the presence or absence of FMCA (0.8 mM). A magnetic stirrer and stirrer bar provided the convective transport during the current−time (I−t) amperometric studies.

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RESULTS AND DISCUSSION Characterization of the PVA-pphTEOS and PVA-Ag/ AuNPs-pphTEOS Composite Films: UV−Visible Spectroscopy. The synthesis of the composite materials with embedded NPs was carried out by the strategies according to reaction schemes 1 and 2. The strategy used consists of forming Ag/AuNPs that are initially synthesized by boiling the PVA solution at 100 °C for 60 min in the presence of AgNO3 or HAuCl4, wherein PVA acts as a reducing agent and as a stabilizer47 (see Figure S-1 of the Supporting Information (SI)). The addition of pphTEOS followed by film casting leads to the formation of NPs embedded in PVA-pphTEOS matrix. The presence of hydroxyl groups in the repeating units of the PVA polymer are expected to produce strong interactions (hydrogen or covalent bonds) with the silanol groups generated from acid catalyzed hydrolysis and polycondensation of TEOS (Figure 1).

Figure 2. UV−vis absorption spectra of PVA films, containing AuNPs prepared from different loadings of HAuCl4 precursor.

absorption spectrum shows that the AuNPs do not agglomerate or become destabilized when in the presence of pphTEOS. Transmission Electron Microscopy. Transmission electron microscopy (TEM) images demonstrate the formation of spherical AgNPs, homogeneously distributed in the PVA and PVA-pphTEOS matrix (SI Figure S-4a and b). It is obvious that PVA possesses a dual function (a) acting as a reducing agent, which reduces the complexed silver ions (Ag+) to elemental silver (Ag0), and (b) functioning as a stabilizer required for the homogeneous distribution of AgNPs in the PVA matrix. Spherical AgNPs with an average diameter of 5.2 ± 1.0 nm (SI Figure S-4a) and 7.5 ± 1 nm (SI Figure S-4 (b)) were measured and their formation was also confirmed in a separate study in our group45 using X-ray diffraction analysis. The histogram illustrates the relatively narrow size distribution of the average number of the particles per unit area of the films (inset of SI Figure S-4a and b). Moreover, energy dispersive Xray (EDX) analysis confirms the formation of the AgNPs by exhibiting a peak at approximately 3 keV, which is typical for the absorption of metallic silver nanocrystallites because of surface plasmon resonance (SI Figure S-4c).45 TEM images also demonstrate the formation of spherical AuNPs as shown in SI Figure S-5a and Figure 3. AuNPs with an average diameter of 4.7 ± 1.0 (SI Figure S-5a) and 10.9 ± 1.0 nm (Figure 3) were measured. The histograms again illustrate the relatively narrow size distribution of the average number of the particles per unit area of the films (inset of SI Figure S-5a and Figure 3). EDX analysis exhibited a peak at approximately 2 keV, again typical for the absorption of metallic gold nanocrystallites (SI S-5b).55 FTIR Spectroscopy. PVA-Ag/AuNPs and PVA-Ag/AuNPspphTEOS composite materials were characterized by FTIR spectroscopy. Figure 4 presents the FTIR spectra of pure PVA films and PVA with pphTEOS films in the absence or presence of Ag/AuNPs. In all cases, a broad absorption peak centered at around 3300 cm−1 is attributed to the O−H stretching vibration of hydrogen bonded hydroxyl groups present in the samples as well

Figure 1. Schematic representation of PVA-Ag/AuNPs-pphTEOS composite (where M refers to Ag or Au).

Si(OC2H5)4 + nH 2O hydrolysis

⎯⎯⎯⎯⎯⎯⎯⎯⎯→ Si(OH)n (OC2H5)4 − n + nC2H5OH

(1)

(HO)3 SiOH + HOSi(OH)3 condensation

⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯→ (HO)3 SiOSi(OH)3 + H 2O

(2)

The formation of AgNPs is evidenced from UV−Vis spectroscopy by the appearance of strong absorption bands at ∼420 nm48,49 when in the form of solution and in the form of films (SI Figure S-2). The appearance of absorption bands at a wavelength of ∼420 nm indicates the formation of spherical AgNPs,50,51 which is in accordance with the TEM observations. Relatively narrow and symmetrical absorption peaks of the liquid samples are indicative of relatively narrow size distribution and good dispersion of the AgNPs in the polymer matrix/film samples.52 It also shows that the absorption peaks of the AgNPs becomes stronger with increase in the Ag content from 50 to 500 mg, but an obvious decrease is shown when a content of 1000 mg AgNO3 is used. This shows that the polymer becomes saturated with the amount of AgNO3 added, therefore restricted C

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that the interaction between O−H groups from PVA and O−H groups from pphTEOS is a result of hydrogen bonding. However, the presence of Ag or AuNPs could not be detected by FTIR spectroscopy and no changes are observed in the absorption spectra from PVA to PVA-Ag/Au and from PVApphTEOS to PVA-pphTEOS-Ag/AuNPs films. Electrochemical Characterization of the PVApphTEOS-GOD Composite Films: Optimization of the Experimental Conditions. The ratio of PVA to pphTEOS in the presence of GOD was optimized. This was achieved by mixing various amounts of pphTEOS (0−0.5 mL) with PVA (1 mL) containing GOD (10 U μL−1) in the ratio of 4:1 by volume respectively, and applying to the electrode surface. The PVA-pphTEOS mixture in the volume ratio of 1:0.3 gave the highest peak current response when mixed with GOD. Ratios lower than this formed a layer that were not acceptably stable and broke down under use causing enzyme leaching. The PVA-GOD layer (1:0) showed an initial good response but upon repeated use led to decrease in glucose response. Upon addition of pphTEOS (1:0.1, 1:0.2 and 1:0.3) the responses gradually increased and on repeated use were much more stable. When a ratio of 1:0.3 was used, leaching of the enzyme lessened drastically and the response stabilized. With further increases in pphTEOS content (1:0.4 and 1:0.5) the current response to glucose rapidly decreased. A lack of diffusion of analyte to the enzyme through the increasingly denser film formation, and/or possible denaturation of GOD could have occurred with increase in amounts of pphTEOS. A ratio of 1:0.3 of PVA to pphTEOS was used for further experimentation as this mixture showed good balance between sensitivity and stability of the response toward glucose. Mechanism of Glucose Determination and Influence of Buffer pH. SI Figure S-6 (curve a) shows the cyclic voltammogram of the PVA-pphTEOS-GOD graphite electrode in 0.2 M PBS where no obvious reaction is taking place. However, in the presence 0.8 mM FMCA, a pair of reversible cyclic voltammetric peaks appeared (curve b). These peaks are assigned to one electron redox reaction of FMCA+/FMCA couple. Upon introducing glucose to the buffer solution containing FMCA, well-defined catalytic waves are observed (curves c−f). The electrocatalytic peak current increases by increasing the concentration of glucose in buffer solution. These results indicate that the immobilized GOD in PVA-pphTEOS retained its electrocatalytic activity for the oxidation of glucose. The reaction can be described by the following mechanism.

Figure 3. TEM image of PVA-AuNPs-pphTEOS film. Inset: Particle size distribution.

Figure 4. FTIR of the differently prepared and optimized PVA and PVA-composite films.

as uncondensed silanols (Si−OH). The obtained silanol groups during hydrolysis of TEOS are also expected to produce secondary interactions with the hydroxyl groups arising from the main PVA chain or with other silanols in the system.56 In all the spectra, there is a strong absorption peak observed at 1730 cm−1 characteristic of the carbonyl groups arising from the acetate groups in the partially hydrolyzed PVA. The peaks at 1430 and 1326 cm−1 are characteristic of the O−H groups and C−H deformation vibration respectively in PVA and are present in all spectra. The absorption peak at 1000−1100 cm−1 can be assigned to the C−O stretching and O−H bending vibrations arising from the PVA chain. In the same region, the asymmetric stretching vibration because of the Si−O−Si linkage as a result of the condensation reaction between hydrolyzed silanol Si−O−H groups as shown in the structures of Figure 1 is also present. The strong absorption at 470 cm−1 in PVA-pphTEOS films is attributed to deformation vibration of Si−O−Si bending and the peak at 960 cm−1 is characteristic of the Si−O−H stretching vibration. The strong absorption peak at 800 cm−1 begins to appear with increasing amounts of pphTEOS within the films indicating

FMCA ⇄ FMCA+ + e−

(3)

GOD(FAD) + glucose k1

XooY GOD(FADglucose) k −1 k2

→ GOD(FADH 2) + glucolactone

(4)

k3

GOD(FADH 2) + 2FMCA+ → GOD(FAD) + 2FMCA (5)

where GOD(FAD/H2) represent the oxidized and reduced forms of glucose oxidase, and FMCA/FMCA+ are the reduced and oxidized forms of ferrocene monocarboxylic acid mediator. Buffer solutions at various pH values were tested to investigate the effect of pH varied between 5.0 and 9.0. Supporting Information Figure S-7 shows that the maximum response was obtained at pH 7.0. Therefore pH 7.0 was selected as the D

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Figure 5. Current−time curves obtained at the PVA-Ag-pphTEOS-GOD (A lower plots) and PVA-Au-pphTEOS-GOD (A upper plots) graphite electrodes for successive additions of 0.5 mM glucose. Conditions: 0.2 M pH 7.0 PBS in the presence of 0.8 mM FMCA; applied potential, 0.55 V (vs Ag/AgCl). Plot B shows the calibration curves of the enzyme electrode as a function of glucose concentrations.

optimum pH and all measurements were performed at this pH. This value is in good agreement with most of the data reported in the literature57−59 and shows that the PVA-pphTEOS composite film did not alter the structure of GOD and still provided an environment similar to that of native GOD. Performance Characteristics of the Enzyme Electrodes. The amperometric I−t responses of the PVA-pphTEOSGOD and PVA-Ag/Au-pphTEOS-GOD electrodes under optimized experimental conditions to successive additions of 0.5 mM glucose is shown in SI Figure S-8A and in Figure 5A respectively. It is clear that rapid and sensitive responses to glucose could be achieved for all electrodes apart from those containing AgNPs. Calibration curves for the electrodes over the glucose concentration range of 0−9 mM are presented in SI data Figure S-8B and Figure 5B. The linear range for the electrode of PVA-pphTEOS-GOD was from 1 to 6 mM. The respective equation of calibration is Ip = 4.3221 × 10−5 Cglucose + 8.5072 × 10−6 with a correlation coefficient of 0.994. The slopes in each case represent the current sensitivity to glucose in Amps mM−1 cm−2. The limit of detection (LOD) for the enzyme electrode was estimated to be 0.35 mM at a signal-to-noise ratio (S/N) of 3. The LOD with the proposed electrode was lower than values obtained with the SPE/PB/SiO2−PVA− GOx, 5.47 mM and the SPE/PB/Ag−SiO2−PVA−GOx, 3.89 mM electrodes proposed by Zuo et al.29 The PVA-pphTEOS-GOD-graphite electrode reached 95% of the steady-state current within an average of 5 s as calculated from each step in the linear region. Before linearity began and as it came to an end, the response times were shorter. However, this response time was extremely fast for the increments of analyte (0.5 mM), and this was measured over within the linear range. Figure 5A shows the amperometric I−t responses of the PVA-Ag-pphTEOS-GOD and PVA-Au-pphTEOS-GOD electrodes, where the latter electrode displayed an extended linear glucose concentration range from 1 to 8 mM. The respective equation of calibration for PVA-Au-pphTEOS-GOD electrode is Ip = 4.9051 × 10−5 Cglucose + 1.5195 × 10−5 with correlation coefficient of 0.989, which means in the presence of AuNPs the sensitivity was improved by 13%. However, the LOD for the enzyme electrode was estimated to be 0.7 mM at a signal-tonoise ratio (S/N) of 3. The slight increase may have been caused by the slightly higher noise levels of the fluctuating value of the steady state background current due to the presence of the NPs. The PVA-Au-pphTEOS-GOD graphite electrodes reached 95% of the steady-state current within an average of 6 s, as calculated from each step in the linear region.

The PVA-Ag/Au-pphTEOS-GOD graphite electrodes were also tested under cyclic voltammetric conditions in PBS over the apparent direct electron transfer potential range for GOD (see SI Figure S-9). Although the glucose detection characteristics were improved with the presence of AuNPs, no response was seen to signify direct electron transfer from GOD as others have reported.24−27,60−62 The improvement in response can be a result from the large active surface areas and the excellent electron transfer ability of the AuNPs, but not great enough to penetrate the enzymes protein shell and develop direct connectivity. The presence of AgNPs showed an extremely degenerative response to glucose. This was suspected to be caused by the heavy metal Ag+ ions unreacted in the silver colloid.31 However, this property held by the AgNP’s was used to our advantage toward the effects of bacteria.45,46 The apparent Michaelis−Menten constant (Kapp m ) can be calculated from the electrochemical version of the LinweaverBurk equation: 1/Iss = 1/Imax + K mapp/Imax × C

where Iss is the steady-state current after the addition of substrate, Imax is the maximum current under saturated substrate conditions, C is the concentration of substrate. The Kapp m value for the PVA-pphTEOS-GOD and PVA-Au-pphTEOS-GOD electrodes was found to be 27 and 23 mM respectively, which are lower than the value of ∼33 mM, reported for the native enzyme.63−65 The smaller value of Kapp m signifies that GOD exhibits higher enzymatic activity and higher affinity for glucose. However, the values of Kapp m for GOD in this work are higher than that obtained at other glucose biosensors66,67 but also comparable.68,69 This can be attributed to the highly robust nature of the interlinked PVA and pphTEOS matrix, which imposes diffusional constraints on the substrate to the enzyme active site. In the presence AuNPs, the value of Kapp m is reduced signifying an improvement in the microenvironment of the enzyme and conductivity within the PVA-pphTEOS matrix. Reproducibility and Stability of the Enzyme Electrode. Several calibration curves were plotted by use of the independently prepared PVA-pphTEOS-GOD electrodes (SI Figure S-8A). The RSD of their sensitivities (gradients of the plots from SI Figure S-8B) was found to be 9.2%. This is highly acceptable when taking into account of multiple electrode preparations and testing. The immobilization of GOD by the entrapment in a PVApphTEOS composite film was relatively stable. When the PVApphTEOS-GOD graphite electrode was scanned continuously in a solution of glucose in the presence of FMCA mediator, the voltammetric response decreased slowly with further increase in E

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voltammetric cycles (SI Figure S-10). The peak current remained around 80% of the initial response after 50 cycles. Table 1 shows a summary of the key characteristics of the PVA-pphTEOS-GOD graphite electrode. Table 1. Summary of the Performance Characteristics at the Amperometric Enzyme Electrodes parameters

PVA-pphTEOS- PVA-Au-pphTEOSGOD GOD

linear range (mM) sensitivity (Amps cm−2 mM−1) R2 Imax (μA) response time (T95%, 0.5 → 1 mM, s) Kapp m (mM) LOD (S/N = 3, mM) reproducibility (RSD)

1−6 4.3221 × 10−5 0.994 65.8 5 27 0.35 9.2%

1−8 4.9051 × 10−5 0.989 86.0 6 23 0.7

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CONCLUSION In this study, GOD was successfully immobilized in a mixture containing silica sol−gel and PVA composite film. The enzyme electrode exhibited good performance for the electrocatalytic oxidation of glucose, such as high sensitivity, low detection limit, short response time, and a linear range that can be increased through further material manipulation and optimization. The favorable results were attributed to the sympathetic nature of PVA and improved stabilizing effect of pphTEOS created in the matrix. The presence of AuNPs in the immobilization matrix not only offers a biocompatible microenvironment but also efficiently improved electron transfer between GOD/FMCA and graphite electrode surface. Kapp m values for both types of electrodes were high, however, a higher loading of AuNPs homogenously dispersed within the PVA-TEOS matrix may further aid in increasing the substrate to enzyme affinity. The enzyme immobilization produced here can serve as a model toward simple and cheap fabrication of biosensors for the immobilization of many other enzymes through further research and development.

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ASSOCIATED CONTENT

S Supporting Information *

Additional material as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors wish to thank EPSRC for the DTA award to U.L. and British Council U.K. funding part of the research work on hybrid thin films in collaboration with Bulgaria. U.L. also wishes to thank SPEME/IMR for the infrastructure support during the course of this research work.

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