studies on glucose biosensors based on nonmediated and mediated electrochemical oxidation of reduced...

Studies on Glucose Biosensors Based on Nonmediated andMediated Electrochemical Oxidation of Reduced GlucoseOxidase Encapsulated Within Organically Modi®ed Sol-GelGlasses

P.C. Pandey,* S. Upadhyay, H.C. Pathak, Ida Tiwari, and V.S. Tripathi

Department of Chemistry, Banaras Hindu University, Varanasi-221005, India

Received: May 27, 1999

Final version: July 8, 1999

Abstract

A new, organically modi®ed sol-gel glass electrode is reported using 3-aminopropyltriethoxy silane and 2-(3,4-epoxycyclohexyl)-ethyl-trimethoxy silane as sol-gel precursors for the construction of electrochemical biosensors. Four different systems of new sol-gel glassmodi®ed glucose electrodes are made in acidic medium having common sol-gel precursors and: 1) glucose oxidase, 2) glucose oxidasealong with polyethylene glycol, 3) glucose oxidase and graphite powder, and 4) glucose oxidase along with polyethylene glycol andgraphite powder. Both nonmediated and mediated electrochemical regeneration of immobilized glucose oxidase within sol-gel glasses arestudied in these four systems. The nonmediated regeneration is achieved in the presence of oxygen as electron donor whereas mediatedregeneration involves soluble ferrocene monocarboxylic acid as electron donor in each system. The electrochemical performance of sol-gelglass based biosensors is compared on the basis of cyclic voltammetry and amperometry. This leads to the observations: i) all four systemsreach a diffusion limited condition associated with the transport of soluble ferrocene monocarboxylic acid as well as for dissolved oxygenwithin the sol-gel matrix, ii) the relative rate of diffusion of these analytes increases from system 1 to system 4, iii) both nonmediated andmediated amperometric responses at suitable potentials are based on the oxidation of H2O2 and enzymatically reduced soluble ferrocenewith relatively ampli®ed electrochemical signal of system 4. Data on the reduction of oxygen at conventional graphite disk electrode and attypical sol-gel glass modi®ed electrode are reported.

Keywords: Organically modi®ed sol-gel glass, Voltammetry, Amperometry, Glucose biosensor, Mediated bioelectrochemistry

1. Introduction

Several examples of the synthesis of sol-gel glasses [1±17]have become available during the last few years. One of thepossible applications of such materials in the development ofsensors is the attachment of the sensing material to the surface ofphysico-chemical transducers. The sol-gel process involves a lowtemperature production of ceramic materials through the hydro-lysis of an alkoxide precursor, followed by copolymerization ofthe hydroxylated monomers [11]. The use of sol-gel glass for thedevelopment of electrochemical biosensors has received greatattention associated with the coupling of biological components toelectrochemical transducers. The development of such biosensorsbased on sol-gel glass is currently restricted mainly due to twomajor problems: 1) the requirement of controlled gelation ofsoluble sol-gel components at ambient conditions, 2) preparationof a sol-gel glass with a smooth surface, controlled thickness andporosity. Apparently the synthesis of suitable biocompatible sol-gel glass of desired thickness and porosity is of considerableinterest. The soluble materials leading to the formation of sol-gelglasses are derivatives of alkoxysilane. The availability of a suit-able reactive group attached to alkoxysilane may provide anadvantage for the cross-linking of the sensing element to the solid-state network. The research work is aimed on these lines.

The sol-gel glasses modi®ed electrodes prepared by introducinggraphite particles (particle size 1±50m) have received great atten-tion [18±21]. The dispersed carbon provides the electrical con-ductivity essential for electrochemical measurements. Thesecarbon-ceramic electrodes (CCEs) can be bulk modi®ers byorganic, inorganic, or biochemical species [6] which can be sub-sequently used in the preparation of Pd-modi®er, enzyme-doped

carbon-Ormosil composite material (organically modi®ed Sol-Gelglasses) and this made it possible to cast silica-carbon matrices invirtually any desired geometrical con®guration, including ¯atlayers spread on insulating or conductive matrices, monolithicdisks or rods [16] or even in the form of miniature CCEs [9].Another way for the construction of modi®ed CCEs which can beused for the large surface area ampli®cation is the use of hydro-philic and hydrophobic silica-forming monomers, such as cya-noethyltri-alkoxysilane as hydrophilic monomer and methyltri-methoxysilane as hydrophobic monomers. When hydrophobicsilica forming monomers are used, the resulting electrodes rejectwater, leaving only segregated islands of carbon at the outermostsurface in contact with electrolyte [16]. Thus the ratio of hydro-philic and hydrophobic monomers in organically modi®ed sol-gelCCEs is crucial in sensor design. Additionally monomers con-taining a Si±C bond and an easily derivatized radicals throughcross-linking such as an amino, vinyl-, epoxy-, or mercapto-groups can be used to prepare readily derivatized xerogels[16] andprovides a convenient method for the production of organicallymodi®ed surfaces [22] which can be used as covalent anchors forspeci®c chelating agents. The lower degree of cross-linking pro-vides an inherent strain relaxation pathway allowing thick (>1mm)silica ®lms to be prepared in one coating step [23]. Another way ofincreasing the exposed surface area (wetted section of sol-gel glass)is by incorporating readily leachable, water-soluble components inthe matrix and dissolving them out by immersing the electrodes inan electrolyte solution. This leaves wide open channels for thepenetrating electrolyte, thereby increasing the wetted section insidethe sol-gel matrix. A typical example is the use of poly(ethyleneglycol) [6]. The present research investigation is aimed to developorganically modi®ed electrodes using 3-aminopropyltriethoxy-

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silane (relatively hydrophilic modi®er) and 2-(3,4-epoxy-cyclohexyl)-ethyltrimethoxy silane (relatively hydrophobicmodi®er) in the presence and absence of poly(ethylene glycol) forthe construction of four different organically modi®ed glucoseCCEs (glucose biosensors).

Mediated biosensors [24±27] have gained wide attention forprobing redox enzyme catalyzed reactions. However, the incor-poration of a redox mediator within sol-gel network may providestrongly diminished electrochemistry in dried xerogel samplesdue to the restricted mobility in a dried sol-gel structure [15, 28].Similarly Pankrato and Lev [29] reported tetrathiafulvalenemediated CCEs, which, however, had only limited storage and in-use stability. On the other hand many organic and inorganicsoluble analytes are small enough to diffuse through the nano- tomicro-porous structure of the sol-gel glass [13].

We have recently described a glucose biosensor based onsandwich con®guration of organically-modi®ed sol-gel glass[30]. The response of the biosensor based on nonmediatedmechanism and subsequently using soluble ferrocene was used tounderstand the heterogeneous mediated electrochemical regen-eration of glucose oxidase has been reported [30].The ferrocenemonocarboxylic acid present in solution may penetrate within thewetted section of sol-gel glass and hence heterogeneous mediatedelectron transfer resulting through the sol-gel encapsulated glu-cose oxidase catalyzed reaction is desirable. The sandwich con-®guration of glucose biosensor was made using a based layer oforganically modi®ed sol-gel glass followed by a layer of adsor-bed glucose oxidase and subsequently another organically mod-i®ed sol-gel glass layer was assembled. This new organicallymodi®ed sol-gel glass was made using 3-aminopropyltriethoxysilane and 2-(3,4-epoxycyclohexyl)-ethyltrimethoxy silane assol-gel main precursors. However, it is necessary to understandthe analytical performance of this new sol-gel glass within whichglucose oxidase is encapsulated within a single sol-gel glasslayer. Based on this approach we developed four different sys-tems using these two silanes as the main sol-gel precursors andsubsequently these four systems were different in the presenceand absence of graphite powder (particle size 1±2m). We haveobserved remarkable ®ndings on the four systems based on theapproach of nonmediated and mediated mode of electrochemicalregeneration of glucose oxidase. A critical comparison con-sidering the water wettability area of sol-gel glass, rate of oxygendiffusion and diffusion limited conditions of soluble ferrocene onthese four systems is reported. The results on cyclic voltammetryand amperometry in the presence and absence of glucose arereported on these four systems. The overall goal of this work is i)to design functional sol-gel precursors based glucose biosensors;ii) to study the variation in the properties of functional sol-gelprecursors sol-gel glass ®lm in the presence and absence ofdopants like polyethylene glycol and graphite powder whichprovide four different systems; iii) to make a critical comparisonof nonmediated and mediated bioelectrochemistry of sol-gelglass based biosensors; iv) to study the stability, reproducibilityand selectivity of sol-gel glass based biosensors.

2. Experimental

2.1. Materials

3-Aminopropyltriethoxy silane was obtained from Aldrich; 2-(3,4-epoxycyclohexyl)-ethyltrimethoxy silane was obtained fromUnited Chemical Technologies, Inc., Petrarch Silanes and Sili-

cones, Bristol, PA, USA; Glucose oxidase was obtained fromSigma. All other chemicals employed were of analytical grade.

The electrode body used for the preparation of composite sol-gel glass modi®ed electrodes was similar to that described in anearlier publication [31] made from Te¯on containing platinumbase with a recessed depth of 2 mm. The new materials of thefour different systems were developed with common sol-gelprecursors; 3-aminopropyltriethoxy silane (70mL), 2-(3,4-epoxy-cyclohexyl)-ethyltrimethoxy silane (20mL), 0.1 M HCl (5 mL),glucose oxidase (4 mg) and in the presence and absence of otheradditives providing four different CCEs; 1) 700 mL double dis-tilled water only 2) 700mL saturated aqueous solution of poly(ethylene glycol) [mol.wt. ca. 6000]; 3) 700mL double distilledwater and 2 mg graphite powder [particle size; 1±2 m], and 4)700mL saturated aqueous solution of poly(ethylene glycol) and2 mg graphite powder. The resulting mixture in each cases wasstirred thoroughly and the desired amount of the homogeneoussolution was added to the well of the specially designed electrodebody. The gelation was allowed to occur at 25 �C for 30 h. Asmooth very thin GOD immobilized ®lm of sol-gel glass givingfour different glucose CCEs which differ in the presence andabsence on polyethylene glycol and graphite particles appearedon the Pt surface. The electrode obtained in this manner waswashed with 0.1 M phosphate buffer, pH 7.0 several times andstored in 0.1 M phosphate buffer pH 7.0 at 4 �C when not in use.Some of the glucose sensors made following such procedureswere stored at room temperature (26 �C).

2.2. Measurements

The electrochemical measurements were performed with aSolartron electrochemical interface (Solartron 1287 Electro-chemical Interface, UK) connected to a PC. A one compartmentcell with a working volume of 4 mL and a sol-gel glass modi®edworking electrode, Ag=AgCl=3 M NaCl reference electrodeseparated from the test solution by a ceramic frit and a platinumfoil auxiliary electrode were used for the measurements. Thesource of chloride ion in the system was a saline phosphatebuffer. The cyclic voltammetry using GOD modi®ed sol-gel glasselectrode was studied between 0±1 V (vs. Ag=AgCl). The non-mediated amperometric measurements using GOD immobilizedsol-gel modi®ed electrode was operated at 0.70 V (vs. Ag=AgCl).The experiments were performed in phosphate buffer (0.1 M, pH7) employing both types of modi®ed electrodes.

The electrochemical measurements based on mediated elec-tron exchange were studied using an aqueous solution of ferro-cene monocarboxylic acid in 0.1 M phosphate buffer pH 7.1.First the electrochemistry of soluble ferrocene on sol-gel glassesmodi®ed biosensors was studied at various scan rates in thepotential range from ÿ0.2 to 0.6 V (vs. Ag=AgCl). Cyclic vol-tammetry in the presence and absence of glucose was also used toidentify the mediated mechanism of electron exchange from theimmobilized glucose oxidase within sol-gel glasses. Theamperometric responses of the sol-gel glasses based glucosebiosensors were studied at 0.35 V (vs. Ag=AgCl). The back-ground current was allowed to decay to a steady state in thestirred solution followed by injection of increasing concentra-tions of glucose. Before the measurements of mediated electronexchange using soluble ferrocene, nitrogen was passed throughthe system to remove oxygen.

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Electroanalysis 1999, 11, No. 17

3. Results and Discussion

3.1. Physical Characteristics of Sol-Gel Glass Based

Biosensor

The physical characteristics of sol-gel glass made from the twosilanes in the absence of polyethylene glycol and graphite powderwas studied in the presence and absence of glucose oxidase.These two silanes result in a very smooth surface of sol-gel glass.Figure 1a show the scanning electron microscopy of the sol-gelglass (system 1) in absence of glucose oxidase at magni®cationsof 1000 and 1500, respectively. The micrographs show a verysmooth surface. Figure1b shows the SEM of sol-gel glass withimmobilized glucose oxidase (system 1) at magni®cations of 500and 2000, respectively. The micrograph shows the sol-gelencapsulated glucose oxidase with a smooth surface. The presentsol-gel glasses, the surface structure of which are shown inFigures 1a and b, were prepared following a very simple one stepgelation process and the sol-gel precursors did not requiresonication for the homogenization of the monomers andadditives suspension. The monomers were mixed by simplestirring and this even provides better sol-gel surface as comparedto relatively complex protocols of gelation reported for the pre-paration of sol-gel glasses by earlier workers [6, 7, 10]. A smoothsol-gel ®lm without cracking and having a better performancewhen used as sensor was obtained using the optimum con-centrations of sol-gel precursors reported in the experimentalsection. The scanning electron microscopy of system 4 shown inFigure 1c obviously shows better porosity with a smooth surface

and evidently better sensing performance as discussed vide infra.The organically modi®ed sol-gel glass matrix inhibits inter-molecular interactions of the encapsulated macromolecules [13].The presence of the rigid silicate cage prevents protein movementand the matrix functions as a solid solution of encapsulatedbiomolecules.

3.2. Effect of Functional Sol-Gel Precursors Composition

on the Physical Properties of Sol-Gel Glass

Biosensors

The physical characteristics of the sol-gel glass have beenexamined using various compositions of 3-aminopropyltriethoxysilane, 2-(3,4-epoxycyclohexyl)-ethyltrimethoxy silane, andenzyme dissolved in water and HCl. The content of 2-(3,4-epoxycyclohexyl)-ethyltrimethoxy silane signi®cantly affect thephysical characteristics of the sol-gel matrix which is a hydro-phobic monomer of organically modi®ed sol-gel glass. We stu-died at three different concentrations of 3-aminopropyltriethoxy-silane (20, 40, 80mL) with the same concentration of 2-(3,4-epoxycyclohexyl)-ethyltrimethoxy silane (50 mL). The physicalcharacteristics of the sol-gel glass made using 80 mL of 3-ami-nopropyltriethoxysilane were relatively better. Similarly, we alsostudied at three different concentrations of 2-(3,4-epoxy-cyclohexyl)-ethyltrimethoxy silane (20, 40, 80mL) with sameconcentration of 3-aminopropyltrimethoxysilane (70 mL). Thesol-gel obtained with a high amount of epoxycyclohexyl-group

Fig. 1. a) Scanning electron microscopy of organically-modi®ed sol-gel glass in absence of enzyme; magni®cation 1) 10006 and 2) 15006. b) In thepresence of enzyme; magni®cation: 3) 5006 and 4) 20006. c) Scanning electron microscopy of organically-modi®ed sol-gel glass (system 4);magni®cation: 5006.

Glucose Oxidase Encapsulated Within Sol-Gel Glasses 1253


resulted in a brittle and cracked surface, while on the other handa low amount (20 mL) of epoxycyclohexyl-group resulted in asmooth sol-gel ®lm. Another important requirement of the sen-sors based on such design is the coupling of the modi®ed sol-gel®lm and the Pt surface, which contributes signi®cantly to theperformance and storage stability of the biosensors. The presentmodi®ed sol-gel ®lm has been found to be strongly attached tothe Pt surface and is not easily removed from the Pt surface.

3.3. Nonmediated Sol-Gel Glasses Based Biosensors

First we studied the electrochemical behavior of sol-gel glassbiosensors of the four different compositions in absence of anyelectron-transfer mediator. The cyclic voltammetric studies wereconducted in between 0±1 V (vs. Ag=AgCl) whereas ampero-metric measurements were made at 0.70 V (vs. Ag=AgCl). Thecyclic voltammetry results for systems 3 and 4 in the presenceand absence of 50 mM glucose are shown in Figure 2a and b,respectively between 0 and 1 V (vs. Ag=AgCl) at the scan rate of5 mV=s in 0.1 M phosphate buffer pH 7.0. at 25 �C. Curve 1 is inthe absence of glucose whereas curve 2 is in the presence ofglucose. On the addition of glucose (curves 2 of Fig. 2a and b) alarge increase in anodic current corresponding to the oxidation ofhydrogen peroxide is shown. The magnitude in the increase ofanodic current in system 4 was greater than that in system 3. Thedata on amperometric response on these four systems are shownin Figure 3. Curves 1, 2, 3, and 4 show the typical amperometricresponses of the biosensor on subsequent addition of glucoserecorded for the systems 1, 2, 3, and 4 respectively. The system 4(curve 4 of Fig. 3) shows the relatively large magnitude of theamperometric response in accordance to the cyclic voltammetricresults. The inset (I) to Figure 3 shows the calibration plots for

Fig. 2. a) Cyclic voltammograms of sol-gel glass modi®ed glucosebiosensor made from system 3 in absence (1) and the presence (2) of50 mM glucose in 0.1 M phosphate buffer pH 7.0 at the scan rate of5 mV=s. b) Cyclic voltammograms of sol-gel glass modi®ed glucosebiosensor made from system 4. Other conditions as in (a).

Fig. 3. Typical amperometric response of sol-gel glass based glucose biosensors of four different compositions : 1) system 1, 2) system 2, 3) system 3,and 4) system 4. The recording shows typical response curves on the addition of subsequent increasing concentrations of glucose. The calibration plotsfor glucose analysis based on nonmediated electrochemical reaction from data are recorded in inset (I). The electrodes were held at 0.70 V (vs.Ag=AgCl). Inset (II) shows the cyclic voltammograms of sol-gel modi®ed electrodes showing the oxygen reduction in 0.1 M phosphate buffer pH 7.0at the scan rate of 5 mV=S; curve 1 was recorded from graphite powder dispersed sol-gel modi®ed electrode without glucose oxidase whereas curve 2was recorded using system (4). Inset (III) shows the cyclic voltammogram showing oxygen reduction recorded using bare graphite disk electrode (ù3 mm).



glucose analysis. The system 1 (curve 1 of Fig. 3) shows thelowest amperometric response.

3.4. Effects of Dopants on the Performance of Sol-Gel

Glass Based Biosensors

The system 1 shows the lowest amperometric responsewhereas system 4 shows a relatively larger amperometricresponse as shown in Figure 3. This is mainly due to a relativelyless concentration of oxygen at the site of the enzymatic reactionrequired for the formation of hydrogen peroxide which can beexplained from the consideration of the exposed surface area ofthe sol-gel glass to the solution and water contact angle. It hasbeen reported that neither highly hydrophobic nor totallyhydrophilic sol-gel matrices are desirable for sensing application[6]. When chemical modi®ers such as metal dispersion, watersoluble polymers and proteins are added to the materials, theresulting electrodes became more hydrophilic and subsequentlyalter the water contact angle which manifests the wettability. Ithas been reported [6] that a blank sol-gel electrode without anyhydrophilic modi®er shows the highest water contact angle (80�)and in turn the lowest wettability (not amenable for nitrogenadsorption analysis) whereas the sol-gel electrode with allhydrophilic modi®ers (carbon, poly(ethylene glycol), and Pd-GOD) shows the lowest water contact angle (42�) and highestwettability (42 m2=g). An increase in the wetted area increasesthe wetted conductive surface accessible to the solution and alsothe corresponding electrochemically active area and capacitivecurrent. On the other hand the unwetted area does not contributeto the capacitive or faradic currents. The results reported inFigure 3 follow the similar arguments. Recently Ingersoll andBright have also reported on the effect of dopant addition timeand used oxygen as the analyte to study sensor performance [32].The variation of oxygen concentrations has also been determinedexperimentally based on direct electrochemical reduction ofoxygen at system 3 without glucose oxidase, system 4 and at thebare Pt surface. These results are shown in insets (II) and (III) ofFigure 3, respectively. The curves 1 and 2 of inset (II) to Figure 3show the oxygen reduction at system 3 without glucose oxidaseand at the surface of system 4 respectively. It is clear that system4 shows a large cathodic peak current as compared to that ofsystem 3 (curve 1) showing a relatively increased rate of oxygendiffusion within the wetted section of system 4. Finally we alsostudied the oxygen reduction at a bare Pt electrode in the sameaqueous medium and the result are shown in inset (III) of Figure3 which verify the two electron transfer as reported earlier [33,34]. These data demonstrate that the rate of oxygen diffusionincreases from system 1 to system 4 in accordance to the dataconcluded from the surface wettability area.

3.5. Electrochemical Performance of Sol-Gel Glass

Biosensors Based on Four Different Systems

It is important to discuss the relative variation of amperometricresponse as given in the calibration plot for glucose analysis inset(I) of Figure 3 on the same concentrations of glucose and similaramount of enzyme with similar speci®c activity. The sol-gelmatrix is a compact network. The enzyme is caged within the sol-gel network hence occurrence of limited mass-transport kinetics atthe solution=sol-gel glass (within the wetted surface area) isdesirable which is also indicated by the results recorded vide infra.The increase in anodic current in all four systems is based on theformation of hydrogen peroxide as a function of the enzymaticreaction. The formation of hydrogen peroxide, keeping all othercomponents of the enzymatic reaction constant, depends on i) theconcentration of dissolved oxygen diffusing solution across thewetted area of sol-gel glass and ii) glucose concentration withinthe sol-gel glass matrix. The results discussed above based on thecontact angle and wetted area and also proved experimentally onoxygen reduction suggest that the diffusion of oxygen is a rate-limiting step. The relative rate of dissolved oxygen diffusionincreases from system 1 to 4 since the wetted area increases due tothe introduction of hydrophilic modi®ers. Although diffusion ofglucose across the wetted area may also be a rate-limiting step,diffusion of glucose will continue until a steady state is reached.This might cause an increase in response time but not in magni-tude of the amperometric response which is also supported by theexperimental data discussed above. Thus the lowest amperometricresponse of system 1 is mainly due to limited concentrations ofdissolved oxygen which in turn is mainly due to relatively lesswetted area in system 1 as compared to that of system 4 having arelatively greater wetted area. Thus although sol-gel glass is agood biocompatible material for enzyme immobilization withbetter stability, its network creates a diffusion controlled processof analytes within the sol-gel matrix and the equilibrium isreached after a longer time. Additionally, the nonmediateddetection of glucose requires oxygen consumption to formhydrogen peroxide that can be electrochemically detected. Theavailability of oxygen at the site of enzymatic reaction is a rate-determining step as evidenced by some more experiments dis-cussed above although, the relative rate of glucose diffusion isdifferent in all four systems due to change in the water contactangle as reported earlier by Sampath and Lev [6]. The results onthe amperometric responses recorded in Figure 3 support thisconclusion. A comparison on the electrochemical performance ofthese four systems is given in Table 1. However, if there is amediated electron exchange, by replacing oxygen using somemediator of well-de®ned electrochemistry, the dependence ofoxygen concentration as a rate-limiting step may be eliminated.Accordingly, the construction of a mediated enzyme electrodewhich has been commercially implemented using ferrocene

Table 1. Comparison of electroanalytical performance of sol-gel glass biosensors based on four different systems.

Nonmediated electrochemistry Mediated electrochemistry

imax Response ibackground Linearity imax Response ibackground LinearitySystem [mA] time [s] [mA] [mM] [mA] time [s] [mA] [mM]

1 1.30 105 0.30 06 44.70 60 7.10 402 3.18 80 0.63 15 72.00 135 7.83 453 6.85 95 0.66 25 43.80 280 4.55 554 12.58 140 0.27 50 95.67 225 15.4 60



modi®ed glucose oxidase [35] is of great interest. The biosensorsbased on sol-gel glass requires the immobilization of glucoseoxidase and mediator together within the sol-gel glass matrix. Wetried to incorporate dimethyl ferrocene together with glucoseoxidase within the sol-gel glass but we could not observe themediated electrochemical signal. These ®ndings indicate that theferrocene molecule immobilized within the sol-gel glass does notallow the reaction between the immobilized glucose oxidase andthe immobilized mediator which is related to the restrictedmobility of the mediator as well as enzyme within the sol-gelmatrix. In the present case we used soluble ferrocene to investi-gate the mediated response from the glucose oxidase which dif-fuses within the reactive surface area of sol-gel matrix and indeedwe observed remarkable results on all four systems which arediscussed below.

3.6. Mediated Bioelectrochemistry of Sol-Gel Glasses


Actually the con®guration of the biosensors based on thepresent approach does not ful®l the requirement of the mediatedglucose biosensor, however, we have found some very interestingobservations on the mediated bioelectrochemistry using solubleferrocene and glucose oxidase immobilized within the sol-gel

glasses of the four different systems. We studied the electro-chemistry of ferrocene monocarboxyic acid at the surface of theorganically modi®ed sol-gel glass based glucose electrodes oftwo different systems (1 and 4). Figures 4a and b show theelectrochemistry of soluble ferrocene monocarboxylic acid usingsystem 1 and 4, respectively, at different scan rates (3, 6, 10, 20,50 and 100 mV=s) between ÿ0.2 and 0.6 V (vs. Ag=AgCl). Thevoltammograms of these systems were recorded in the sameferrocene solution. Before the start of the measurements eachenzyme immobilized sol-gel glass electrode was allowed toincubate for 2 h in ferrocene solution to attain a steady-statemass-transfer kinetics within the wetted area of sol-gel glass. Theinset in each ®gures shows the plot of anodic peak current vs.square root of scan rates. The results shown in the inset followthe same trends as described by Wang et al. [21] that show alinear relation between peak current and square root of the scanrates although the linear line does not pass through the originwhich shows that the systems are not very well diffusion con-trolled. The peak separation increases with scan rates. However,the peak separation decreases from system 1 to system 4 from84 mV to 60 mV, respectively, at the same scan rate. Wang et al.[21] has also reported the peak separation of 57 to 65 mV in twosol-gel systems and linear lines obtained from the plots of peakcurrent vs. square root of the scan rate also do not pass throughorigin. The voltammograms shows reversible electrochemistry offerrocene in each system with diffusion limited conditions ineach case as evidenced from the results shown in insets. The data

Fig. 4. a) Cyclic voltammograms of 5 mM ferrocene monocarboxylicacid in 0.1 M phosphate buffer pH 7.1 on sol-gel glass modi®ed elec-trode made from system 1 at 25 �C at the scan rate of 3, 6, 10, 20, 50, and100 mV=s. The inset shows the plot of anodic peak current vs. squareroot of scan rate. b) Cyclic voltammograms of ferrocene monocarboxylicacid ion sol-gel glass modi®ed electrode made from system 4. Otherconditions as in (a).

Fig. 5. a) Cyclic voltammogram of GOD immobilized composite sol-gelglass (system 1) in absence (1) and presence (2) of 150 mM glucose0.1 M phosphate buffer pH 7.1 containing 5 mM ferrocene mono-carboxylic acid at the scan rate of 5 mV=s. b) Cyclic voltammogram ofGOD immobilized composite sol-gel glass (system 4). Other conditionsas in (a).



in Figure 4 show that peak current is relatively high in system 4(Fig. 4b) associated to relatively faster diffusion of ferrocene ascompared to system 1 to 4 in accordance to the conclusion drawnbased on the nonmediated mode of the electrochemical reactiondiscussed above. Subsequently we examined the relativelymediated electrochemical response of the four systems in thepresence and absence of 150 mM glucose. The cyclic voltam-mograms using systems 1 and 4 in aqueous solution of ferrocenemonocarboxylic acid between ÿ0.2 to 0.6 V (vs. Ag=AgCl) atthe scan rate of 5 mV=s are shown in Figures 5a and 5b,respectively. Curve 1 of each voltammogram shows the recordingin absence of glucose whereas curve 2 shows the recording in thepresence of glucose. There is a large increase in anodic currenton the addition of glucose in each system showing the mediatedelectron exchange from the immobilized glucose oxidase. Hereagain the variation in the magnitude of anodic peak current ismuch larger in system 4 (Fig. 5b) again supporting our conclu-sion discussed above. In this case the rate of glucose diffusion isrelatively more rapid and apparently, keeping the second orderrate constant for the reaction between reduced glucose oxidaseand oxidized ferrocene in all four systems. The concentration ofglucose in system 4 at the site of enzymatic reaction is greaterthan all the other systems (1±3). In order to have a deeper insight

of the mediated electrochemical reaction between soluble ferro-cene and immobilized glucose oxidase, we studied the ampero-metric responses of all four systems in the presence of a constantconcentration of soluble ferrocene monocarboxylic acid. Figure6a shows the typical amperometric response curves of systems 1±4 at the constant potential of 0.35 V (vs. Ag=AgCl). The responsetimes recorded for systems 1 and 2 are very large (Fig. 6a; curves1 and 2) as compared to response time recorded for systems 3and 4 (Fig. 6a, curves 3 and 4). This suggests that a steady-statevalue, which is a function of the diffusion rate of glucose, wouldbe reached relatively more rapidly in systems 3 and 4 as com-pared to systems 1 and 2. Figure 6b shows the typical hetero-geneous mediated response of system 3 and 4, respectively. Theinset of Figure 6b shows the calibration plots for glucose analysisbased on mediated bioelectrochemistry of system 3 and 4. Sys-tem 4 shows high amperometric responses (Fig. 6a, curve 4; Fig.6b, curve D) both in nonmediated and mediated mode of thebioelectrochemical reaction. Thus incorporation of polyethyleneglycol and graphite powder signi®cantly affect the morphologyof the sol-gel glasses based biosensor that is related to theintroduction of hydrophilic modi®ers in order to control thethickness of the wetted section of the electrode (reaction layerthickness). Additionally the incorporation of graphite particlesnot only increases the wetted surface area of the electrode, it alsofacilitate the electron transfer within the sol-gel matrix as a resultof increased electronic conductivity of the electrode. A com-parison of mediated amperometric response of these four systemsis shown in Table 1.

3.7. Stability and Reproducibility of Sol-Gel Glass


The stability of these sol-gel glass based biosensors of fourdifferent types is determined under two conditions. In the ®rstcase the enzyme electrode (each system) was stored in 0.1 Mphosphate buffer pH 7.0 at 4 �C whereas in the second case theenzyme electrode was stored at room temperature in the samebuffer. The stability of the enzyme electrode stored under the ®rstcondition is relatively better, as compared to that of earlier sol-gelglass based glucose sensors under similar conditions, withoutloss of the amperometric response after 2 months. Under thesecond storage condition the response is consistent without lossfor 20 days. The reproducibility of the sensor design wasinvestigated using ten sets of a symmetrical Te¯on electrodebody and using a constant amount of sol-gel precursors anddopants. In each case 16mL of homogenous precursors solutionwas added in to the well of the electrode body with a recesseddepth of 2 mm. This leads to the production of ca. 0.3 mmthickness of a sol-gel layer under 24 h drying at (25 �C).Although the nature of individual events is random and thegeometry and pore-size distribution of the product gel are dif®-cult to determine, the nature of polymeric gel can be regulated toa certain extent by controlling the rates of the individualsteps [13].

3.8. Selectivity of Sol-Gel Glass Based Biosensors

Finally it is most important to study the selectivity of theglucose oxidase encapsulated within the organically modi®edsol-gel glasses. The selectivity of the encapsulated GOD withinsol-gel glass was determined using sucrose, fructose, and b-D-

Fig. 6. a) Typical response curves of the sol-gel glass modi®ed enzymeelectrodes made from systems 1±4, respectively, on the subsequentaddition of constant concentrations of glucose recorded in 0.1 M phos-phate buffer, pH 7.1 containing 5 mM ferrocene at 0.35 V (vs.Ag=AgCl.) b) Typical response curves of the sol-gel glass modi®edenzyme electrode made from systems 3 and 4 respectively, on the sub-sequent addition of increasing concentrations of glucose recorded in0.1 M phosphate buffer, pH 7.1 containing 5 mM ferrocene at 0.35 V (vs.Ag=AgCl). The inset shows the calibration plots for glucose analysis: Cfor system 3 and D for system 4.



glucose. Figure 7 shows the response of the two sets of sol-gelbased biosensors made from system 4 which differ in thethickness of the sol-gel glass. The arrows indicate the addition ofsucrose (a, a0), fructose (b, b0) and glucose (c, c0). There was noresponse on the addition of 100 mM sucrose and fructosewhereas good amperometric response was observed on theaddition of glucose (curve 1). Curve 2 shows the same results onthe evaluation of the selectivity however, the response time of thesensor is relatively much larger which is due to the relativelythicker ®lm of the sol-gel glass. The sol-gel ®lm (curve 2) wasmade using 32mL of the sol-gel precursors which is twice theamount as compared to the sol-gel ®lm giving the responserecorded in curve 1. The inset of Figure 7 shows the ampero-metric response of the sol-gel modi®ed electrode made from sol-gel precursors and graphite powder with no GOD. The arrowsshow the addition of sucrose (1), glucose (2) and standard per-oxide solution [10 mM] (3). The results show no response toglucose or sucrose whereas an amperometric signal is recordedon the addition of peroxide with a relatively longer responsetime.

4. Conclusion

We report the construction of new organically modi®ed sol-gelglass electrode of four different systems using 3-aminopropyl-triethoxysilane and 2-(3,4-epoxycyclohexyl)-ethyltri-methoxysilane, in the presence of distilled water and HCl as commonprecursors. These four systems differ among each other in thepresence and absence of dopants such as polyethylene glycol andgraphite powder. The enzyme encapsulated sol-gel glass ®lm ofsystem 4 which is comprised of both polyethylene glycol andgraphite powder shows relatively much better sensor perfor-mance as compared to the other three systems. These dopantsprovide controlled pore size distribution and increase in reactionlayer thickness (wetted surface area) as compared to early sol-gel

glucose biosensors. The comparative bioelectro-chemistry basedon both mediated and nonmediated approaches of these foursystems is reported. The results reported on these systems openfuture projections in designing mediated sol-gel glass basedbiosensor by co-immobilizing both the mediator and redoxenzyme.

5. Acknowledgement

The authors are thankful to UGC, New Delhi for ®nancialassistance.

6. References

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Fig. 7. Typical amperometric response of sol-gel modi®ed electrode onthe addition of sucrose, fructose, and glucose. Curve 1 was recordedfrom system 4 whereas curve 2 was also recorded from system 4 withdouble thickness of the sol-gel ®lm. The arrows show the addition ofsucrose (a, a0), fructose (b, b0), and glucose (c, c0). The inset shows theamperometric response of the graphite powder dispersed sol-gel mod-i®ed electrode without glucose oxidase. The arrows show the subsequentaddition of sucrose (1), glucose (2) and H2O2 (3) [10 mM]. In each casethe modi®ed electrodes were operated in 0.1 M phosphate buffer pH 7.0at 0.7 V (vs. Ag=AgCl).



studies on glucose biosensors based on nonmediated and mediated electrochemical oxidation of reduced...

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