A New Glucose Biosensor Based on Sandwich Con®guration ofOrganically Modi®ed Sol-Gel Glass

P. C. Pandey,* S. Upadhyay, and H. C. Pathak

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

Received: August 20, 1998

Final version: October 15, 1998

Abstract

A new glucose biosensor was developed based on the sandwich con®guration of organically modi®ed sol-gel glasses. The new sol-gel glasswas developed using 3-aminopropyltrimethoxy silane and 2-(3,4-epoxycyclohexyl)-ethyltrimethoxy silane. Two types of sol-gel glasseswere used to develop glucose biosensors that differ in absence (A) and the presence of graphite powder [particle size 1±2 m] (B). Anadditional additive (polyethylene glycol, Mol. wt. 6000) was also incorporated in both types of the upper sol-gel glass layer. The new sol-gelmatrix with immobilized glucose oxidase was analyzed by scanning electron microscopy (SEM).The sandwich con®guration was developedusing a bilayer of sol-gel glasses having a layer of glucose oxidase in between the bilayer. This electrode with special con®guration was usedto form a layer of sol-gel glass of ca. 0.2 mm thickness. The performance of sol-gel glasses (A & B) was analyzed based on cyclicvoltammetry using ferrocene monocarboxylic acid. The results show a diffusion limited condition of ferrocene across the sol-gel matrix.The characterization of sol-gel glass based biosensor was recorded based on the cyclic voltammograms in absence and presence of glucose.The results show an increase in anodic current which is also characteristic of hydrogen peroxide oxidation in both cases (A & B). Theresponses of the sol-gel glasses based biosensors were analyzed based on chronoamperometric measurements. An ampli®ed signal on theaddition of the same concentrations of glucose was recorded with the B-type sol-gel glass electrode which was attributed to its relativelyhigh porosity and better conductivity of the graphite loaded sol-gel glass. These observations were in accordance with the results on thediffusion of ferrocene and the magnitude of anodic current resulting from hydrogen peroxide oxidation. The calibration plots for glucoseanalysis using both type of sensors are reported. Data on the mediated electrochemical oxidation of glucose oxidase using soluble ferrocenewere also reported based on cyclic voltammograms and amperometric measurement.

Keywords: Glucose biosensor, Sandwich con®guration of organically modi®ed sol-gel glass, Ferrocene

1. Introduction

Recent reports on the synthesis of sol-gel glasses [1±6] havereceived widespread attention because of their application invarious directions. One of the potent applications of such mate-rials is in the development of sensors particularly for attachingthe sensing material to the surface of physico-chemical trans-ducers. A number of publications are available in the literature onthe applications of sol-gel glass for the development of opticaland electrochemical sensors since the sol-gel process involves alow temperature production of ceramic materials through thehydrolysis of the alkoxide precursor, followed by co-poly-merization of the hydroxylated monomers [7]. Wang et al. [8, 9]developed biogel-based carbon inks that display compatibilitywith the screen-printing device to develop microband electrodes.The development of an electrochemical biosensor involvesthe coupling of biological components with polarizable or non-polarizable electrodes. The use of sol-gel glass for the develop-ment of electrochemical biosensors have received great attentionbecause of their possible applications in commercialization. Thedevelopment of such biosensors based on sol-gel glass iscurrently restricted mainly due to two major problems: 1) therequirement of controlled gelation of the soluble sol-gelcomponents at ambient conditions, 2) preparation of a sol-gelglass of smooth surface, controlled thickness and porosity.Additionally the stability of the biological element within the sol-gel network is another need to develop such sensors atcommercial scale. Apparently the synthesis of suitable biocom-patible sol-gel glass of desired thickness and porosity is ofconsiderable interest. The soluble materials leading to the

formation of sol-gel glasses are the derivatives of alkoxysilane.These alkoxysilanes in acidic and sometime basic mediumgenerate a solid network whose physical structure can becomparable to conventional glass. However, research is neededto synthesize such sol-gel glasses suitable for better performanceas sensors and reactors of practical signi®cance. The applicationof these glasses in sensors designing requires a control synthesisof the solid-state network with the desired porosity and thickness.Additionally, the availability of a suitable group within the solid-state network provides an advantage for the cross-linking of thesensing element to the solid-state network.

Wang et al. [10] recently reported a novel network of sol-gel-derived gold composite electrodes and its application in theconstruction of electrochemical biosensors representing the ®rstexample of glass-ceramic sensing electrodes and of the bulkmodi®cation of metallic working electrodes. Zink et al. exten-sively studied silicate glasses obtained by the sol-gel methodwhich can provide such a host matrix that biomolecules immo-bilized by this method retain their functional characteristics tolarge extent [11, 12]. They also reviewed more than 35 differenttypes of hybrid biochemical-bioceramic materials [13]. Furtherstudies by Zink et al. demonstrated the biomolecular materialsbased on sol-gel encapsulated proteins [14, 15].

Our work on sol-gel glass starts from tetramethoxysilane, itscontrolled hydrolysis followed by copolymerzation of the sol-gelnetwork [4]. The resulting sol-gel network although it showsa solid-state biocompatible network its physical characteristicsare not well suited for the development of sol-gel glass ofcommercial based products because of two reasons mainly: 1)relatively fragile properties of the sol-gel glass; 2) shrinkage

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behavior of the sol-gel network with time that restrict one toobtain constant diffusion limited kinetics across the sol-gel glassinterface. Subsequently our attentions were focused to developcomposite sol-gel glasses that show the better aspects indesigning sol-gel glass based sensors=biosensors. Accordingly,we have developed two types of composite sol-gel glass using amixture of two silanes: 1) trimethoxysilane and 3-glycidoxy-propyltrimethoxysilane [16], and 2) 3-aminopropyltrimethoxy-silane and 2-(3,4-epoxycyclohexyl)-ethyltrimethoxy silane [17].The sol-gel network obtained from these silanes provided aremarkable microstructure, biocompatibility and long termstability. In all these cases the protein was homogeneouslydistributed within the sol-gel network. Subsequently, we alsocoupled ferrocene carboxaldehyde to the sol-gel networkobtained from the composition (2) and developed a novel solid-state potassium ion sensor [18]. The cross-linked ferrocenewithin the sol-gel glass network provides a stable open circuitpotential at the sensor interface. The ferrocene incorporatedwithin the sol-gel matrix does not show reversible electro-chemistry mainly due to the restricted translational degree ofmotion which is essentially required in the development ofmediated enzyme biosensors. The enzyme distributed throughoutthe sol-gel matrix in these kinds of preparations have shown theleast porosity which restricts the substrate diffusion as well asoxygen through the sol-gel matrix. This is essentially required forthe better performance of the glucose oxidase enzyme basednonmediated biosensor where the response of the sensor is afunction on the formation of hydrogen peroxide and its subse-quent electrochemical oxidation. Accordingly, it was planned tomake a sol-gel matrix of controlled porosity followed by puttingan enzyme layer and subsequently over that another layer of sol-gel glass of controlled porosity. Such type of arrangement inwhich the protein is in between two sol-gel glass layers is alsoreferred to as a sandwich con®guration. Here again the co-immobilization of mediator together with glucose oxidase did notpermit the occurrence of a mediated electrochemical reactionagain associated to the restricted mobility of the mediator co-immobilized within the sol-gel glasses. Such type of sandwichcon®guration based on nonmediated glucose sensors haveadvantages over other sol-gel glass based sensors within whichthe enzyme is homogeneously distributed mainly due to ; a)relatively rapid diffusion of substrate and other required analytesthrough the upper sol-gel glass layer having relatively controlledporosity and b) shorter enzymatic reaction layer within the sol-gel matrix along with high concentration of active enzyme.

In the present investigation we propose to develop a compositesol-gel glucose biosensor based on a sandwich con®gurationusing 3-aminopropyltrimethoxysilane and 2-(3,4-epoxycyclo-hexyl)-ethyltrimethoxy silane. The lower layer of the compositesol-gel glass is prepared in absence of polyethylene glycolwhereas the upper layer (above the glucose oxidase) is preparedwith incorporation of polyethylene glycol. Two types of sand-wich con®guration based glucose biosensors were developedbased on the similar scheme that differs only in the absence andpresence of graphite particle (1±2 mm) suspended with themonomers precursors. Results based on scanning electronmicroscopy of glucose oxidase immobilized composite sol-gelglass, cyclic voltammogramms of these two types of glucosebiosensors showing the diffusion limited condition of the ferro-cene, cyclic voltammograms of these glucose biosensors inabsence and in presence of glucose, typical response curves andcalibration plots of the sol-gel glass based glucose biosensors arereported.

2. Experimental

2.1. Reagents

3-Aminopropyltrimethoxy silane was obtained from Aldrich;2-(3,4-epoxycyclohexyl)-ethyltrimethoxy silane was obtainedfrom United Chemical Technologies, Inc., Petrarch Silanes andSilicones, Bristol, PA, USA; Glucose oxidase was obtained fromSigma. All other chemical employed were of analytical grade.

2.2. Preparation of Composite Sol-Gel Modi®ed

Electrodes

The electrode's body used for the preparation of compositesol-gel glass modi®ed electrodes was similar to that as describedin an earlier publication [19] made from Te¯on containing aplatinum base with a recessed depth of 2 mm. A platinum diskplaced on the threaded aluminum rod was ®tted in a hollowTe¯on cylinder in such a manner that the platinum surface wasexposed to the solution on the top from where the cavity of 2 mmdepth starts. Before placing the platinum disk on the aluminumrod its surface was polished with a ®ne alumina slurry followedby washing and drying. The composition of the sol-gel mono-mers and the additives were similar to those described in Table 1in preparing the two types of sol-gel glases based glucosebiosensors. The components of the sol-gel glass (Table 1) weremixed thoroughly by stirring and 20 mL of the homogenizedsolution was allowed to form sol-gel glass (®rst layer). Theglucose oxidase solution 20 mg=mL (5 mL) was added to the baselayer of the sol-gel glass and allowed to incubate overnight at4 �C. On the next day the second sol-gel layer (Table 1) was thenassembled over the adsorbed glucose oxidase on the ®rst layerof the sol-gel glass followed by gelation for 24 h to form abilayer of sol-gel glass. This con®guration of the sandwich sol-gel glass was made in the presence and absence of graphiteparticles (1±2m).

2.3. Electrochemical Measurements

The electrochemical measurements were performed with aSolartron Electrochemical Interface (Solartron 1287), electroderesponses were recorded and plotted with a printer. A onecompartment cell with a working volume of 4 mL and a sol-gelglass modi®ed working electrode, Ag=AgCl reference electrodeand a platinum foil auxiliary electrode were used for themeasurements. Cyclic voltammetry using a GOD modi®ed sol-gel glass electrode was studied between 0 ±1 V (vs. Ag=AgCl).The amperometric measurements using a GOD immobilized sol-gel modi®ed electrode was operated at 0.70 V (vs. Ag=AgCl).The experiments were performed in phosphate buffer (0.1 M,pH 7) employing both types of organically modi®ed sol-gel glasselectrodes.

The speci®c activity of glucose oxidase was determined to be80 U=mg before its immobilization on the sol-gel matrix. Scan-ning electron micrograph (SEM) measurements were made usinga JEOL-JSM 840A Scanning Electron Microscope.

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60 P. C. Pandey et al.

3. Results and Discussion

3.1. Physical Characteristics of the Composite Sol-Gel

Glass

The composite sol-gel glass obtained, following the abovedescribed procedures, results in a smooth surface solid-matrixwith homogeneous distribution of the polymer network. Thecolor of the sol-gel glass was white. The scanning electronmicroscopy of the composite sol-gel glass within which glucoseoxidase was distributed along with the precursors solution isshown in Figure 1. The microgaph shows better distribution ofenzyme and pores while retaining enzymatic activity as shown bythe activity measurement of the immobilized enzyme-substratereaction product and subsequently proved by electrochemicalmeasurements.

3.2. Electrochemistry of Ferrocene Monocarboxylic Acid

on Both Types of Sol-Gel Glass Modi®ed Glucose

Sensors

The bilayer arrangement leading to a sandwich con®gurationof a glucose biosensor is shown in Figure 2. The glucose oxidase(layer B) is sandwiched between two layers (A & C) of the sol-gel glass. The glucose biosensors developed, based on sandwichcon®guration, have been examined for the transport behavior offerrocene monocarboxylic acid across the composite sol-gelmatrix. Figure 3 shows cyclic voltammograms of these two typesof glucose biosensors (A & B) in the presence of 6 mM ferrocenemonocarboxylic acid at different sweep rates. Figure 3a showsthe results recorded using the glucose biosensor designed inabsence of graphite powder (system A) whereas Figure 3b showsthe results of the graphite powder doped glucose biosensor. Theinsets of Figures 3a and b show the plot of anodic peak currentversus square root of the scan rate. A linear relation betweenpeak current and square root of scan rate shows a diffusionlimited transport of ferrocene at the solution sol-gel interface.The values of the peak current was greater in the case of thegraphite doped sol-gel based glucose biosensor.

3.3. Electrochemistry of Glucose Oxidase Immobilized

Sol-Gel Glass Modi®ed Electrodes

The glucose biosensors (system A & B) were examined fortheir performance in absence and presence of 50 mM glucose toinvestigate the nature of the amperometric response of theglucose biosensors. These results are shown in Figure 4a and b,respectively. There is an increase in anodic current correspondingto the electrochemical oxidation of hydrogen peroxide. Theseresults suggest that the response of the sensor follows the aerobic

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Fig. 1. Scanning electron micrograph of glucose oxidase immobilizedsol-gel glass. The glucose oxidase was dissolved with sol-gel precursorsand allowed to form a sol-gel network for 24 h at 28 �C.

Table 1. Composition of sandwich con®gurations of sol-gel based glucose sensors.

System A: Sol-gel glass composition without graphite powder.

i) Base sol-gel layer3-APTMS[a] EETMS[b] Distilled water Polyethylene glycol Graphite powder HCl (0.1N)

[mL] [mL] [mL] [mg=mL] [mg=mL] [mL]70 20 700 ± ± 5

ii) Enzyme layer: 20 mg=mL glucose oxidase solution was allowed to incubate for 24 h over base sol-gel layer

iii) Upper sol-gel layer

3-APTMS EETMS Distilled water Polyethylene glycol Graphite powder HCl (0.1N)[mL] [mL] [mL] [mg=mL (Mol.Wt.

� 6000)][mg=mL] [mL]

70 20 700 2 ± 5

System B: Sol-gel glass composition with graphite powder

iv) Base sol-gel layer

3-APTMS EETMS Distilled water Polyethylene glycol Graphite powder HCl (0.1N)[mL] [mL] [mL] [mg=mL] [mg=mL] [mL]70 20 700 ± 2 5

v) Enzyme layer: 20 mg=mL glucose oxidase solution was allowed to incubate for 24 h over base sol-gel layer

vi) Upper sol-gel layer

3-APTMS EETMS Distilled water Polyethylene glycol Graphite powder HCl (0.1N)[mL] [mL] [mL] [mg=mL

(Mol.Wt. � 6000)][mg=mL] [mL]

70 20 700 2 2 5

[a] 3-APTMS� 3-Aminopropyltrimethoxy silane; [b] EETMS� 2-(3, 4-Epoxycyclohexyl)-ethyltrimethoxy silane.

Glucose Biosensor Based on Sandwich Con®guration 61

regeneration of glucose oxidase. Here again the difference in themagnitude of the anodic current was in accordance to the resultsrecorded in Figure 3a and b.

We also tried to investigate the mediated mechanism of theelectron transport from the glucose oxidase sandwiched withinthe bilayer of sol-gel glass. For this experiment we used 6 mMferrocene monocarboxylic acid as described for experiments 3.Figure 5 shows the cyclic voltammograms of soluble ferrocene inabsence (1) and presence (2) of 50 mM glucose. There was anincrease in anodic current on the addition of glucose showing theheterogeneous mediated reaction for the regeneration of glucoseoxidase.

3.4. Amperometric Response of the Enzyme Immobilized

Sol-Gel Glasses Based Glucose Sensor

Typical chronoamperometric responses at 0.7 V (vs.Ag=AgCl) of the glucose biosensors (system A & B) arerecorded in Figure 6. The response time of these biosensors wasreasonably impressive as compared to the diffusion limitedcondition of glucose as well as dissolved oxygen at the sensorinterface. Figure 7 shows the calibration plot for glucoseanalysis. The sensitivity of system B is again higher than system

Fig. 2. Schematic diagram of the sol-gel: glucose oxidase: sol-gelsandwich ®lm: I) Schematic diagram of the electrode body made fromhollow Te¯on cylinder equipped with Pt disk placed on a screwed alu-minum rod electrical connector; II) sandwich con®guration of sol-gelbased glucose sensor.

Fig. 3. Cyclic voltammograms of 6 mM ferrocene monocarboxylic acidin 0.1 M phosphate buffer pH 7.0 on: a) system A and b) system B. Thevoltammograms were recored at 25 �C and at different scan rates.

Fig. 4.a) Cyclic voltammogram of glucose biosensors based on asandwich con®guration (system A) in absence (1) and presence (2) of50 mM glucose in 0.1 M phosphate buffer pH 7.0 at the same scan rates.b) Cyclic voltammogram of glucose biosensors based on sandwichcon®guration (system B) in absence (1) and presence (2) of 50 mMglucose acid in 0.1 M phosphate buffer pH 7.0 at the same scan rates.

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62 P. C. Pandey et al.

A in accordance to the results recorded in Figures 3, 4 and 6.Curve 1 shows the responses of the biosensor (system A)whereas curve 2 is the response of the system B. The greaterresponses as recorded in Figures 3, 4, 6, and 7 for system B aremainly due to relatively good conductivity and better porosity ofthe composite sol-gel ®lm obtained from the loading of ®negraphite particles within the sol-gel network.

We also made an effort to study the mediated electrochemicalresponse of the sandwich con®guration based sol-gel glassbiosensor using soluble ferrocene monocarboxylic acid on theaddition of varying concentrations of glucose. The resultsrecorded in Figure 5 show that there was a mediated electrontransport between the sol-gel sandwiched glucose oxidase andthe soluble ferrocene. The results reported in Figure 8 show theamperometric mediated response of sol-gel based glucosebiosensor in the presence of 6 mM ferrocene monocarboxylicacid at 0.35 V (vs. Ag=AgCl) on the addition of varyingconcentrations of glucose. The results recorded in Figure 8 arevery interesting. The response time at lower substrate concen-trations (0.25 mM; 1.25 mM) was relatively faster than the

response time recorded at higher substrate concentrations(100 mM) possibly associated to the relatively high concentrationof active enzyme and slow diffusion of the substrate saturation.The maximum amplitude of the anodic current based on themediated electrochemical reaction was 12 fold greater (ca.36mA) than the amperometric response of the sensor based onhydrogen peroxide oxidation (3 mA). Such a large difference inmaximum anodic current at substrate saturation indicate that theenzyme sandwiched within the two sol-gel layers was highlyactive. The low response of the sensor at the substrate saturation(ca. 3mA) based on hydrogen peroxide oxidation was mainlydue to the kinetic limitation conditions prevailing at thesensor=solution interface. The formation of hydrogen peroxidewithin the sandwiched layer was kinetically controlled due to thelimited diffusion of oxygen across the upper layer of the sol-gelglass that limits the regeneration of the highly active glucoseoxidase within the sandwiched layer. On the other hand Figures 5and 8 indicate that ferrocene shows good electrochemistry toregenerate glucose oxidase and accordingly soluble ferroceneprovides a several fold enhanced response depending on theactivity of the immobilized enzyme. The relatively large responsetime of the biosensor based on hydrogen peroxide oxidation(Fig. 6) also suggest that the response was kinetically controlledcaused by the restricted diffusion of dissolved oxygen across thesol-gel layer. However, the response recorded in Figure 8 wasbased on the mediated reaction between the sol-gel cagedenzyme and mobile ferrocene. Here again the response time wasrelatively much larger (Fig. 8) mainly due to the slow secondorder kinetics between the soluble mobile ferrocene and immo-bilized glucose oxidase caged within the sol-gel glass and alsothe diffusion limited condition of ferrocene within the sol-gellayer (Fig. 3a, b). The slower second order rate constant betweenimmobilized glucose oxidase and mobile ferrocene could also bequalitatively analyzed from the data recorded in Figure 5. Theinset to Figure 8 shows the calibration plot of glucose analysisbased on the mediated mechanism.

The stability of these glucose biosensors was determinedunder two conditions. In the ®rst case the enzyme electrode wasstored in dry condition at 4 �C whereas in the second case theenzyme electrode was stored at room temperature in the same

Fig. 6. Typical response curves of a sandwich con®guration based glu-cose sensors on the subsequent addition of increasing concentrations ofglucose: 1) 0.25 mM; 2) 1.25 mM; 3) 2.5 mM; 4) 12.5 mM; 5) 25 mM: I)recorded from system A; II) recorded from system B. The enzymeelectrode was held at 0.70 V (vs. Ag=AgCl).

Fig. 5. Cyclic voltammogram of glucose biosensors based on sandwichcon®guration in absence (1) and presence (2) of 50 mM glucose in thepresence of 6 mM ferrocene monocarboxylic acid in 0.1 M phosphatebuffer pH 7.0 with a scan rate of 10 mV=s. Fig. 7. Calibration plots of the glucose biosensors. The values were

calculated from curves 1 and 2 of Figure 6. 1) for system A (s); 2) forsystem B (d).

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Glucose Biosensor Based on Sandwich Con®guration 63

buffer. The stability of the enzyme electrode stored under the ®rstcondition was relatively very good without loss of the ampero-metric response after 2 months. Under the second storagecondition the response was consistent without loss of any activityfor 20 days.

The physical characteristics of the sol-gel glass has beenexamined using various compositions of 3-aminopropyltri-methoxy silane, 2-(3,4-epoxycyclohexyl)-ethyltrimethoxy silane,enzyme dissolved in water and HCl. The content of 2-(3,4-epoxycyclohexyl)-ethyltrimethoxy silane signi®cantly affect thephysical characteristics of the sol-gel matrix. The color of theenzyme immobilized sol-gel ®lm was yellow whereas the colorwas white in the absence of enzyme. The smooth sol-gel ®lmwithout cracking and having a better performance when used as asensor was obtained using the optimum concentrations reported inthe experimental section. Another important requirement of thesensors based on such design was the coupling of the modi®ed sol-gel ®lm and Pt surface which contribute signi®cantly the perfor-mance and storage stability of the biosensors. The present modi®edsol-gel ®lm has been found to be strongly attached and is not easilyremoved from the Pt surface.

4. Conclusion

We described the construction of new composite sol-gelglasses glucose biosensors based on a sandwich con®guration.Two types of glucose biosensors were designed based on absenceand presence of graphite particles (1±2m). The graphite particleincorporated glucose biosensor shows an ampli®ed amperometricsignal attributed to good conductivity and a better porosity of thesol-gel network obtained from the graphite loading. The glucosebiosensors were made using 3-aminopropyltrimethoxysilane and

2-(3,4-epoxycyclohexyl)-ethyltrimethoxy silane, in the presenceof distilled water and HCl based on a sandwich con®guration.The resulting silane provides a very smooth surface with a rigidporous structure. The enzyme modi®ed sol-gel glass constructionof an amperometric biosensor for glucose is described. Underoptimum composition of the sol-gel glass ingredients, a verysmooth and thin closely attached to Pt surface was obtained withbetter porosity and regular distribution of enzyme within thesolid-state network which contributes to the better performanceof the biosensors as discussed above. The mediated response ofthe biosensor using soluble ferrocene was also studied. Theamperometric signal based on the mediated reaction was 12 foldgreater (ca. 36 mA) than the amperometric response of the sensorbased on hydrogen peroxide oxidation (ca. 3 mA) mainly due tothe availability of oxygen at the site of the enzymatic reaction.The mediated reaction between mobile ferrocene and immobi-lized glucose oxidase was also very slow which was mainly dueto the diffusion limited condition of ferrocene and a slow secondorder reaction between the enzyme caged in the sol-gel glass andthe mobile ferrocene as evidenced by the relatively largerresponse time of the sensor based on the mediated mechanism.

5. Acknowledgement

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

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Fig. 8. Typical response curves on the additions of varying concentra-tions of glucose (1: 0.25 mM; 2: 1.25 mM; 3: 12.5 mM; 4: 100 mM) of asandwich con®guration based glucose sensor based on mediated reactionbetween mobile (soluble) ferrocene and immobilized glucose oxidasesandwiched between two layers of sol-gel glasses. The inset shows thecalibration plot from data recorded at different concentrations.

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64 P. C. Pandey et al.