a novel ormosil based electrocatalytic biosensor for glucose/ethanol based on dehydrogenase modified...
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A Novel Ormosil Based Electrocatalytic Biosensor forGlucose=Ethanol Based on Dehydrogenase Modi®ed Electrode
P. C. Pandey,* S. Upadhyay, Ida Tiwari, and V. S. Tripathi
Department of Chemistry, Banaras Hindu University, Varanasi-221005, India; e-mail: [email protected]
Received: June 8, 2000
Final version: September 14, 2000
Abstract
A novel ormosil material for designing electrocatalytic biosensors for glucose and ethanol based on dehydrogenase catalyzed reactions isreported. The electrode material is prepared using palladium-linked glycidoxypropyltrimethoxysilane, ferrocene monocarboxylic acid,trimethoxysilane and HCl. The ormosil prepared from these ingredients shows reversible electrochemistry of ormosil encapsulated fer-rocene. The electrocatalytic oxidation of NADPH=NADH and subsequently novel dehydrogenase based biosensors for glucose and ethanolare developed. The results based on cyclic voltammetry and amperometry are reported. The enzyme sensors are developed based on themodi®cation of electrode material by dehydrogenase enzymes. The modi®cation of electrode material is made by two approaches of enzymeimmobilization: immobilization of dehydrogenase within polyvinyl alcohol and sandwiching the dehydrogenase within two layers oformosils among which the ®rst layer being the electrode material itself and the second layer of ormosil prepared without palladium linkageprecursor and ferrocene. The biosensor made by the second approach shows high stability and much better reproducibility of enzymeelectrode performance. The results on glucose and alcohol sensing based on electrochemical measurements are reported.
Keywords: Organically modi®ed sol-gel glass, Ferrocene encapsulated sol-gel glass, Amperometry, Biosensor, Voltammetry
1. Introduction
The developments of mediated electrochemical biosensors[1±7] have received wide spread attentions for precise probing ofenzyme catalyzed reactions. These biosensors incorporate theintroduction of an electron transfer mediator that shuttles theelectrons from the active center of the redox enzyme=biomoleculesto the electrode surface. The introduction of an electron transfermediator in the construction of an electrochemical biosensor ulti-mately involves the occurrence of coupled catalytic reactions, the®rst one being associated with the regeneration of the enzymaticactivity and the second one with the regeneration of the mediatorelectrochemically. We have studied the mediated electrochemicalregeneration of glucose oxidase [7], peroxidase [8], cholineoxidase [9] and NADH [10] using mediator and respective enzymemodi®ed graphite paste electrodes in detail. In order to designsensors without further treatment of the electrode surface withrelatively high stability applications of sol-gel glass=ormosilmaterial have received great attention. Several examples of thesynthesis of sol-gel glasses [11±30] have become available duringthe last few years. One of the possible applications of such mate-rials in the development of sensors is the attachment of the sensingmaterial to the surface of physicochemical transducers. The use ofsol-gel glass for the development of electrochemical biosensors hasreceived great attention associated with the coupling of biologicalcomponents to electrochemical transducers. The development ofsuch biosensors based on sol-gel glass is currently restricted mainlydue to two major problems: 1) the requirement of controlledgelation of soluble sol-gel components at ambient conditions,2) preparation of a sol-gel glass with a smooth surface, controlledthickness and porosity. Apparently the synthesis of a suitablebiocompatible sol-gel glass of desired thickness and porosity is ofconsiderable interest. The soluble materials leading to the forma-tion of sol-gel glasses are derivatives of alkoxysilane. The avail-ability of a suitable reactive group attached to alkoxysilane mayprovide an advantage for the cross-linking of the sensing compo-nents to the solid-state network.
The encapsulation of redox material within sol-gel glass hasgained signi®cant share of attention for sensors designing.Pankratov and Lev [24] reported tetrathiafulvalene mediatedcarbon ceramic electrode (CCEs) with limited storage and in-usestability. Several other reports on ferrocene encapsulated sol-gelglasses including those of Lev et al. [21±23] are available.Audebert et al. [25] reported modi®ed electrodes from organic-inorganic hybrid gels containing ferrocene unit covalentlybonded inside a silica network and modi®ed electrodes fromorganic-inorganic hybrid gels formed by hydrolysis-poly-condensation of some trimethoxysilylferrocenes [26]. Collinsonet al. [27] reported electroactivity of redox probes encapsulatedwithin sol-gel derived silicate ®lm based on anionic, i.e.,
[Fe�CN�3ÿ=4ÿ6 ]; [IrCl
2ÿ=3ÿ6 ] and cationic, i.e., ferrocenemethanol
[FcCH2OH0=�] gel-doped probes. Other reports on ferrocenebased sol-gel sensors are also available [28]. There is greatpotential to study ferrocene encapsulated=linked sol-gel glassesfor mediated biosensors applications. We have recently reported aglucose biosensor [19, 20, 30] based on a sol-gel matrix ofcontrolled porosity followed by putting an enzyme layer andsubsequently over that another layer of sol-gel glass of controlledporosity using 3-aminopropyltriethoxysilane and 2-(3,4-epoxy-cyclohexyl)-ethyltrimethoxy silane as sol-gel glass precursors[9]. We studied both mediated and nonmediated response of theglucose biosensor. The mediated response was based on the useof soluble ferrocene for the regeneration of reduced glucoseoxidase. However, co-immobilization of mediator together withglucose oxidase does not permit the occurrence of a mediatedelectrochemical reaction associated to a restricted degree of thetranslational degree of motion which is essentially required inthe development of mediated enzyme biosensors. Accordingly,immobilization of ferrocene derivatives within sol-gel glassnetwork with good electrochemistry is an attractive requirement.Recently we have developed novel material [31] with encapsu-lated ferrocene having redox electrochemistry similar to thatobserved using soluble ferrocene in solution. The application ofthis material in biosensor construction is reported.
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Relatively sparse literature on electrochemical biosensorinvolving glucose dehydrogenase (GDH) enzyme which in-corporate the participation of NAD�=NADP� and subsequentelectrochemical oxidation of these cofactors are available [32±35]. The present research investigation is undertaken focussingon two objectives: 1) to study immobilization of dehydrogenaseenzymes over ferrocene encapsulated ormosil material and 2) tostudy the oxidation of NADPH=NADH and subsequently sensingof glucose=ethanol generated through dehydrogenases (glucosedehydrogenase or alcohol dehydrogenase) catalyzed reactionsinvolving the participation of new material. The immobilizationof dehydrogenase on new electrode material was carried outbased on two different approaches. In the ®rst approach theenzyme was immobilized in polyvinyl alcohol matrix and in thesecond approach the enzyme was adsorbed on new electrodematerial followed by making another layer of ormosil withoutpalladium reaction with glymo-precursor and ferrocene. In thesecond approach the enzyme was sandwiched within new elec-trode material and another layer of ormosil. The performance,characterization of these ormosil based enzyme electrodes arereported based on electrochemical measurements.
2. Experimental
2.1. Reagents
Trimethoxysilane, ferrocene monocarboxylic acid and palla-dium chloride were obtained from Aldrich Chemical Co.3-Glycidoxypropyltrimethoxysilane was obtained from UnitedChemical Technologies, Inc., Petrarch Silanes and Silicones,Bristol, PA, USA. Glucose dehydrogenase, EC 1.1.1.47 alcoholdehydrogenase EC 1.1.1.1, NAD� and NADP were obtainedfrom Sigma Chem. Co. The aqueous solutions were prepared indouble distilled deionized water. All other chemical employedwere of analytical grade.
2.2. Construction of Ferrocene Encapsulated Pd-Linked
Ormosil
The new material was prepared as follows: palladium chloride(1 mg) and 4 mg ferrocene monocarboxylic acid were dissolvedin 500mL distilled water. This solution was added into 3-glyci-doxypropyltrimethoxy silane (70 mL) which results in a blacksolution. The resulting reaction product was mixed with trime-thoxy silane (30mL), and 0.1 N HCl (5 mL). The resulting reac-tion mixture was stirred thoroughly up to 5 min at 25 �C and thedesired amount of the homogeneous solution ranging between70mL was added to the well of the specially designed electrodebody. The gelation was allowed to occur at 25 �C for 30 h.A smooth, very thin ferrocene encapsulated palladium-linkedormosil appeared on the Pt surface.
2.3. Construction of Ferrocene Encapsulated Pd-Linked
Ormosil Based Electrocatalytic NADPH=NADH
Biosensor Based on Two Different Approaches
2.3.1. Immobilization of Glucose Dehydrogenase Within PVAMatrix
Glucose dehydrogenase (GDH) 20 units (50 mL) was pouredinto 10 % polyvinyl alcohol (PVA) solution (100 mL). The enzyme
solution in PVA (35mL) was added at the surface of new ormosiland allowed to be solidify overnight at 4 �C. A Nucleoporemembrane was mounted over the immobilized enzyme withinan O-ring for better contact of the immobilized matrix withelectrode material. The performance of resulting NADPH=glucose biosensor based on bioelectrocatalysis was studied.
2.3.2. Sandwiching of Alcohol Dehydrogenase Within TwoLayers of Ormosil
The modi®ed electrode having encapsulated ferrocene was®rst prepared as described above. Alcohol dehydrogenase20 units (20mL) was allowed to be adsorbed overnight on aferrocene encapsulated ormosil modi®ed electrode. The secondlayer of ormosil was made on the adsorbed enzyme using3-glycidoxypropyltrimethoxy silane (70 mL), trimethoxy silane(30mL), distilled water (50mL) and 0.1 N HCl (5 mL) followed byallowing it to form ormosil at 25 �C for 15 h.
2.4. Electrochemical Measurements
The electrochemical measurements were performed with aSolartron electrochemical interface (Solartron 1287 electro-chemical interface). A one compartment cell with a workingvolume of 4 mL and new ormosil based NADPH=glucosebiosensor as working electrode, Ag=AgCl reference electrode anda platinum foil auxiliary electrode were used for the measure-ments. The cyclic voltammetry using a GDH modi®ed ormosilelectrode was studied between 70.2±0.6 V (vs. Ag=AgCl). Theamperometric measurements using the ormosil based biosensorwere operated at 0.30 V (vs. Ag=AgCl). The experiments wereperformed in phosphate buffer (0.1 M, pH 7.8) employing the newormosil based electrocatalytic NADPH=NADH biosensor. All themeasurements were made at 25 �C. Before each set of measure-ment the working solution was degassed by passing nitrogen for15 min.
2.5. Speci®c Activity of Glucose Dehydrogenase
The speci®c activity of glucose dehydrogenase (GDH) andalcohol dehydrogenase was determined to be 50 U=mg and80 U=mg, respectively, before assembling the immobilizedenzyme over the new ormosil modi®ed electrode. The activityof GDH was monitored by measuring the rate of formation ofNADPH using glucose as substrate and PMS, iodonitro-tetrazolium as reagents. The activity of ADH was monitoredusing ethanol as substrate and monitoring the rate of NADHformation at 340 nm. The total protein in both cases was moni-tored by Ohnishi and Barr's modi®cation of the Lowry method.
3. Results and Discussion
3.1. Electrochemical Characterization of Ferrocene
Encapsulated Within Ormosil and Glucose
Dehydrogenase Modi®ed Enzyme Electrode
The spectroscopic characterization suggest the structure ofglymo-Pd precursor [31] shown in Scheme 1.
When the Pd-linked glymo-precursor was added to trimeth-oxysilane, HCl, ferrocene monocarboxylic acid solution and
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allowed to form ormosil at 30 �C, a novel ferrocene encapsulatedPd-linked ormosil was formed [31]. We studied the electro-chemistry of the enzyme electrode made by immobilizing GDHwithin PVA on ferrocene encapsulated palladium-linked ormosilmaterial followed by protecting the immobilized enzyme matrixusing a Nucleopore membrane. The resulting biosensor wascharacterized electrochemically. Figure 1 shows the cyclicvoltammograms of the new ferrocene encapsulated ormosil basedbiosensor at different scan rates. The electrochemistry ofencapsulated ferrocene within ormosil is reversible. The linearrelation and peak separation to the order of 57±65 mV suggesta novel reversible electrochemistry of encapsulated ferrocenewithin ormosil which is one of the novel ®ndings of the presentmetal-ormosil material.
Figure 2 shows the cyclic voltammogramms of ormosil basedbiosensor in absence (1) and the presence (2) of 60 mM glucose.The variation in peak current suggests regeneration of NAD(P)�.
Figure 3 shows the typical response of ormosil based glucosebiosensor. The calibration plot of the glucose analysis is shownin Figure 4. The sensitivity of the analysis is found to be0.012� 0.0001 mA=mM (r 2� 0.962) calculated from three setsof enzyme electrodes of ®rst analytical data. Two possiblemechanisms can be considered for the electron exchange fromsoluble NADPH generated as a function of enzymatic (GDH)reaction and electrode surface; a) mediated mechanism andb) electrocatalytic mechanism. The geometry of the presentbiosensor shows that a macromolecule like glucose dehydro-genase is immobilized over Pd-linked ferrocene encapsulatedormosil. From the morphology of ormosil and the molecular
Scheme 1. Structural formula of glymo-Pd precursor.
Fig. 1. Cyclic voltammetry of ferrocene encapsulated Pd-linked ormosilmodi®ed glucose biosensor, made from immobilized glucose dehy-drogenase within polyvinyl alcohol matrix, at scan rates of 5, 10, 20, and50 mV=s in 0.1 M phosphate buffer pH 7.8 at 25 �C.
Fig. 2. Cyclic voltammetry of ferrocene encapsulated Pd-linked ormosilbased glucose biosensor, made from immobilized glucose dehy-drogenase within polyvinyl alcohol matrix, in absence (1) and the pre-sence (2) of 60 mM glucose in 0.1 M phosphate buffer pH 7.8 at 25 �C.
Fig. 3. Typical amperometric responses of the ferrocene encapsulatedpalladium-linked ormosil based glucose biosensor, made from immobi-lized glucose dehydrogenase within polyvinyl alcohol matrix, in 0.1 Mphosphate buffer pH 7.8 at 25 �C on the addition of increasing con-centrations of glucose (0.5, 1.25, 2.5, 5, 12.5, 25, and 50 mM).
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weight of GDH, it is not expected to be a homogeneous orheterogeneous reaction between the GDH catalyzed formation ofNADPH and ferrocene encapsulated within the nanoporousgeometry of ormosil. However, NADPH generated as a functionof the enzymatic reaction may diffuse within the ferroceneencapsulated ormosil and a mediated reaction between NADPHand encapsulated ferrocene can be expected. On the otherhand the response time as shown in Figure 3 which is < 25 ssuggests electrocatalytic oxidation of NADPH instead of amediated mechanism. Further investigation on this aspect isneeded, which is undergoing to identify the mechanistic approachof bioelectrochemical reaction. It appears that palladium linkedto the geometry of ormosil is playing a crucial role in electronexchange from enzymatically reduced NADPH and the electrodesurface. Since the enzyme was immobilized within PVAmatrix over the electrode material, close contact of immobilizedenzyme and electrode material is doubtful. In order to attainbetter contact between enzyme, cofactor and electrode material,the enzyme was adsorbed on the electrode material followed byformation of another ormosil layer originated from the sameormosil precursors without ferrocene and palladium linkage. Theresulting enzyme electrode con®guration results in a betterconnection between enzymatic components and the electrodematerial and does not require protection by a membrane. Theresults based on alcohol dehydrogenase enzyme are reported anddiscussed below.
3.2. Ferrocene Encapsulated Ormosil and Sandwiched
Alcohol Dehydrogenase Modi®ed Enzyme Electrode
In order to develop better con®guration of a dehydrogenasemodi®ed enzyme electrode, the immobilization protocol involvesa bilayer of ormosil based sensor since the PVA matrix on a newormosil based electrode material shows poor contact and requirethe support of a membrane ®lter. Since the enzyme is sandwiched
within two layer of ormosil, mass-transfer kinetics is relativelyslower across the second layer of ormosil as compared to acrossthe Nucleopore membrane and PVA matrix. The electrochemistryof encapsulated ferrocene on a bilayer based enzyme electrodecontaining NAD� in solution is shown in Figure 5 based oncyclic voltammetry at different scan rates. The encapsulatedferrocene again shows reversible electrochemistry (Figure 5). Theperformance of enzyme electrode was examined before and afterthe addition of 8 mM alcohol (ethanol) was similar to thatobserved in the case of GDH based biosensor immobilizedwithin PVA matrix.
Figure 6 shows the typical response plot of alcohol biosensorbased on the sandwiched con®guration of ormosils. Excellentreproducible response is observed with faster response time asobserved in the case of GDH catalyzed reaction. Figure 7 showsthe calibration plot for ethanol analysis. The sensitivity of theanalysis is found to be 0.139� 0.001mA=mM (r 2� 0.9885)calculated from three sets of enzyme electrodes from ®rst ana-lytical data.
3.3. Relative Stability of Biosensors Based on
Immobilization Protocol
It is important to study the stability and reproducibility ofenzyme electrode response for practical applications. The dehy-drogenase biosensors, we studied, were developed following twoapproaches of enzyme immobilization. In the ®rst approach theenzyme was entrapped within the PVA matrix over new ormosilmaterial followed by mounting the immobilized matrix using amembrane. However, in the second approach, the enzyme was®rst adsorbed on the electrode material followed by laying downanother layer of ormosil. The relative performance of these twoelectrodes were studied. The electrochemistry of encapsulatedferrocene of these two enzyme electrode con®guration is shown
Fig. 4. The calibration plots of glucose analysis using ferroceneencapsulated Pd-linked ormosil based glucose biosensor made fromimmobilized glucose dehydrogenase within polyvinyl alcohol matrix.
Fig. 5. Cyclic voltammetry of ferrocene encapsulated Pd-linked ormosilmodi®ed alcohol biosensor, made from sandwiched alcohol dehy-drogenase within two layer of ormosils, at scan rates of 5, 10, 20, 50, and100 mV=s in 0.1 M phosphate buffer pH 7.8 at 25 �C.
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in Figure 1 and Figure 5, respectively. The results clearly indicatechange in the electrochemical behavior of the electrode material,however, reversible electrochemistry of ferrocene was observedin both cases. Figure 8 shows the relative stability of these twoapproaches based enzyme electrode. The enzyme electrode loses70 % of its initial response after 150 runs when immobilizedwithin the PVA matrix, whereas, retaining 95 % response when
immobilized in the sandwiched con®guration even after 150 runs.It is obvious from the immobilization protocol that another layerof ormosil giving the sandwich con®guration facilitates theenzyme stability as compared to enzyme stability in the PVAmatrix. However, the PVA matrix is more porous and facilitatesfaster diffusion of analyte across the immobilized matrix.
3.4. Role of Palladium Linked to Ormosil Structure in
Electron Exchange Between NADPH=NADH and the
Electrode Surface
Since one of the precursors of ormosil is linked to palladium,the geometrical orientation of ormosil constituting the nanopor-ous structure within which ferrocene is caged having goodreversible electrochemistry suggests the interaction of palladiumand ferrocene in an electron exchange. The encapsulation offerrocene like a small molecule within a nanoporous structure oformosil is also examined; 1) ferrocene molecules caged withinormosil do not leach out from the ormosil matrix; 2) theconcentration of ferrocene in working solution was not detect-able; 3) the peak current of the cyclic voltammograms recorded atdifferent scan rate shows a linear relation and the peak currentat the same scan rate shows 99 % reproducibility for at least50 cycles a day. Additionally the Pd±C linkage has alreadyproved to be a good catalyst for several chemical=biochemicalinteractions. It appears that the NADPH=NADH generated as afunction of enzymatic reaction ®rst communicates from palla-dium which is expected from the close association ofNADPH=NADH and Pd-linked ormosil. Subsequently ferroceneencapsulated within nanopores of ormosil electrochemicallycommunicate from palladium which is ultimately followed byagain better electron hopping sites and reversible redox electro-chemistry of ferrocene till the electrode surface. Accordingly itappears that in the present case electrical communicationbetween NADPH=NADH and the electrode surface is maintained
Fig. 6. Typical amperometric responses of the ferrocene encapsulatedpalladium-linked ormosil based alcohol biosensor, made from sand-wiched alcohol dehydrogenase within two layer of ormosils, in 0.1 Mphosphate buffer pH 7.8 at 25 �C on the addition of increasing con-centrations of alcohol (0.2, 0.4, 0.8, 1,2, 1.6, and 8 mM).
Fig. 7. Calibration plot of alcohol analysis using ferrocene encapsulatedPd-linked ormosil based alcohol biosensor based on sandwiched alcoholdehydrogenase within two layers of ormosils.
Fig. 8. Relative stability of GDH=ADH based biosensor made based ontwo approaches of enzyme immobilization on the electrode; 1) immo-bilization within polyvinyl alcohol matrix and 2) sandwiching theenzyme within two layer of ormosils.
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through palladium-ferrocene-palladium and subsequently theelectrode surface.
4. Conclusions
A novel ferrocene encapsulated palladium linked ormosil isreported. The electrochemistry of encapsulated ferrocene isreversible and the material is promising for the development of adehydrogenase enzyme based biosensor. Electrocatalytic biosen-sors for glucose=ethanol based on dehydrogenase catalyzedreactions are examined. The new NADPH=NADH=glucose=ethanol biosensor has fast response time with high reproducibilityand operational stability. The reversibility of encapsulated ferro-cene and its bioelectrocatalytic electron exchange suggest thebehavior of the new material as solid solution.
5. Acknowledgement
Thanks to CSIR, New Delhi for ®nancial assistance.
6. References
[1] A. Heller, J. Phys. Chem. 1992, 96, 3579.[2] L. Bifulco, C. Cammaroto, J.D. Newman, A.P.F. Turner, Anal. Lett.
1994, 27, 1443.[3] A.E.G. Cass, G. Davis, G.D. Francis, H.A.O. Hill, W.J. Aston,
I.J. Higgins, E.V. Plotkin, D.L. Scott, A.P.F. Turner, Anal. Chem.1984, 56, 667.
[4] S.P. Hendry, A.P.F. Turner, Horm. Metab. Res. Suppl. 1988, 20, 37.[5] J. Wang, Anal. Chem. 1995, 67, R487.[6] P.C. Pandey, A.M. Kayastha, V. Pandey, Appl. Biochem. Biotech.
1992, 33, 139.[7] P.C. Pandey, S. Upadhyay, B. Upadhyay, Anal. Biochem. 1997,
252, 136.[8] P.C. Pandey, S. Upadhyay, H.C. Pathak, C.M.D. Pandey, Anal. Lett.
1998, 31, 2327.
[9] P.C. Pandey, S. Upadhyay, H.C. Pathak, C.M.D. Pandey, Ida Tiwari,Sens. Actuators 2000, B62, 109.
[10] P.C. Pandey, S. Upadhyay, B.C. Upadhyay, H.C. Pathak, Anal.Biochem. 1998, 260, 195.
[11] V. Glezer, O. Lev, J. Am. Chem. Soc. 1993, 115, 2533.[12] J.I. Zink, J.S. Valentine, B. Dunn, New J. Chem. 1994, 18, 1109.[13] J. Wang, P.V.A. Pamidi, D.S. Park, Anal. Chem. 1996, 68, 2705.[14] J. Wang, P.V.A. Pamidi, D.S. Park, Electroanalysis 1997, 9, 52.[15] J. Wang, P.V.A. Pamidi, Anal. Chem. 1997, 69, 4490.[16] P. Audebert, C. Demaille, C. Sanchez, Chem. Mater. 1993, 5, 911.[17] K. Kimura, T. Sunagawa, M. Yokoyama, Anal. Chem. 1997, 69,
2379.[18] L.M. Ellerby, C.R. Nishida, F. Nishida, S.A. Yamanaka, B. Dunn,
J.S. Valentine, J.I. Zink, Science 1992, 255, 1113.[19] P.C. Pandey, S. Upadhyay, H.C. Pathak, Electroanalysis 1999, 11,
59.[20] P.C. Pandey, S. Upadhyay, H.C. Pathak, Sens. Actuators 1999,
B60, 83.[21] O. Lev, Z. Wv, S. Bharathi, V. Glezer, A. Modestov, J. Gun,
L. Ravinovich, S. Sampath, Chem. Mater. 1997, 9, 2354.[22] G. Gun, M. Tsionsky, O. Lev, Anal. Chim. Acta 1994, 294, 61.[23] J. Gun, O. Lev, Anal. Lett. 1996, 29, 1933.[24] B. Pankratov, O. Lev, J. Electroanal. Chem. 1993, 393, 35.[25] P. Audebert, P. Calas, G. Cerveau, R.J.P. Corriv, N. Costa,
J. Electroanal. Chem. 1994, 372, 275.[26] P. Audebert, G. Cerveau, R.J.P. Corriv, N. Costa, J. Electroanal.
Chem. 1996, 413, 89.[27] M.M. Collinson, C.G. Rausch, A. Voigt, Langmuir 1997, 13, 7245.[28] S.L. Chut, J. Li, S. Tan, Analyst 1997, 122, 431.[29] P.C. Pandey, S. Upadhyay, H.C. Pathak, C.M.D. Pandey, Electro-
analysis 1999, 11, 950.[30] P.C. Pandey, S. Upadhyay, H.C. Pathak, I. Tiwari, V.J. Tripathi,
Electroanalysis 1999, 11, 1251.[31] P.C. Pandey, S. Upadhyay, I. Tiwari, G. Singh, V.J. Tripathi, Elec-
troanalysis 2001, in press.[32] T. Yao, in Analytical Applications of Immobilized Enzyme Reaction
(Eds: S. Lam, G.B. Malikin), Glasgow, 1994, p. 231.[33] W. Jin, F. Bier, F.W. Scheller, Biosens. Bioelect. 1995, 10, 823.[34] M. Hedemmo, A. Narvaez, E. Dominguez, I. Katakis, Analyst
(Lond.) 1996, 121, 1891.[35] H.-Z. Bu, S.R. Mikkelsen, A.M. English, Anal. Chem. 1998, 70,
4320.
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