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Biosensors and Bioelectronics 20 (2004) 204–210

A bioelectrochemical polypyrrole-containing Fe(CN)63− interface for the design of a NAD-dependent reagentless biosensor
Pierre Gros∗ , Maurice Comtat
Laboratoire de Génie Chimique UMR CNRS 5503, Université Paul Sabatier, 118 route de Narbonne, 31062 Toulouse Cedex, France Received 11 July 2003; received in revised form 10 February 2004; accepted 10 February 2004 Available online 5 May 2004

Abstract Ferricyanide ions were immobilized on a platinum electrode surface by means of an electrochemically grown polypyrrole film. The entrapped Fe(CN)6 3− /Fe(CN)6 4− redox system displayed a high heterogeneous electron transfer rate. The resulting modified electrode was efficient for the ferricyanide-mediated NADH oxidation catalyzed by a diaphorase. The bioelectrochemical interface was applied to the design of a reagentless amperometric d-lactate biosensor. A weakly polarized two polypyrrole-containing Fe(CN)6 3− modified electrode system was involved without any reference. An enzymatic solution containing d-lactate dehydrogenase, diaphorase and NAD-dextran was further confined on the sensing electrode using a semi-permeable membrane. The sensitivity and the response time of the reagentless biosensor were similar to those of the analogous sensor working with soluble mediator and cofactor, i.e. 25 A mM−1 cm−2 and 120 s, respectively. The other analytical performances were less satisfactorily: the detection limit was 5 × 10−2 mmol L−1 and the linearity range was comprised between 0.1 and 0.5 mmol L−1 . © 2004 Elsevier B.V. All rights reserved.
Keywords: Reagentless amperometric biosensor; Polypyrrole-containing Fe(CN)6 3− ; NAD-dependent dehydrogenase; d-Lactate assay

1. Introduction The pioneering works of Clark and Lyons (1962) on glucose electrode have lead to the development of electrochemical biosensors over the last four decades (Wang, 2001). This success is in great part due to the specificity of the biological catalysts and the selectivity of the electrochemical transduction (Turner et al., 1987; Scheller and Schubert, 1992). The successful coupling of both enzymatic and electrochemical reactions as well as the interest in designing miniaturized reagentless devices requires the confinement of the biocatalyst in the vicinity of the electrode surface. This procedure is now well established for oxidase-based first generation biosensors needing naturally available oxygen as electron acceptor. Among the numerous methods of enzyme immobilization, Coulet et al. (1974) particularly showed the opportunity offered by collagen membranes to the covalent binding of enzymes; glucose electrodes (Thevenot et al., 1978,

Corresponding author. Tel.: +33-5-6155-8269; fax: +33-5-6155-6139. E-mail address: (P. Gros). 0956-5663/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2004.02.023

1982) and devices for the determination of oxidase activities (Blum et al., 1983) have been designed in this way. If glucose electrodes play undoubtedly a leading role in numerous application fields such as clinical biology, bioprocess monitoring or food industry, biosensors involving NAD-dependent dehydrogenase have been the subject of tremendous attention since more than 250 enzymes have been identified (Lobo et al., 1997). In the later case the fabrication of the bioelectrochemical interface is not so easy since NAD cofactor has to be regenerated. The direct electrochemical oxidation of NAD(P)H requiring a high overpotential and thus generating possible interferences, additional redox mediator or even a second enzymatic system are necessary. Design a reagentless and selective NAD-dependent biosensor requires therefore the immobilization of the enzyme(s), the cofactor and the mediator(s). In this way electropolymerized conducting films like polypyrrole or polyaniline were widely used (Bartlett and Cooper, 1993; Cosnier, 1999). Firstly the polymers can be prepared in a one-step process from aqueous solution by oxidation of monomers occurring at potentials which avoid the evolution of oxygen (Asavapiriyanont et al., 1984). Secondly the resulting films are generally homogeneous and adhere strongly to the

This last property allowed the immobilization of redox mediators such as ferricyanide (Zeng et al. 2000. control of cellular culture reactors.A. l-malic acid (Gilis et al. This modified electrode architecture makes the amperometric biosensor completely reagentless. Rover Junior et al.0 buffer were used as supporting electrolytes. etc. 1986). The characteristics of the resulting biosensor were defined and discussed. 2001) or formate (Zhao et al. All the electrochemical experiments were carried out at room temperature with either an EG&G PAR Model 362 potentiostat/galvanostat connected to a Sefram x–y recorder or an EG&G PAR Model 283 interfaced to a Dell XPS-R350 microcomputer and using the Corrware 2.. 2002). 1996) or l-alanine and pyruvate ion (Gilis et al. Synthesis and study of the electrochemical behavior of the polypyrrole modified electrode were performed with a three-electrode system consisting in a 2 mm or a 2 cm diameter platinum disk as working electrode.and d-lactate ions (Durliat et al. 1997.28. Cosnier et al.P. General Diaphorase from Clostridium kluyveri (E.C. On the other hand we showed that a .1-dimethyl-2-hydroxyethyl)amino]-2-hydroxy-propanesulfonic acid) 0. 1991) can be roughly controlled by means of the electrolysis conditions..8... 1997) or on a nylon mesh (Tzang et al. 1. Biocatalysts were dissolved in a reaction chamber delimited by the electrode surface and a semi-permeable membrane where the biochemical reaction of substrate recognition takes place: Substrate + NAD(P)+ → Product + NAD(P)H + H+ (1) polypyrrole-containing Fe(CN)6 3− modified platinum electrode could be used as pseudo-reference since the immobilized Fe(CN)6 3− /Fe(CN)6 4− redox system still displayed a high heterogeneous electron transfer rate (Gros et al. Works realized in our laboratory since 30 years are devoted to the design and the development of amperometric biosensors involving NAD-dependent dehydrogenase.. rinsing with distilled water and cycling in H2 SO4 0.1. 1976.. 2002).4. Between experiments the pyrrole solution was stored under nitrogen in the dark to avoid oxidation by air.. Recent papers described the co-immobilization of dehydrogenase. This principle has been successfully applied for the assay of various substrates such as l.. The resulting modified electrode was tested for the ferricyanide-mediated NADH oxidation. Amphiphilic (Cosnier et al. ferrocyanide (Garcia et al.0 buffer or an AMPSO (3[(1.) 177 U mg−1 . NAD+ -dextran (molecular weight 40. 1993).. only a weakly polarized two electrode system was used in order the working electrode potential to belong to the ferrocyanide oxidation plateau. thus involving a reference.. and in the case where Fe(CN)6 3− was added in the analyzed sample in high concentration compared to that of Fe(CN)6 4− produced by reaction (2). 1988). A weakly polarized two polypyrrole-containing Fe(CN)6 3− modified electrode system was used with enzymes and cofactor confined in the vicinity of the sensing electrode by means of a semi permeable membrane (Fig. 2001) by means of glutaraldehyde.. a large platinum grid counter electrode and a saturated calomel reference electrode (SCE) against which all potentials were measured.1.4 U mg−1 ... l-glutamate ion (Montagné et al. The bioelectrochemical interface was then applied to the design of a reagentless amperometric d-lactate biosensor.. For the design of the biosensor. Inc. 1997) for different applications such as sports medicine.C. The goal of the present work is to design a bioelectrochemical interface by combining these two approaches. 1a). 1b).) 6.).1 mol L−1 pH 7. Nevertheless a three-electrode potentiostatic system was used in both cases..1. 2. Montagné et al. M.5 mol L−1 between Ferricyanide was used as electron acceptor to regenerate the oxidized form of the coenzyme NAD by a reaction catalyzed by a diaphorase: NADH+2Fe(CN)6 3− → NAD+ +2Fe(CN)6 4− + H+ (2) Given the high rate of the heterogeneous electron transfer reaction between the Fe(CN)6 3− /Fe(CN)6 4− system and a platinum electrode. Thirdly they are conducting and their thickness (Almeida et al. Unless otherwise indicated. Gros.000) and all other chemicals were purchased from Sigma. No reference electrode is then necessary (Fig. NAD+ and redox mediator inside an electropolymerized matrix for the assay of glycerol (Eftekhari. 2000) or deposited on it (Milagres et al. Nevertheless in all these studies NAD(P)+ was introduced in solution. respectively.1-A software (S.. Biosensor device All platinum electrodes were firstly treated by successively polishing the surface with diamond paste. d-lactate dehydrogenase from Lactobacillus leichmannii (E. 1993) as well as the amount of enzyme entrapped (Fortier and Bélanger. 2000). 2000).. 1. Experimental 2. 1998) have also been synthesized. Ferricyanide ions were immobilized on the electrode surface by means of an electrochemically grown polypyrrole film.5 mm diameter and a 5 mm diameter platinum disk electrodes were used as sensing and pseudo-reference. a 0. The activity of the enzymatic solutions and the spectrophotometric assay of NADH were determined using a 8451 HP spectrophotometer.2. Comtat / Biosensors and Bioelectronics 20 (2004) 204–210 205 electrode surface. either a phosphate 0. 2. Several modified electrodes were thus proposed where dehydrogenase was either co-immobilized within the polymer (Curulli et al. control of alcoholic and malo-lactic fermentations during wine making. 1993). 1997) or pre-functionalized conducting polymers containing covalent attached mediator (Chaubey et al. 1998) or osmium complex (Vilkanauskyte et al.1 mol L−1 pH 9.. Finally the oxidized form of the polymers is positively charged and requires the incorporation of doping anions in order to maintain its electroneutrality (Diaz and Bargon. l-carnitine (Comtat et al.1..

e. These concentrations ensured a minimal response time of the biosensor (Gilis et al.1 mol L−1 .3 and 1.0 solution containing diaphorase 1 mg mL−1 and d-lactate dehydrogenase 0.1 mol L−1 pH 9.0 stirred buffer solution.8 V to enable the oxidation of Fe(CN)6 4− into Fe(CN)6 3− and the electropolymerization of pyrrole to occur simultaneously at the electrode surface.1.1 V (b) BiosensorIII Fig. 1988). No other supporting electrolyte was added to avoid competitive incorporation with ferrocyanide in the polymer backbone. three configurations were adopted for the biosensor: • Biosensor I (Fig. 1a): 2 L of AMPSO 0.1 mg mL−1 were dipped on the working electrode. M. Polymerization was performed potentiostatically at 0.4 V for 10 min.. Fe(CN)6 3− and NAD+ 10 mmol L−1 were introduced in the AMPSO solution.1 V was applied between a 0..1 mol L−1 and either perchlorate or Fe(CN)6 4− ions 0. Schematic representation of the d-lactate biosensor and the enzymatic reaction sequences. 2000). (b) Reagentless biosensor III. Adopting these experimental conditions the polymer remained roughly in its conductive state. Gros. The polypyrrole modified electrode was then immersed in the deaerated phosphate buffer solution and its potential was held at 0. Fe(CN)6 4− or ClO4 − .2. Briefly polypyrrole films were prepared by anodic electropolymerization of 10 mL distilled water containing pyrrole 0.1 V (a) Biosensor I PPY Fe(CN)63e D-lactate pyruvate membrane NADH PPY Fe(CN 36 e D-LDH Diaph Fe(CN)64- D-lactate NAD+ dextran Fe(CN)64- E = 0.1 mol L−1 pH 9.2. • Biosensor II: The auxiliary electrode was modified with a 9 m thick polypyrrole-containing Fe(CN)6 3− film and . 1984). The enzymatic solution was retained by means of a semi-permeable membrane maintained on the top of the electrode using an o-ring. A chronoamperogram was recorded during electrolysis. 2. i. It was assumed that the thickness of the film was the same whatever the dopant anion used. −0.3 V at 50 mV s−1 until recording reproducible typical voltammograms (Angerstein-Kozlowska. (a) Biosensor I: NAD+ and Fe(CN)6 3− are dissolved in solution. The thickness of the film was controlled by means of the electrolysis time and was calculated from the amount of charge consumed in the case were ClO4 − ions were used as dopant anion (Holdcroft and Funt. 1. Polypyrrole film synthesis The protocol of the polypyrrole film synthesis has been described and discussed previously (Gros et al. 2. In order to analyze the functionalities of the polypyrrole-containing Fe(CN)6 3− modified electrode for the sensing and the auxiliary electrode separately. 1996). Comtat / Biosensors and Bioelectronics 20 (2004) 204–210 membrane Fe(CN)63e D-lactate NAD+ Fe(CN)63Fe(CN)64D-lactate NAD+ Fe(CN)64D-LDH Diaph pyruvate NADH Fe(CN)63e ∆ E = 0. Biosensors design 0.5 mm diameter working electrode and a 5 mm diameter auxiliary electrode immersed in AMPSO 0.2.206 P. The solution was deaerated and a nitrogen flux was maintained during the electrolysis.

Diaphorase 1 mg mL−1 . well-defined oxidation and reduction peaks appeared at 0. The sensing electrode was the same as for biosensor I and was immersed in AMPSO solution containing Fe(CN)6 3− and NAD+ 10 mmol L−1 . although 1 10 0.2 0.14 and 0. • Biosensor III (Fig. Electrode potential: 0. M.. Both these results display the reversible electrochemical properties of the Fe(CN)6 3− /Fe(CN)6 4− system entrapped in the polymer.. Comtat / Biosensors and Bioelectronics 20 (2004) 204–210 207 immersed in AMPSO solution.0 buffer solution containing NADH 10 mmol L−1 and diaphorase 1 mg mL−1 were electrolyzed on a 2 cm diameter platinum disk electrode modified with a 9 m thick polypyrrole-containing Fe(CN)6 3− ( ) or ClO4 − ( ). 3. (1991).8 5 I / µA 0. 3. As ferricyanide ions were incorporated in the polymer as counter ions. 1b): The sensing electrode was previously modified with a 9 m thick polypyrrole-containing Fe(CN)6 3− film. Contrary to work of Schuhmann et al. A similar. Potentialities of the polypyrrole-containing Fe(CN)6 3− modified electrode for the oxidation of NADH catalyzed by a diaphorase were also investigated. Both electrodes were immersed in the same AMPSO solution without any other added compound. allowing the oxidation of ferrocyanide produced by reaction (2).. values close to the redox potential of Fe(CN)6 3− /Fe(CN)6 4− system. Electrochemical properties of the polypyrrole modified electrode Fig.09 V respectively. were similar. Two milliliters stirred phosphate 0.1 mol L−1 pH 7. The difference between both studies could arise from the protocol adopted: in the former case conclusions were deduced from the absence of an amperometric wave when plotting a cyclic voltammogram.. 3 shows the variation of NADH concentration with time in the case where the polypyrrole film was doped with Fe(CN)6 3− and with ClO4 − . Cyclic voltammetry performed in a deaerated unstirred phosphate 0.6 -5 0. Electrolysis was performed by holding the electrode potential at 0.7 0 0. The auxiliary electrode was the same as for biosensor II. polypyrrole film had to be kept in its oxidative conductive state in order to avoid leaching of the redox mediator from the matrix (Zagorska et al. respectively. SCE) 0. the results clearly demonstrate an effective biocatalyzed electron transfer between NADH and immobilized ferricyanide ions since concentration of NADH continuously decreased in time.0 C cm−2 .2 mV s−1 . calculated after reduction of the charge measured from the cyclic voltammogram recorded with the same polymer synthesized with ClO4 − (dashed line).3 and 3.1.2 0 1 2 3 4 5 6 0 E / (V vs. 1991).1 mol L−1 pH 7.15 V.5 -10 -0. In this case the modified electrode conserved its electrochemical properties at least during 2 months with an assay every 2 days.4 time / hour Fig. d-lactate dehydrogenase 0. 3. Results and discussion 3.P. Fig. Previous works in our laboratory (Gros et al. Furthermore the amounts of charge under both peaks. 2 shows the cyclic voltammogram obtained with a 2 mm diameter platinum disk electrode modified with a 9 m (electrolysis time 4 min) thick polypyrrole-containing Fe(CN)6 3− film and immersed in the deaerated unstirred phosphate buffer solution (solid line).0 buffer solution with a 2 mm diameter disk electrode modified with a 9 m thick polypyrrole film synthesized with Fe(CN)6 4− (—) or with ClO4 − (. Potential sweep rate: 0. Variation of the concentration of a NADH solution with time.1 mg mL−1 and NAD-dextran 10 mmol L−1 were then confined in the reaction chamber. 2000) showed that this was possible if the potential of the modified electrode was not shifted to values lower than −0. 1987).-).3 V. Gros.9 [NADH] / [NADH]˚ 0. .3 V. 2. An ionic connection was established between both compartments by means of an agar–agar bridge containing ammonium nitrate. Fig. As previously shown (Breen et al. A 2 cm diameter platinum disk electrode modified with a 9 m thick polypyrrole was immersed in 2 mL of stirred phosphate buffer solution containing NADH 10 mmol L−1 and diaphorase 1 mg mL−1 . In our case the modified electrode was in contact with the NADH solution during several hours.

Consequently the detection limit.208 P. Fig.01–1 5 × 10−3 7 100 2 Biosensor II 35 0. 1993).05–2 10−2 12 100 2 Biosensor III 25 0. NAD-dextran and Fe(CN)6 3− .. However. Biosensors performances Table 1 summarizes the analytical performances of the three biosensors for the assay of d-lactate. decreasing slightly the electronic conductivity of the film.e. reached 5 × 10−2 mol L−1 . the slight diminution can be attributed to the variation of the reaction chamber thickness (Durliat et al. ascorbate (curve b) and urate (curve c) 0. overoxidation of the polypyrrole matrix by Fe(CN)6 3− ions as already suggested (Lian and Dong. This could be due to the progressive Table 1 Analytical performances of the amperometric biosensors for the assay of d-lactate Biosensor I Sensitivity ( A L mmol−1 cm−2 ) Linearity range (mmol L−1 ) Detection limit (mmol L−1 ) Residual current (nA) Response time (90%) (s) Relative standard deviation (%) 37 0. Gros. 1989). This last value is lower than that of the free enzyme relating to d-lactate substrate.1–0. i.3 mmol L−1 . 2 × 10−2 mol L−1 (Barman. All these results consequently demonstrate that ferricyanide was actually electrochemically regenerated at the modified electrode. respectively.2. value very close to that of biosensors I and II.5 5 × 10−2 25 120 10 Fig.9997 for n = 5. Fig.0 stirred solution.5 [d-lactate] (mmol L−1 ) with a correlation coefficient of 0. Nevertheless the discrepancy between NADH concentrations of both experiences increased with time. M. it can be reasonably assumed that this reaction takes place only at the polypyrrole-solution interface owing to the size of diaphorase and even NADH which prevents them from diffusing inside the polymer film (Gros et al. However.. The maximum current response Imax and the apparent app Michaelis–Menten constant KM were calculated from the intercept and slope of the straight line. 4. Lobo et al..1 mmol L−1 . diaphorase. The values were 76 nA and 0.1 V was applied between a 0. The equation of the curve in the linearity range was I(nA) = 24. The response time corresponding to 90% of the maximal current was 120 s. Comtat / Biosensors and Bioelectronics 20 (2004) 204–210 less important. Design of each biosensor is detailed in the text. In the same way. This last result highlights once again the high electron transfer reaction rate inside the polypyrrole-containing Fe(CN)6 3− film. 1993. 0.2 + 46. 1969). value of the same order of magnitude as for the others biosensors. 5 shows the calibration curve obtained with the biosensor III. The apparent kinetic parameters of the immobilized enzyme were determined from the electrochemical Lineweaver–Burk form of the Michaelis–Menten equation. A drifting baseline was also observed on the current – time curve. 1976) by means of the polymer deposit. These results are certainly due to the polypyrrole deposit on the sensing electrode inducing a significant capacitive current. 4 represents the response curve obtained with the biosensor III for addition of d-lactate 0.5 mm diameter membrane-covered polypyrrole modified working electrode containing d-LDH. This can be explained by a possible electrocatalytic activity of the redox polypyrrole backbone for NADH oxidation as previously shown with several others polymers (Persson et al.1 mol L−1 pH 9. 1996).. and a 5 mm diameter auxiliary electrode modified with a 9 m thick polypyrrole-containing Fe(CN)6 3− film immersed in buffered AMPSO 0. On the contrary the residual current was almost four-fold the value previously recorded. evolution of NADH concentration was observed when Fe(CN)6 3− was replaced by ClO4 − . 1997). Performances recorded with the biosensor I working with the mediator and cofactor dissolved in the sample were similar to those of previous biosensors fabricated in the laboratory (Montagné et al. the sensitivity was therefore 25 A L mmol−1 cm−2 . 3. . a relatively high noise was observed for the amperometric response. Design of the biosensor is detailed in the text. corresponding to the concentration of d-lactate for which the current was twice the background noise.. Response curve of the biosensor III to addition of d-lactate (curve a). proving that the biolectrocatalytic reaction was efficient during the whole experience. it should be noted that these experimental kinetic parameters characterize the modified electrode rather than the enzyme itself.1 mmol L−1 . Considering the electrode surface area.

The resulting reagentless biosensor showed similar sensitivity and response time than the device working with dissolved mediator and cofactor.5 mm diameter membrane-covered polypyrrole modified working electrode containing d-LDH. The linearity range of biosensor III was sensibly restricted.1 mol L−1 pH 9. The precision is lesser than that observed with biosensor I.5 mmol L−1 . . The reduced linearity range could rather be attributed to the biosensor design. Fig. 0.S.2 mmol L−1 .3 V. Secondly the linearity range obtained with biosensor II was greatly more widen.1 20 0 0. 10%). In the later case no amperometric response was recorded while the current observed for ascorbate represented 12% of that obtained with d-lactate. it can be assumed that the electron transfer at the interface between the polypyrrole-containing Fe(CN)6 3− film and the enzymatic solution was lower than with free mediators and cofactors. 0. Calibration curve ( ) and variation of the potential ( ) of the amperometric biosensor for the assay of d-lactate. The relatively small amperometric response obtained could be explained both by the mass transport barrier imposed by the polymer film and the membrane and by the high negative charge density due to the immobilization of ferricyanide in the polymer. N.1 V was applied between a 0. The diffusion of d-lactate through the membrane was the rate limiting step and controlled therefore the amperometric response. Beckman. Bioeng. 42. M.1 V between the working and the auxiliary modified electrodes. was too low in order the electrochemical oxidation of urate at the platinum electrode to occur with a high electronic transfer rate. The precision of the measurements was evaluated by performing triplicate measurements in a solution of d-lactate 0.. In the same way. The device is based on a weakly polarized two-electrode system. SCE) 209 I / nA 0.6 0 0.. Conclusion A reagentless electrochemical biosensor involving NAD-dependent dehydrogenase has been designed. i.4 [D-lactate] / mmol L -1 0. The diffusivity of NAD+ in the reaction chamber certainly decreased when attached to dextran molecules. Biotechnol. Fig.8 Fig. Immobilization of glucose oxidase in thin polypyrrole films: influence of polymerization conditions and film thickness on the activity and stability of the immobilized enzyme.J.0 stirred solution. the proposed device allowed the detection of d-lactate without any added compound in the sample. Actually a current–potential curve of urate 10 mmol L−1 performed with the biosensor III showed that the limiting current was obtained for potential higher than 0. This assumption was supported by the fact that the amperometric response for ascorbate recorded with the biosensor III represented less than 5% of the current obtained with the same unmodified platinum bare (result not shown). In the case of biosensor III.F.P. Finally ascorbate and urate ions were tested as possible interferents.1 mmol L−1 . M. Effective electron transfer between NADH and immobilized ferricyanide was highlighted.2 0.5 nA with a standard deviation of 0. certainly due to the more complex architecture of the electrode sensing. 2000). Both electrodes were modified with a polypyrrole-containing Fe(CN)6 3− film. was 8. The average amperometric response.2 40 30 0. 4. 4 shows the response curve of the biosensor III for the addition of both species at 0.6 V (result not shown). This did not result from a significant deviation of the sensing electrode potential which would no longer correspond to the ferrocyanide oxidation plateau. after subtraction of the residual. 5. 1993. References Almeida.3 V simply by applying 0.8 nA (R. Gros. In the case of biosensors I and II. E. the sensing electrode potential. This actually made the solution more viscous. comprised between 5×10−2 and 2 mmol L−1 . the semi permeable membrane represented the only mass transport barrier.. and a 5 mm diameter auxiliary electrode modified with a 9 m thick polypyrrole-containing Fe(CN)6 3− film immersed in buffered AMPSO 0.M.1 V. Works are in progress to improve the analytical performances of the reagentless biosensor before being broadened to other NAD-dependent enzymatic systems..D. In this later case the auxiliary polypyrrole-containing ferricyanide modified electrode was also used as pseudo-reference and allowed the potential of the sensing electrode to be monitored satisfactorily (Gros et al.1 and 0. On the contrary the oxidation of ascorbate was effective at potential as low as −0. Comtat / Biosensors and Bioelectronics 20 (2004) 204–210 60 0. NAD-dextran and Fe(CN)6 3− . The sensing electrode was further covered with a solution containing enzymes and cofactor by means of a semi-permeable membrane. The auxiliary was used as pseudo-reference since the immobilized Fe(CN)6 3− /Fe(CN)6 3− exhibited reversible electrochemical properties. the enzymatic solution contained not only the biocatalysts but also NAD-dextran. Although the detection limit and the linearity range were less satisfactorily. Design of the biosensor is detailed in the text. 1037–1045. 5 clearly shows that the later was fixed at 0.e. comprised between 0.3 50 E / (V vs. In these conditions the diffusion of d-lactate through the membrane was not the only parameter which influenced the amperometric response and thus the linearity range of the proposed biosensor. Ataai. In the case of urate. diaphorase.

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