Biosensors and Bioelectronics 20 (2004) 204210

A bioelectrochemical polypyrrole-containing Fe(CN)63 interface for the design of a NAD-dependent reagentless biosensor
Pierre Gros , Maurice Comtat
Laboratoire de Gnie Chimique UMR CNRS 5503, Universit 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 lm. The entrapped Fe(CN)6 3 /Fe(CN)6 4 redox system displayed a high heterogeneous electron transfer rate. The resulting modied electrode was efcient 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 modied electrode system was involved without any reference. An enzymatic solution containing d-lactate dehydrogenase, diaphorase and NAD-dextran was further conned 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 mM1 cm2 and 120 s, respectively. The other analytical performances were less satisfactorily: the detection limit was 5 102 mmol L1 and the linearity range was comprised between 0.1 and 0.5 mmol L1 . 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 specicity 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 connement of the biocatalyst in the vicinity of the electrode surface. This procedure is now well established for oxidase-based rst 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: gros@chimie.ups-tlse.fr (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 elds 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 identied (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 lms 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 lms are generally homogeneous and adhere strongly to the

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electrode surface. Thirdly they are conducting and their thickness (Almeida et al., 1993) as well as the amount of enzyme entrapped (Fortier and Blanger, 1991) can be roughly controlled by means of the electrolysis conditions. 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, 1986). This last property allowed the immobilization of redox mediators such as ferricyanide (Zeng et al., 2000), ferrocyanide (Garcia et al., 1998) or osmium complex (Vilkanauskyte et al., 2002). Several modied electrodes were thus proposed where dehydrogenase was either co-immobilized within the polymer (Curulli et al., 1997; Rover Junior et al., 2000) or deposited on it (Milagres et al., 1997) or on a nylon mesh (Tzang et al., 2001) by means of glutaraldehyde. Amphiphilic (Cosnier et al., 1997) or pre-functionalized conducting polymers containing covalent attached mediator (Chaubey et al., 2000; Cosnier et al., 1998) have also been synthesized. Nevertheless in all these studies NAD(P)+ was introduced in solution. Recent papers described the co-immobilization of dehydrogenase, NAD+ and redox mediator inside an electropolymerized matrix for the assay of glycerol (Eftekhari, 2001) or formate (Zhao et al., 2002). This modied electrode architecture makes the amperometric biosensor completely reagentless. Nevertheless a three-electrode potentiostatic system was used in both cases, thus involving a reference. Works realized in our laboratory since 30 years are devoted to the design and the development of amperometric biosensors involving NAD-dependent dehydrogenase. 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 modied 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., 2000). The goal of the present work is to design a bioelectrochemical interface by combining these two approaches. Ferricyanide ions were immobilized on the electrode surface by means of an electrochemically grown polypyrrole lm. The resulting modied electrode was tested for the ferricyanide-mediated NADH oxidation. The bioelectrochemical interface was then applied to the design of a reagentless amperometric d-lactate biosensor. A weakly polarized two polypyrrole-containing Fe(CN)6 3 modied electrode system was used with enzymes and cofactor conned in the vicinity of the sensing electrode by means of a semi permeable membrane (Fig. 1b). The characteristics of the resulting biosensor were dened and discussed.

2. Experimental 2.1. General Diaphorase from Clostridium kluyveri (E.C. 1.8.1.4.) 6.4 U mg1 , d-lactate dehydrogenase from Lactobacillus leichmannii (E.C. 1.1.1.28.) 177 U mg1 , NAD+ -dextran (molecular weight 40,000) and all other chemicals were purchased from Sigma. Between experiments the pyrrole solution was stored under nitrogen in the dark to avoid oxidation by air. Unless otherwise indicated, either a phosphate 0.1 mol L1 pH 7.0 buffer or an AMPSO (3[(1,1-dimethyl-2-hydroxyethyl)amino]-2-hydroxy-propanesulfonic acid) 0.1 mol L1 pH 9.0 buffer were used as supporting electrolytes. All the electrochemical experiments were carried out at room temperature with either an EG&G PAR Model 362 potentiostat/galvanostat connected to a Sefram xy recorder or an EG&G PAR Model 283 interfaced to a Dell XPS-R350 microcomputer and using the Corrware 2.1-A software (S.A. Inc.). Synthesis and study of the electrochemical behavior of the polypyrrole modied electrode were performed with a three-electrode system consisting in a 2 mm or a 2 cm diameter platinum disk as working electrode, a large platinum grid counter electrode and a saturated calomel reference electrode (SCE) against which all potentials were measured. For the design of the biosensor, a 0.5 mm diameter and a 5 mm diameter platinum disk electrodes were used as sensing and pseudo-reference, respectively. The activity of the enzymatic solutions and the spectrophotometric assay of NADH were determined using a 8451 HP spectrophotometer. 2.2. Biosensor device All platinum electrodes were rstly treated by successively polishing the surface with diamond paste, rinsing with distilled water and cycling in H2 SO4 0.5 mol L1 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, 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), only a weakly polarized two electrode system was used in order the working electrode potential to belong to the ferrocyanide oxidation plateau. No reference electrode is then necessary (Fig. 1a). This principle has been successfully applied for the assay of various substrates such as l- and d-lactate ions (Durliat et al., 1976; Montagn et al., 1993), l-glutamate ion (Montagn et al., 1993), l-carnitine (Comtat et al., 1988), l-malic acid (Gilis et al., 1996) or l-alanine and pyruvate ion (Gilis et al., 1997) for different applications such as sports medicine, control of alcoholic and malo-lactic fermentations during wine making, control of cellular culture reactors, etc. On the other hand we showed that a

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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 = 0,1 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 V

(b)

BiosensorIII

Fig. 1. Schematic representation of the d-lactate biosensor and the enzymatic reaction sequences. (a) Biosensor I: NAD+ and Fe(CN)6 3 are dissolved in solution. (b) Reagentless biosensor III.

0.3 and 1.3 V at 50 mV s1 until recording reproducible typical voltammograms (Angerstein-Kozlowska, 1984). 2.2.1. Polypyrrole lm synthesis The protocol of the polypyrrole lm synthesis has been described and discussed previously (Gros et al., 2000). Briey polypyrrole lms were prepared by anodic electropolymerization of 10 mL distilled water containing pyrrole 0.1 mol L1 and either perchlorate or Fe(CN)6 4 ions 0.1 mol L1 . No other supporting electrolyte was added to avoid competitive incorporation with ferrocyanide in the polymer backbone. The solution was deaerated and a nitrogen ux was maintained during the electrolysis. Polymerization was performed potentiostatically at 0.8 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. A chronoamperogram was recorded during electrolysis. The thickness of the lm 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, 1988). It was assumed that the thickness of the lm was the same whatever the dopant anion used, i.e. Fe(CN)6 4 or ClO4 . The polypyrrole modied electrode was then immersed in

the deaerated phosphate buffer solution and its potential was held at 0.4 V for 10 min. Adopting these experimental conditions the polymer remained roughly in its conductive state. 2.2.2. Biosensors design 0.1 V was applied between a 0.5 mm diameter working electrode and a 5 mm diameter auxiliary electrode immersed in AMPSO 0.1 mol L1 pH 9.0 stirred buffer solution. In order to analyze the functionalities of the polypyrrole-containing Fe(CN)6 3 modied electrode for the sensing and the auxiliary electrode separately, three congurations were adopted for the biosensor: Biosensor I (Fig. 1a): 2 L of AMPSO 0.1 mol L1 pH 9.0 solution containing diaphorase 1 mg mL1 and d-lactate dehydrogenase 0.1 mg mL1 were dipped on the working electrode. These concentrations ensured a minimal response time of the biosensor (Gilis et al., 1996). The enzymatic solution was retained by means of a semi-permeable membrane maintained on the top of the electrode using an o-ring. Fe(CN)6 3 and NAD+ 10 mmol L1 were introduced in the AMPSO solution. Biosensor II: The auxiliary electrode was modied with a 9 m thick polypyrrole-containing Fe(CN)6 3 lm and

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immersed in AMPSO solution. 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 L1 . An ionic connection was established between both compartments by means of an agaragar bridge containing ammonium nitrate. Biosensor III (Fig. 1b): The sensing electrode was previously modied with a 9 m thick polypyrrole-containing Fe(CN)6 3 lm. Diaphorase 1 mg mL1 , d-lactate dehydrogenase 0.1 mg mL1 and NAD-dextran 10 mmol L1 were then conned in the reaction chamber. The auxiliary electrode was the same as for biosensor II. Both electrodes were immersed in the same AMPSO solution without any other added compound. 3. Results and discussion 3.1. Electrochemical properties of the polypyrrole modied electrode Fig. 2 shows the cyclic voltammogram obtained with a 2 mm diameter platinum disk electrode modied with a 9 m (electrolysis time 4 min) thick polypyrrole-containing Fe(CN)6 3 lm and immersed in the deaerated unstirred phosphate buffer solution (solid line). As previously shown (Breen et al., 1991), well-dened oxidation and reduction peaks appeared at 0.14 and 0.09 V respectively, values close to the redox potential of Fe(CN)6 3 /Fe(CN)6 4 system. Furthermore the amounts of charge under both peaks, calculated after reduction of the charge measured from the cyclic voltammogram recorded with the same polymer synthesized with ClO4 (dashed line), were similar, respectively, 3.3 and 3.0 C cm2 . Both these results display the reversible

electrochemical properties of the Fe(CN)6 3 /Fe(CN)6 4 system entrapped in the polymer. As ferricyanide ions were incorporated in the polymer as counter ions, polypyrrole lm had to be kept in its oxidative conductive state in order to avoid leaching of the redox mediator from the matrix (Zagorska et al., 1987). Previous works in our laboratory (Gros et al., 2000) showed that this was possible if the potential of the modied electrode was not shifted to values lower than 0.15 V. In this case the modied electrode conserved its electrochemical properties at least during 2 months with an assay every 2 days. Potentialities of the polypyrrole-containing Fe(CN)6 3 modied electrode for the oxidation of NADH catalyzed by a diaphorase were also investigated. A 2 cm diameter platinum disk electrode modied with a 9 m thick polypyrrole was immersed in 2 mL of stirred phosphate buffer solution containing NADH 10 mmol L1 and diaphorase 1 mg mL1 . Electrolysis was performed by holding the electrode potential at 0.3 V, allowing the oxidation of ferrocyanide produced by reaction (2). Fig. 3 shows the variation of NADH concentration with time in the case where the polypyrrole lm was doped with Fe(CN)6 3 and with ClO4 . Contrary to work of Schuhmann et al. (1991), the results clearly demonstrate an effective biocatalyzed electron transfer between NADH and immobilized ferricyanide ions since concentration of NADH continuously decreased in time. 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. In our case the modied electrode was in contact with the NADH solution during several hours. A similar, although
1

10

0.9

[NADH] / [NADH]

0.8

5 I / A

0.7

0.6

-5
0.5

-10 -0.2

0 E / (V vs. SCE)

0.2

0.4

time / hour
Fig. 3. Variation of the concentration of a NADH solution with time. Two milliliters stirred phosphate 0.1 mol L1 pH 7.0 buffer solution containing NADH 10 mmol L1 and diaphorase 1 mg mL1 were electrolyzed on a 2 cm diameter platinum disk electrode modied with a 9 m thick polypyrrole-containing Fe(CN)6 3 ( ) or ClO4 ( ). Electrode potential: 0.3 V.

Fig. 2. Cyclic voltammetry performed in a deaerated unstirred phosphate 0.1 mol L1 pH 7.0 buffer solution with a 2 mm diameter disk electrode modied with a 9 m thick polypyrrole lm synthesized with Fe(CN)6 4 () or with ClO4 (- - -). Potential sweep rate: 0.2 mV s1 .

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less important, evolution of NADH concentration was observed when Fe(CN)6 3 was replaced by ClO4 . 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., 1993; Lobo et al., 1996). Nevertheless the discrepancy between NADH concentrations of both experiences increased with time, proving that the biolectrocatalytic reaction was efcient during the whole experience. However, 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 lm (Gros et al., 1997). All these results consequently demonstrate that ferricyanide was actually electrochemically regenerated at the modied electrode. 3.2. Biosensors performances Table 1 summarizes the analytical performances of the three biosensors for the assay of d-lactate. 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 (Montagn et al., 1993). Fig. 4 represents the response curve obtained with the biosensor III for addition of d-lactate 0.1 mmol L1 . The response time corresponding to 90% of the maximal current was 120 s, value very close to that of biosensors I and II. This last result highlights once again the high electron transfer reaction rate inside the polypyrrole-containing Fe(CN)6 3 lm. On the contrary the residual current was almost four-fold the value previously recorded. In the same way, a relatively high noise was observed for the amperometric response. These results are certainly due to the polypyrrole deposit on the sensing electrode inducing a signicant capacitive current. Consequently the detection limit, corresponding to the concentration of d-lactate for which the current was twice the background noise, reached 5 102 mol L1 . A drifting baseline was also observed on the current time curve. 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 mmol1 cm2 ) Linearity range (mmol L1 ) Detection limit (mmol L1 ) Residual current (nA) Response time (90%) (s) Relative standard deviation (%) 37 0.011 5 103 7 100 2 Biosensor II 35 0.052 102 12 100 2 Biosensor III 25 0.10.5 5 102 25 120 10

Fig. 4. Response curve of the biosensor III to addition of d-lactate (curve a), ascorbate (curve b) and urate (curve c) 0.1 mmol L1 . 0.1 V was applied between a 0.5 mm diameter membrane-covered polypyrrole modied working electrode containing d-LDH, diaphorase, NAD-dextran and Fe(CN)6 3 , and a 5 mm diameter auxiliary electrode modied with a 9 m thick polypyrrole-containing Fe(CN)6 3 lm immersed in buffered AMPSO 0.1 mol L1 pH 9.0 stirred solution. Design of the biosensor is detailed in the text.

Design of each biosensor is detailed in the text.

overoxidation of the polypyrrole matrix by Fe(CN)6 3 ions as already suggested (Lian and Dong, 1989), decreasing slightly the electronic conductivity of the lm. Fig. 5 shows the calibration curve obtained with the biosensor III. The equation of the curve in the linearity range was I(nA) = 24.2 + 46.5 [d-lactate] (mmol L1 ) with a correlation coefcient of 0.9997 for n = 5. Considering the electrode surface area, the sensitivity was therefore 25 A L mmol1 cm2 , value of the same order of magnitude as for the others biosensors; the slight diminution can be attributed to the variation of the reaction chamber thickness (Durliat et al., 1976) by means of the polymer deposit. The apparent kinetic parameters of the immobilized enzyme were determined from the electrochemical LineweaverBurk form of the MichaelisMenten equation. The maximum current response Imax and the apparent app MichaelisMenten constant KM were calculated from the intercept and slope of the straight line. The values were 76 nA and 0.3 mmol L1 , respectively. This last value is lower than that of the free enzyme relating to d-lactate substrate, i.e. 2 102 mol L1 (Barman, 1969). However, it should be noted that these experimental kinetic parameters characterize the modied electrode rather than the enzyme itself.

P. Gros, M. Comtat / Biosensors and Bioelectronics 20 (2004) 204210

60 0.3 50 E / (V vs. SCE)

209

I / nA

0.2 40

30

0.1

20 0 0.2 0.4 [D-lactate] / mmol L

-1

0.6

0 0.8

Fig. 5. Calibration curve ( ) and variation of the potential ( ) of the amperometric biosensor for the assay of d-lactate. 0.1 V was applied between a 0.5 mm diameter membrane-covered polypyrrole modied working electrode containing d-LDH, diaphorase, NAD-dextran and Fe(CN)6 3 , and a 5 mm diameter auxiliary electrode modied with a 9 m thick polypyrrole-containing Fe(CN)6 3 lm immersed in buffered AMPSO 0.1 mol L1 pH 9.0 stirred solution. Design of the biosensor is detailed in the text.

The linearity range of biosensor III was sensibly restricted, comprised between 0.1 and 0.5 mmol L1 . This did not result from a signicant deviation of the sensing electrode potential which would no longer correspond to the ferrocyanide oxidation plateau. Fig. 5 clearly shows that the later was xed at 0.3 V simply by applying 0.1 V between the working and the auxiliary modied electrodes. Secondly the linearity range obtained with biosensor II was greatly more widen, comprised between 5102 and 2 mmol L1 . In this later case the auxiliary polypyrrole-containing ferricyanide modied electrode was also used as pseudo-reference and allowed the potential of the sensing electrode to be monitored satisfactorily (Gros et al., 2000). The reduced linearity range could rather be attributed to the biosensor design. In the case of biosensors I and II, the semi permeable membrane represented the only mass transport barrier. The diffusion of d-lactate through the membrane was the rate limiting step and controlled therefore the amperometric response. In the case of biosensor III, the enzymatic solution contained not only the biocatalysts but also NAD-dextran. This actually made the solution more viscous. The diffusivity of NAD+ in the reaction chamber certainly decreased when attached to dextran molecules. In the same way, it can be assumed that the electron transfer at the interface between the polypyrrole-containing Fe(CN)6 3 lm and the enzymatic solution was lower than with free mediators and cofactors. In these conditions the diffusion of d-lactate through the membrane was not the only parameter which inuenced the amperometric response and thus the linearity range of the proposed biosensor. The precision of the measurements was evaluated by performing triplicate measurements in a solution of d-lactate 0.2 mmol L1 . The average amperometric response, after

subtraction of the residual, was 8.5 nA with a standard deviation of 0.8 nA (R.S.D. 10%). The precision is lesser than that observed with biosensor I, certainly due to the more complex architecture of the electrode sensing. Finally ascorbate and urate ions were tested as possible interferents. Fig. 4 shows the response curve of the biosensor III for the addition of both species at 0.1 mmol L1 . In the later case no amperometric response was recorded while the current observed for ascorbate represented 12% of that obtained with d-lactate. In the case of urate, the sensing electrode potential, i.e. 0.3 V, was too low in order the electrochemical oxidation of urate at the platinum electrode to occur with a high electronic transfer rate. Actually a currentpotential curve of urate 10 mmol L1 performed with the biosensor III showed that the limiting current was obtained for potential higher than 0.6 V (result not shown). On the contrary the oxidation of ascorbate was effective at potential as low as 0.1 V. The relatively small amperometric response obtained could be explained both by the mass transport barrier imposed by the polymer lm and the membrane and by the high negative charge density due to the immobilization of ferricyanide in the polymer. 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 unmodied platinum bare (result not shown). 4. Conclusion A reagentless electrochemical biosensor involving NAD-dependent dehydrogenase has been designed. The device is based on a weakly polarized two-electrode system. Both electrodes were modied with a polypyrrole-containing Fe(CN)6 3 lm. The auxiliary was used as pseudo-reference since the immobilized Fe(CN)6 3 /Fe(CN)6 3 exhibited reversible electrochemical properties. The sensing electrode was further covered with a solution containing enzymes and cofactor by means of a semi-permeable membrane. Effective electron transfer between NADH and immobilized ferricyanide was highlighted. The resulting reagentless biosensor showed similar sensitivity and response time than the device working with dissolved mediator and cofactor. Although the detection limit and the linearity range were less satisfactorily, the proposed device allowed the detection of d-lactate without any added compound in the sample. Works are in progress to improve the analytical performances of the reagentless biosensor before being broadened to other NAD-dependent enzymatic systems.

References
Almeida, N.F., Beckman, E.J., Ataai, M.M., 1993. Immobilization of glucose oxidase in thin polypyrrole lms: inuence of polymerization conditions and lm thickness on the activity and stability of the immobilized enzyme. Biotechnol. Bioeng. 42, 10371045.

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P. Gros, M. Comtat / Biosensors and Bioelectronics 20 (2004) 204210 Gros, P., Gibson, T., Bergel, A., Comtat, M., 1997. Permeability enhancement of electropolymerized thin organic lms. J. Electroanal. Chem 437, 125134. Gros, P., Durliat, H., Comtat, M., 2000. Use of polypyrrole lm containing Fe(CN)6 3 as pseudo-reference electrode: application for amperometric biosensors. Electrochim. Acta 46, 643650. Holdcroft, S., Funt, B.L., 1988. Preparation and electrocatalytic properties of conducting lms of polypyrrole containing platinum microparticulates. J. Electroanal. Chem. 240, 89103. Lian, G., Dong, S., 1989. Electrochemical behaviour of Fe(CN)6 3/4 redox ions in a polypyrrole lm. J. Electroanal. Chem. 260, 127136. Lobo, M.J., Miranda, A.J., Lopez-Fonseca, J.M., Tunon, P., 1996. Electrocatalytic detection of nicotinamide coenzymes by poly(o-aminophenol) and poly(o-phenylenediamine) modied carbon paste electrodes. Anal. Chim. Acta 325, 3342. Lobo, M.J., Miranda, A.J., Tunon, P., 1997. Amperometric biosensors based on NAD(P)-dependent dehydrogenase enzymes. Electroanalysis 9, 191202. Milagres, B., Oliveira Neto, G., Kubota, L., Yamanaka, H., 1997. A new amperometric biosensor for salicylate based on salicylate hydroxylase immobilized on polypyrrole lm doped with hexacyanoferrate. Anal. Chim. Acta 347, 3541. Montagn, M., Durliat, H., Comtat, M., 1993. Simultaneous use of dehydrogenases and hexacyanoferrate (III) ion in electrochemical biosensors for l-lactate, d-lactate and l-glutamate ions. Anal. Chim. Acta 278, 2533. Persson, B., Lan, H.L., Gorton, L., Okamoto, Y., Hale, P.D., Boguslavsky, L.I., Skotheim, T., 1993. Amperometric biosensors based on electrocatalytic regeneration of NAD+ at redox polymer modied electrodes. Biosens. Bioelectron. 8, 8188. Rover Junior, L., Oliveira Neto, G., Fernandes, J., Kubota, L., 2000. Determination of salicylate in blood serum using an amperometric biosensor based on salicylate hydroxylase immobilized in a polypyrroleglutaraldehyde matrix. Talanta 51, 547557. Scheller, F.W., Schubert, F., 1992. Biosensors. Elsevier, Amsterdam. Schuhmann, W., Lammert, R., Hmmerle, M., Schmidt, H.-L., 1991. Electrocatalytic properties of polypyrrole in amperometric electrodes. Biosens. Bioelectron. 6, 689697. Thevenot, D.R., Coulet, P.R., Sternberg, R., Gautheron, D.C., 1978. A highly sensitive glucose electrode using glucose oxidase collagen lm. Bioelectrochem. Bioenerg. 5, 548553. Thevenot, D.R., Sternberg, R., Coulet, P.R., 1982. A glucose electrode using high-stability glucose oxidase collagen membranes. Diabetes Care 5, 203206. Turner, A.P.F., Karube, I., Wilson, G.S., 1987. Biosensors, Fundamentals and Applications. Oxford University Press, Oxford. Tzang, C.H., Yuan, R., Yang, M., 2001. Voltammetric biosensors for the determination of formate and glucose-6-phophate based on the measurement of dehydrogenase-generated NADH and NADPH. Biosens. Bioelectron. 16, 211219. Vilkanauskyte, A., Erichsen, T., Marcinkeviciene, L., Laurinavicius, V., Schuhmann, W., 2002. Reagentless biosensors based on co-entrapment of a soluble redox polymer and an enzyme within an electrochemically deposited polymer lm. Biosens. Bioelectron. 17, 10251031. Wang, J., 2001. Glucose biosensors: 40 years of advances and challenges. Electroanalysis 13, 983988. Zagorska, M., Wycislik, H., Przyluski, J., 1987. Electrochemical studies of polypyrrole doped with ferrocianide anions. Synth. Met. 20, 259268. Zeng, K., Tachikawa, H., Zhu, Z., Davidson, V., 2000. Amperometric detection of histamine with a methylamine dehydrogenase polypyrrole-based sensor. Anal. Chem. 72, 22112215. Zhao, H., Yuan, Y., Adeloju, S., Wallace, G.G., 2002. Study on the formation of the Prussian blue lms on the polypyrrole surface as a potential mediator system for biosensing applications. Anal. Chim. Acta 472, 113121.

Angerstein-Kozlowska, H., 1984. Surfaces, cells and solutions for kinetics studies. In: Yeager, E., Bockris, J.O.M., Conway, B.E., Sarangapani, S. (Eds.), Comprehensive Treatise of Electrochemistry, vol. 9. Plenum Press, New York, p. 28. Asavapiriyanont, S., Chandler, G.K., Gunawardena, G.A., Pletcher, D., 1984. The electrodeposition of polypyrrole lms from aqueous solutions. J. Electroanal. Chem. 177, 229244. Barman, T.E., 1969. Enzyme Handbook, vol. 1. Springer-Verlag, New York, p. 49. Bartlett, P.N., Cooper, J.M., 1993. A review of the immobilization of enzymes in electropolymerized lms. J. Electroanal. Chem. 362, 112. Blum, L.J., Bertrand, C., Coulet, P.R., 1983. Amperometric sensor and immobilized enzyme electrode for the determination of enzymatic activities. Anal. Lett. 16, 525540. Breen, W., Cassidy, J.F., Lyons, M.E.G., 1991. An electrochemical study of cation permeability into polypyrrole-containing [Fe(CN)6 ]4 . J. Electroanal. Chem. 297, 445460. Chaubey, A., Gerard, M., Singhal, R., Singh, V.S., Malhotra, B.D., 2000. Immobilization of lactate dehydrogenase on electrochemically prepared polypyrrolepolyvinylsulphonate composite lms for application to lactate biosensors. Electrochim. Acta 46, 723 729. Clark, L.C., Lyons, C., 1962. Electrode systems for continuous monitoring in cardiovascular surgery. Ann. N.Y. Acad. Sci. 102, 2945. Comtat, M., Galy, M., Goulas, P., Souppe, J., 1988. Amperometric bienzyme electrode for l-carnitine. Anal. Chim. Acta 208, 295300. Cosnier, S., Fontecave, M., Innocent, C., Niviere, V., 1997. An original electroenzymatic system: avin reductase-riboavin for the improvement of dehydrogenase-based biosensors. Application to the amperometric detection of lactate. Electroanalysis 9, 685688. Cosnier, S., Decout, J.-L., Fontecave, M., Frier, C., Innocent, C., 1998. A reagentless biosensor for the amperometric determination of NADH. Electroanalysis 10, 521525. Cosnier, S., 1999. Biomolecule immobilization on electrode surfaces by entrapment or attachment to electrochemically polymerized lms. A review. Biosens. Bioelectron. 14, 443456. Coulet, P.R., Julliard, J.H., Gautheron, D.C., 1974. A mild method of general use for covalent coupling of enzymes to chemically activated collagen lms. Biotechnol. Bioeng. 16, 10551068. Curulli, A., Carelli, I., Trischitta, O., Palleschi, G., 1997. Enzyme electrode probes obtained by electropolymerization of monomers with PMS and selected dehydrogenase enzymes. Talanta 44, 1659 1669. Diaz, A.F., Bargon, J., 1986. Electrochemical synthesis of conducting polymers. In: Skotheim, T.A. (Ed.), Handbook of Conducting Polymers. Marcel Dekker, New York, pp. 81115. Durliat, H., Comtat, M., Mahenc, J., Baudras, A., 1976. Recherche des conditions optimales de fonctionnement dune lectrode enzyme spcique du lactate. Application au dosage dans le sang. Anal. Chim. Acta 85, 3140. Eftekhari, E., 2001. Glycerol biosensor based on glycerol dehydrogenase incorporated into polyaniline modied aluminium electrode using hexacyanoferrate as mediator. Sens. Actuators B 80, 283289. Fortier, G., Blanger, D., 1991. Characterization of the biochemical behavior of glucose oxidase entrapped in a polypyrrole lm. Biotechnol. Bioeng. 37, 854858. Garcia, C.A.B., Oliveira Neto, G., Kubota, L., 1998. New fructose biosensors utilizing a polypyrrole lm and d-fructose 5-dehydrogenase immobilized by different processes. Anal. Chim. Acta 374, 201208. Gilis, M., Durliat, H., Comtat, M., 1996. Electrochemical biosensors for assays of l-malic and d-lactic acids in wines. Am. J. Enol. Vitic. 47, 1116. Gilis, M., Durliat, H., Comtat, M., 1997. Amperometric biosensors for l-alanine and pyruvate assays in biological uids. Anal. Chim. Acta 355, 235240.