Anal. Chem. 1994,66, 3198-3201
Amperometric Detection of Nitrate via a Nitrate Reductase Immobilized and Electrically Wired at the Electrode Surface Serge Cosnler,'gt Chrlstophe Innocent,+ and Yves Jouannead Laboratoire d'Electrochimie Organique et de Photochimie R ~ ~ oURA x , CNRS 12 10, Universit6 Joseph Fourier Grenoble 1, BP 53, 3804 1 Grenoble Cedex 9, France, and Laboratoire de Biochimie Microbienne, Dhpartement de Biologie Moficuhire et Structurale, Centre d'Etudes Nuclhaires de Grenoble, 38054 Grenoble Cedex 9. France
The electropolymerization of a nitrate reductase-amphiphilic pyrrole viologen mixture preadsorbed on the electrode surface provides the immobilization of the enzyme in a N-substituted viologen polypyrrole film. Furthermore, the electrogenerated redox polymer simultaneollslyentrapsthe enzyme and connects it electrically with the electrode. The immobilized enzyme catalyzes the reduction of nitrate to nitrite mediated by the viologen redox couple (V2+/*+). This enzyme immobilization has been accomplished on electrode surfaces previously coated by a polypyrrole-viologen film, leading to a bilayer electrode configuration. The sensitivity and the detection limit of this biosensor are 13.8 mA M-l cm-2 and 4 X M, respectively. Nitrate is a well-known contaminant of ground- and streamwater. The widespread pollution with nitrate is an environmental problem and a health hazard.' Several methods have been employed for nitrate determination including spectrophotometric determinations,2 ion-selective chromatographic techniques,3 and electrochemical methods such as polarographic, voltammetric, and potentiometric determinat i o n ~ .A ~ ,bioassay ~ has been described involving an enzymatic reduction of nitrate to nitrite followedby a colorimetric analysis of the nitrite formed.6 However, these methods are either tedious and time consuming or not sufficiently specific. Guilbault and co-workers have developed an enzymatic system associated with a potentiometric ammonia electrode for the specific and rapid determination of nitrate.' However, this electrochemical method exhibits a poor sensitivity and requires cofactors in the sample solution. Owing to some advantages over other methods, including high selectivity and simple use, biosensors are used in many applications in both synthetic and analytical chemistry. Hence, nitrate analysis could be usefully carried out using amperometric biosensors, whose functioning principle is based on the UniversitC Joseph Fourier Grenoble 1. Centre d'Etudes Nuclbires de Grenoble. (1) Mirvish, S.Nafure 1985, 325, 461-2. (2) Taras, H. J. Colorometric Determination ofNonmefals;Interscience Publishers: New York, 1958; pp 135-7. (3) Fritz, J. S.;Gjerde, D. T.; Pohlandt, C. Ion Chromatography; Huthig: Heidelberg, 1982; p 84. (4) Davenport, R. J.; Johnson,D. C. Anal. Chem. 1973,45,1979-80,andreferences ated therein. ( 5 ) Hussein, W. R.; Guilbault, G.G.Anal. Chim. Acta 1974, 72, 381-90. (6) Gromes, R. S.;Chartz, H.; Heinrich, M.; Johannssen, W. Appl. Microbiol. Biotechnol. 1991, 35, 491-5. (7) Kiang, C. H.; Kuan, S.S.;Guilbault, G.0 .Anal. Chem. 1978.50.1319-22. 8
3198
Analy7icalChemlstry, Vol. 66, No. 19, October 1, 1994
use of a biological component for recognition. In particular, the development of a specific enzyme electrode could provide an inexpensiveand convenient system to directly detect nitrate in liquid samples with reduced cofactor requirements. Willner and co-workers previously reported the immobilization of the enzyme nitrate reductase (EC 1.6.6.2. from Aspergillus niger) in a polyacrylamide gel functionalized by methyl viologen MV2+(Nflt-dimethyl-4,4'-bipyridinium).8 In addition, they have demonstrated that an electrical communication can be established between the entrapped nitrate reductase (NR) and the chemically or photochemically generated viologen radical cation, MV'+, allowing the enzymatic reduction of nitrate to nitrite? 2MV"
+ NO; + 2H+
-
NR
NO;
+ 2MV2++ H20
They have also described the bioelectrocatalyzed reduction of nitrate by poly(thiophene vio1ogen)-modified electrodes containing nitrate reductase.1° However, these enzyme electrodes do not exhibit catalytic cathodic current at the reduction wave of the viologen groups and thus cannot be used as amperometric biosensors. This was attributed to the very low activity of the commercial enzyme and/or to the small amount of NR immobilized in the different electrogenerated polymers.1° Recently, we reported a novel procedure of enzyme immobilization which allowed the designing of sensitive and fast responsive amperometric biosensors.' 1*12 This biosensor construction is based on the electropolymerization of a pyrrole amphiphilic monomer-enzyme mixture previously adsorbed on an electrode surface.'3 Compared to classical electrochemical enzyme entrapments, this method offers the possibility of controlling the composition of the enzyme-polymer layer and allows the easy determination of both the amount and the activity of the immobilized enzyme.14 As an application of our technology to the design of new biosensors, we describe here the construction and characterization of the (8) Willner, I.; Riklin, A.; Lapidot, N. J. Am. Chem. Soc. 1990, 222, 6438-9. (9) Willner, I.; Eichen, Y.; Frank, A. J.; Fox, M. A. J. Phys. Chem. 1993, 97, 7264-7 1. (10) Willner, I.; Katz, E.; Lapidot, N. Bioelectrochem. Bioenerg. 1992,29,29-45. (11) Cosnier, S.;Innocent, C. J. Elecfroanal.Chem. 1992, 328, 3616. (12) Cosnier, S.;Innocent, C. Bioelectrochem. Bioenerg. 1993, 32, 1 4 7 6 0 . (13) Coche-Guerente, L.; Deronzier, A.; Galland, B.; LabM, P.; Moutet, J.-C.; Reverdy, G . J. Chem. Soc., Chem. Commun. 1991, 386-8. (14) Coche-Guerente, L.; Cosnier, S.;Innocent, C.; Mailley, P.; Moutet, J.-C.; Morelis, R. M.; Leca, B.; Coulet, P. R. Electroanalysis 1993, 5, 647-52.
0003-2700/94/0386-3198$04.50/0
0 1994 American Chemical Society
first, to our knowledge, nitrate amperometric biosensor. The strategy adopted here relies on the adsorption on the electrode surface of a nitrate reductase-amphiphilic pyrrole viologen (C12V2+)followed by the electropolymerizationof the resulting adsorbed layer. The enzyme immobilizationwas accomplished on electrodes coated by a polypyrrolicfilm containingviologen groups (C3V2+). This bilayer configuration (C/poly-C3V2+/ poly-C12V2+-NR electrode) appeared to be an efficient nitrate biosensor. In the following, for C12V2+,n = 12, and for C3V2+, n = 3.
thoroughly rinsed in distilled water and incubated 30 min in stirred buffered electrolyte at 20 OC before use. Electrochemical MeasuremeaQ. The electrochemicalequip ment has been described previ0us1y.l~ All experiments were carried out in a conventional three-electrode cell under an argon atmosphere. An Ag/Ag+ 10 mM in CHsCN electrode was used as reference electrode in acetonitrile electrolyte for the electrochemicalpolymerization of C3V2+,and an aqueous saturated calomel electrode (SCE) was used for the electrochemical polymerization of C12V2+and for the amperometric determination of nitrate. The working electrode was a glassy carbon disk (diameter 5 mm) polished with diamond paste. Assays. The electrochemical measurements were performed under an argon atmosphere in a thermostated cell at 25.0 f 0.2 OC. The bioelectrode response to nitrate was monitored amperometrically by measuring the steady-state current for C12V2+reduction at -0.7 V vs SCE in 0.1 M stirred phosphate buffer, pH 7.5. The NR activity was measured by following the oxidation of reduced N,N’-dimethylbipyridiniumradical cation (MV’+) coupled to the reduction of nitrate to nitrite. Assays were carried out under an argon atmosphere ( 0 2
