A Nitrite Sensor Based on a Highly Sensitive Nitrite Reductase

A similar characteristic was found for methyl viologen, but for comparability with other works it was selected.15-17 Furthermore, phenosafranin and sa...
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Anal. Chem. 1996, 68, 807-816

A Nitrite Sensor Based on a Highly Sensitive Nitrite Reductase Mediator-Coupled Amperometric Detection Beate Strehlitz,*,† Bernd Gru 1 ndig,‡ Wolfram Schumacher,§ § Peter M. H. Kroneck, Klaus-Dieter Vorlop,| and Heiner Kotte†

Umweltforschungszentrum Leipzig-Halle GmbH, Permoserstrasse 15, D 04318 Leipzig, Germany, SensLab GmbH, Leonhard-Frank-Strasse 7, D 04318 Leipzig, Germany, Fakulta¨ t fu¨ r Biologie, Universita¨ t Konstanz, Universita¨ tsstrasse 10, D 78434 Konstanz, Germany, and Bundesforschungsanstalt fu¨ r Landwirtschaft, Institut fu¨ r Technologie, Bundesallee 50, D38116 Braunschweig, Germany

Highly sensitive nitrite sensors have been developed for the first time based on mediator-modified electrodes. Tetraheme cytochrome c nitrite reductase from Sulfurospirillum deleyianum and cytochrome cd1 nitrite reductase from Paracoccus denitrificans are able to accept electrons from artificial electron donors, which simultaneously act as electron mediators between the enzyme and an amperometric electrode. In addition to methyl viologen, redox-active compounds such as phenazines (phenosafranin, safranin T, N-methylphenazinium, 1-methoxy-N-methylphenazinium) and triarylmethane redox dyes (bromphenol blue and red) were selected from a range of redox compounds exhibiting the most efficient performance for nitrite detection. After precipitation, the electron mediators were incorporated in a graphite electrode material. Enzyme immobilization is performed by entrapment in a poly(carbamoyl sulfonate) (PCS) hydrogel. Diffusion coefficients and apparent heterogeneous rate constants of the mediators as well as homogeneous rate constants of nitrite sensors were determined by chronoamperometry and cyclic voltammetry. The phenosafranin-modified electrode layered with the PCS hydrogel immobilization of tetraheme cytochrome c nitrite reductase yielded linear current responses up to 250 µM nitrite with a sensitivity of 446.5 mA M-1 cm-2. The detection limit of the enzymatic nitrite sensor was found to be 1 µM nitrite. The quantitative determination of nitrite concentrations is of rapidly increasing interest, especially for the supervision of the quality of drinking water sources, waste water treatment, and the food industry and for the control of remediation procedures. Simple and highly selective and sensitive methods suitable for fast and reliable field measurements are desired in all these cases. Besides DIN and EPA photometric standard methods,1-4 ion chromatography,5 or the use of ion-selective electrodes,6-8 amperometric enzyme electrodes could be an alternative method for the determination of nitrite. †

Umweltforschungszentrum Leipzig-Halle GmbH. SensLab GmBH. § Universita ¨t Konstanz. | Bundesforschungsanstalt fu ¨ r Landwirtschaft. (1) Deutsche Einheitsverfahren zur Wasser-, Abwasser- und Schlammuntersuchung Band II; Physikalische, chemische, biologische und bakteriologische Verfahren (Gruppe D); DIN EN 26777sD 10; Bestimmung von Nitrit; VCH: Weinheim, Germany, 1993. ‡

0003-2700/96/0368-0807$12.00/0

© 1996 American Chemical Society

Kiang et al.9,10 developed enzyme electrodes for the determination of nitrate and nitrite by applying reductases which sequentially reduce nitrate via nitrite to ammonia in the presence of chemically reduced methyl viologen. The ammonia produced was measured by a potentiometric air-gap electrode. It is well-known that nitrite reductases (NiR) are able to use artificial electron donors11-17 such as reduced redox dyes. In this way, nitrite reductase can be coupled with an amperometric redox electrode modified with a suitable redox dye. This cathodically reduced electron donor shuttles electrons from the electrode surface to the reductase in the presence of the enzyme substrate (Figure 1). The resulting cathodic current is proportional to the (2) Deutsche Einheitsverfahren zur Wasser-, Abwasser- und Schlammuntersuchung Band II; Physikalische, chemische, biologische und bakteriologische Verfahren (Gruppe D); DIN 38 405sD 10; Bestimmung des Nitrit-Ions; VCH: Weinheim, Germany, 1981. (3) Methods for chemical analysis of water and wastes; EPA manual 625/676/003a, method 000630: Nitrogen, Nitrate-Nitrite (Cadmium Reduction Method and Automated Cadmium Reduction Method); Environmental Monitoring and Support Laboratory, Environmental Research Center, Cincinnati, OH, July 1976; pp 201-14. (4) Methods for chemical analysis of water and wastes; EPA manual 625/676/003a, method 000615: Nitrogen, Nitrite; Environmental Monitoring and Support Laboratory, Environmental Research Center, Cincinnati, OH, July 1976; pp 215-16. (5) Deutsche Einheitsverfahren zur Wasser-, Abwasser- und Schlammuntersuchung Band II; Physikalische, chemische, biologische und bakteriologische Verfahren (Gruppe D); DIN 38 405sD 19; Bestimmung der Anionen Flourid, Chlorid, Nitrit, Phosphat (ortho-), Bromid, Nitrat und Sulfat in wenig belasteten Wa¨ssern mit der Ionenchromatographie; VCH: Weinheim, Germany, 1988. (6) Schulthess, P.; Ammann, D.; Kra¨utler, B.; Caderas, C.; Stepa´nek, R.; Simon, W. Anal. Chem. 1985, 57, 1397-401. (7) Stepa´nek, R.; Kra¨utler, B.; Schulthess, P.; Lindemann, B.; Ammann, D.; Simon, W. Anal. Chim. Acta 1986, 182, 83-90. (8) Yang, S. T.; Bachas, L. G. Talanta 1994, 41, 963-8. (9) Kiang, C. H.; Kuan, S. S.; Guilbault, G. G. Anal. Chim. Acta 1975, 80, 20914. (10) Kiang, C. H.; Kuan, S. S.; Guilbault, G. G. Anal. Chim. Acta 1978, 50, 131922. (11) Zumft, W. G.; Sheer, B. F.; Payne, W. J. Biochem. Biophys. Res. Commun. 1979, 88, 1230-6. (12) Robinson, M. K.; Markinkus, K.; Kennelly, P. J.; Timkovich, R. Biochemistry 1979, 18, 3921-6. (13) Campbell, W. H.; Kinghorn, R. Trends Biochem. Sci. 1990, 15, 315-9. (14) Mellor, R. B.; Ronnenberg, J.; Campbell, W. H.; Diekmann, S. Nature 1992, 355, 717-9. (15) Iwasaki, Y.; Nishiyama, M.; Horinouchi, S.; Beppu, T.; Kadoi, H.; Uchiyama, S.; Suzuki, S.; Suzuki, M.; Tamiya, E.; Karube, I. Biocatalysis 1992, 6, 23545. (16) Moreno, C.; Costa, C.; Moura, I.; Le Gall, J.; Liu, M. Y.; Payne, W. J.; van Dijk, C.; Moura, J. J. G. Eur. J. Biochem. 1993, 212, 79-86. (17) Strehlitz, B.; Gru ¨ ndig, B.; Vorlop, K.-D.; Bartholmes, P.; Kotte, H.; Stottmeister, U. Fresenius J. Anal. Chem. 1994, 349, 676-8.

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Figure 1. Working principle of the mediator-modified enzyme sensor for nitrite.

nitrite concentration. For enzymatic-electrochemical nitrite removal from contaminant ground water, Mellor et al.14 selected methyl viologen, safranine T, azur A, neutral red, bromphenol blue, and Cibacron blue as suitable electron donors of a crude NiR from a Rhodopseudomonas species. The electron donor was immobilized in a polymer matrix on the surface of the cathode. For electrochemical studies, Iwasaki et al.15 used dissolved 1-methoxy-5-methylphenazine (M-NMP+), Meldola blue, and methylene blue for a copper-containing NiR from an Alcaligenes faecalis strain, and Moreno et al.16 described the use of methyl viologen for a hexaheme NiR from Desulfovibrio desulfuricans. In a recent work, we studied the electron-mediating properties of about 25 artificial electron donors for the cytochrome cd1 NiR from Paracoccus denitrificans. Nevertheless, data available from analytical applications of artificial electron donor-NiR couples indicate a detection limit between 5 × 10-5 M nitrite10 and 4.4 × 10-6 M nitrite.17 Therefore, we have continued the studies with a novel cytochrome c NiR from Sulfurospirillum deleyianum, comparing the results with the cytochrome cd1 NiR. Cytochrome cd1 NiR accepts one electron from the artificial donor, reducing nitrite to nitric oxide according to its in vivo reaction in the catabolic pathway of denitrifying bacteria.18 cathode

Medox + 1 e- 98 Medred NiRox + Medred f NiRred + Medox NiRred + NO2- + 2H+ f NiRox + NO + H2O

(1)

The cytochrome c NiR from S. deleyianum, which was recently isolated and characterized,19-21 reduces nitrite to ammonia, accepting successively six electrons from the artificial donor:19 cathode

6Medox + 6e- 98 6Medred NiRox + 6Medred f NiRred + 6Medox NiRred + NO2- + 8H+ f NiRox + NH4+ + 2H2O (2) Known artificial electron donors for the dissimilatory cytochrome cd1 NiR and tetraheme cytochrome c NiR are N,N,N′,N′-tetramethyl-p-phenylenediamine22 and methyl viologen,19 respectively. (18) Ye, R. W.; Averill, B. A.; Tiedje, J. M. Appl. Environ. Microbiol. 1994, 60, 1053-8. (19) Schumacher, W.; Kroneck, P. M. H. Arch. Microbiol. 1991, 156, 70-4. (20) Schumacher, W.; Kroneck, P. M. H. Arch. Microbiol. 1992, 157, 464-70. (21) Schumacher, W.; Kroneck, P. M. H., Pfennig, N. Arch. Microbiol. 1992, 158, 287-93.

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These highly selective enzymes combined with electron donormodified redox electrodes could be very attractive for nitrite determination. Suitable amperometric redox electrodes can be fabricated by incorporating slightly water-soluble electron mediators into a graphite-epoxy composite.23,24 The immobilization of the enzymes in a self-adhesive poly(carbamoyl sulfonate) (PCS) hydrogel25 seems to be a promising way toward entrapment of the delicate reductases which are then stabilized by cross-linking. Based on selected artificial redox mediators’ highly efficient electron donation to the nitrite reductases, novel amperometric nitrite electrodes are designed and characterized in this report. EXPERIMENTAL SECTION Chemicals. 3′,3′′,5′,5′′-Tetrabromophenolsulfonephthalein (bromphenol blue), 1,1′-dibenzyl-4,4′-bipyridinium, dichloride salt (benzyl viologen), 5′,5′′-dibromophenolsufonephthalein (bromphenol red), 3′,3′′,5′,5′′-tetrabromophenolsulfonephthalein, sodium salt (bromphenol blue, water soluble), 1,1′-dimethyl-4,4′-bipyridinium dichloride (methyl viologen), 1-methyl-1′-tetradecyl-4,4′-bipyridinium dichloride (1-methyl-1′-tetradecylviologen), 3,7-diamino2,8-dimethyl-5-phenylphenazinium chloride (safranin T; CI 50240), 3,7-diamino-5-phenylphenazinium chloride (phenosafranin; CI 50200), 3-(diethylamino)-7-[[4-(dimethylamino)phenyl]azo]-5-phenylphenazinium chloride (Janus green B; CI 11050), 5,5′-indigosulfonic acid, disodium (indigo carmine; CI 73015), 5,5′,7-indigo trisulfonic acid (indigo trisulfonate), 3-amino-7-(dimethylamino)5-phenothiazinium chloride (azure A, CI 52005), 7-(dimethylamino)-3-(methylamino)-5-phenothiazinium chloride (azure B; CI 52010), a mixture of azure I and methylene blue in equal amounts (azure II; CI 52010/52015), 7-(dimethylamino)-4-hydroxy-3-oxophenoxazine-1-carbonic acid (gallocyanine; CI 51030), N-methylphenazine (NMP+) methosulfate, N,N,N′,N′-tetramethyl-p-phenylenediamine dihydrochloride (N,N,N′,N′-TMPD), potassium ferrocyanide, and potassium ferricyanide were obtained from Fluka, Neu-Ulm, Germany. Flavin adenine dinucleotide disodium salt (FAD), tetrarhodanatodiammine chromate (Reinecke salt), graphite powder, sodium hydrosulfite (sodium dithionite), potassium chloride, sodium nitrite, disodium hydrogen phosphate, and sodium dihydrogen phosphate were purchased from Merck, Darmstadt, Germany. 1-Methoxy-5-methylphenazine (M-NMP+) methosulfate and poly(ethylenimine) (PEI) were supplied from Serva, Heidelberg, Germany. Anthraquinone-2,6-disulfonic acid was from Aldrich, Steinheim, Germany. Prepolymer of the PCS hydrogel was obtained from SensLab GmbH, Leipzig, Germany. Dialysis membrane (Cuprophan 80 M) was a free sample from AKZO Faser AG, Wuppertal, Germany. Enzymes. Cytochrome cd1 NiR from P. denitrificans. NiR was isolated from P. denitrificans (DSM 1403) according to the procedure of Kucˇera and Skla´dal26 after fermentation in a medium according to Alefounder and Ferguson27 and Witholt et al.28 The (22) Alefounder, P. R.; Greenfield, A. J.; McCarthy, J. E. G.; Ferguson, S. J. Biochim. Biophys. Acta 1983, 724, 20-39. (23) Gru ¨ ndig, B.; Strehlitz, B.; Krabisch, C.; Thielemann, H.; Kotte, H.; Gomoll, M.; Kopinke, H.; Pitzler, J. In GBF Monographs 17; Biosensors: Fundamentals, Technologies and Applications; Schmid, R. D., Scheller, F., Eds.; VCH: Weinheim, Germany, 1992; pp 275-85. (24) Gru ¨ ndig, B.; Wittstock, G.; Ru ¨ del, U.; Strehlitz, B. J. Electroanal. Chem. 1995, 395, 143-157. (25) Kotte, H.; Gru ¨ ndig, B.; Vorlop, K.-D.; Strehlitz, B.; Stottmeister, U. Anal. Chem. 1995, 67, 65-70. (26) Kucˇera, I.; Skla´dal, P. J. Basic Microbiol. 1990, 30, 515-22.

enzyme was purified as a homodimer (Mr 67 kDa per subunit).29 Cytochrome c NiR from S. deleyianum. The periplasmic cytochrome c nitrite reductase was isolated from the soluble fraction of S. deleyianum (DSM 6946T) according to Schumacher et al.30 The pure enzyme was a monomer (Mr 54 ( 2 kDa) with four covalently linked heme groups. An apparent specific activity of 1070 units mg-1 and an extinction coefficient 553(reduced) ) 89 000 M-1 cm-1 were obtained. Protein was determined with bicinchoninic acid according to Smith et al.31 using bovine serum albumin as the standard. Enzyme Immobilization. Using a bisulfite-blocked hydrophilic isocyanate prepolymer based on the isomer 2,4- and 2,6-tolylene diisocyanate 80:20 (TDI) relation and polyole,32 an enzyme-PCS hydrogel layer was formed according to a modified procedure:25 150 mg of prepolymer was suspended in 0.45 mL of doubly distilled water. PEI (2.5% w/v in water) was added dropwise to adjust the pH to between 4.5 and 5.0. The pH was followed by Merck special indicator paper. A 30 µL aliquot of the blank hydrogel matrix was mixed with 30 µL of enzyme solution (5-20 µL of NiR from S. deleyianum diluted with doubly distilled water to 30 µL). A 3-5 µL volume of this enzyme gel solution was placed on a dialysis membrane (Cuprophan 80 M). The immobilization occurred overnight at 277 K. Construction of Graphite and Mediator-Modified Electrodes. Graphite electrodes were fabricated by homogeneously mixing graphite powder and epoxy resin (0.9:0.1 w/w) and processing to rods at high pressure. The construction of mediator-modified electrodes was carried out as described.23, 24 Apparatus. Cyclic voltammetry and chronoamperometry for electrochemical characterization of electron donors and for the determination of the heterogeneous and homogeneous rate constants was performed using a PAR 273 A (EG&G GmbH Princeton Applied Research, Mu¨nchen, Germany) potentiostat. A three-electrode measuring system was used in a 5 mL (0.5 mL for the determination of the homogeneous rate constants) thermostated, stoppered cell. For the characterization of the artificial electron donors and for the determination of the rate constants, a graphite electrode fabricated as described above was used as the working electrode. The working electrodes were cleaned before each measurement by mechanical polishing with kaolin and rinsing with doubly distilled water. For constructing enzyme electrodes, the native NiR or the immobilized PCS-enzyme layer were mounted on the surface of the working electrode and fixed by a dialysis membrane (Cuprophan 80 M). The counterelectrode and the reference electrode were a platinum foil and a saturated calomel electrode K 10 (Sensortechnik Meinsberg, Meinsberg, Germany), respectively. All potentials are quoted relative to the saturated calomel electrode (SCE). Measurements were carried out at 298 K. The signals from the amperometric steady-state measurements were recorded with a BD 112 two-channel flat-bed recorder (Kipp (27) Alefounder, P. R.; Ferguson, S. J. Biochem. J. 1980, 192, 231-40. (28) Witholt, B.; Boekhout, M.; Brock, M.; Kingma, J.; van Heerikhuizen, H.; de Leij, L. Anal. Biochem. 1976, 74, 160-70. (29) Haalck, L. Institute of Chemical and Biochemical Research (ICB), Mu ¨ nster, 1993, unpublished work. (30) Schumacher, W.; Hole, U.; Kroneck, P. M. H. Biochem. Biophys. Res. Commun. 1994, 205, 911-6. (31) Smith, P. K.; Krohn, R. I.; Hermanson, G. T.; Mallia, A. K.; Gartner, F. H.; Provenzano, M. D.; Fujimoto, E. K.; Goeke, N. M.; Olson, B. J.; Klenk, D. C. Anal. Biochem. 1985, 150, 76-85. (32) Vorlop, K.-D.; Muscat, A.; Beyersdorf, J. Biotechnol. Tech. 1992, 6, 483-8.

& Zonen Deutschland GmbH, Solingen, Germany). Amperometric steady-state measurements were performed at 303 K. The amount of active enzyme immobilized in the gel was determined spectrophotometrically on a photometer SQ 118 (Merck). The thickness of the immobilized enzyme layer was measured by use of an eddy-current measuring method with the minitest 3001 (Electro-Physik, Ko¨ln, Germany). Procedures. N2-Saturated phosphate buffer (0.125 M, pH 7.4) was used for all measurements. Nitrogen was purged through the measuring solution for 10 min before each measurement. Nitrogen bubbling facilitated stirring in amperometric steady-state measurements. Measurements were carried out under an atmosphere of N2, thus preventing any contribution from dissolved oxygen. The most suitable potential-step regimes for chronoamperometric measurements were selected by reference to the cyclic voltammograms. The potential had to be stepped from an initial potential E1, where the reduction process to be studied could be neglected compared with the step potential E2 (usually an overpotential of about 200 mV with respect to the cathodic peak potential of the CV was applied) and where the reduction current of the mediator was diffusion controlled. The electrode was conditioned for 60 s at E1 to eliminate disturbances due to convection, then the electrode potential was stepped to E2, and the current was measured for 10 s. Electrochemical Characterization of the Electron Donors. The peak potentials and peak currents of the cyclic voltammograms were measured at a scan rate of ν ) 50 mV s-1. The switching potentials were selected according to the potentials for oxidation and reduction of the respective donor. The background current of the graphite electrode was measured in 0.125 M phosphate buffer. The cyclic voltammogram of the mediator was recorded in each case after the addition of the mediator solution (final concentration 1.0 or 2.0 mM). The background current was subtracted by the EG&G Model 270 electrochemical analysis system software before determining the peak potentials and peak currents. Determination of the Electrochemically Active Electrode Surface and Diffusion Coefficients of Mediators. Using the Cottrell equation, the electrochemically active electrode surface of the working electrode was estimated by chronoamperometric measurements;33 5 and 10 mM potassium ferrocyanide and potassium ferricyanide dissolved in phosphate buffer with 1 M KCl with known diffusion coefficients of 0.632 × 10-5 and 0.763 × 10-5 cm2 s-1, respectively,34 were used for this procedure. Based on the value of the electroactive area of the electrode surface, diffusion coefficients of selected electron mediators were determined analogously. First, the background current was measured in phosphate buffer and then different mediator concentrations (3, 4, and 5 mmol/L) were added. The background current was subtracted as described above, eliminating the residual current mainly caused by double layer charging.35 The values of these diffusion coefficients were used to determine the apparent heterogeneous standard rate constants kS.36 (33) Adams, R. N. Electrochemistry of solid electrodes; Marcel-Dekker: New York, 1969, Chapter 8, p 213 ff. (34) Adams, R. N. Electrochemistry of solid electrodes; Marcel-Dekker: New York, 1969; p 219. (35) Ryan, M. D.; Wie, J. F.; Feinberg, B. A.; Lau, Y.-K. Anal. Biochem. 1979, 96, 326-33.

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Table 1. Formal Potentials (E°′), Peak Potential Separations (∆Ep), Ratios of the Peak Currents |ia/ic| of Electron Mediators Used as Artificial Electron Donors for Cytochrome cd1 Nitrite Reductase from P. denitrificans and Cytochrome c Nitrite Reductase from S. deleyianum, and Amperometric Steady-State Current Responses to 1 mM NO2-a amperometric steady-state measc electron donor bromphenol blue benzyl viologen bromphenol red bromphenol blue, sodium salt methyl viologen 1-methyl-1′-tetra-decylviologen safranin T phenosafranin Janus green B FADH2 indigo carmine anthraquinone-2,6-disulfonic acid indigo trisulfonate azure A azure B azure II gallocyanine M-NMP+ NMP+ N,N,N′,N′-TMPD

CV characteristicsb E°′ (mV) ∆Ep (mV) -739 -721 -709 -697 -675 -489 -480 -437 -424 -386 -382 -356 -282 -171 -170 -170 -113 -102 -70 +16

169.0 184.0 105.0 126.0 106.0 77.0 70.5 72.0 58.0 96.0 58.0 160.0 88.1 82.6 59.8 68.3 51.2 72.8 96.7 63.3

NiR

|ia/ic|

polarization voltage (mV vs SCE)

P. denitrificans

S. deleyianum

0.84 1.67 0.94 0.88 1.03 1.03 0.89 1.19 1.16 1.22 1.19 1.09 0.98 1.41 1.28 1.03 1.01 1.63 2.20 0.94

-800 -800 -800 -800 -750 -650 -600 -600 -600 -600 -500 -500 -500 -300 -300 -300 -300 -200 -100 -100

1.000 0.083 0.375 0.500 0.500 0.275 0.038 0.113 0.113 0.250 0.055 0.050 0.113 0.240 0.045 0.105 0.050 0.440 0.375 0.050

0.712 0.630 0.368 0.503 0.988 0.395 0.834 1.000 ndd 0.605 0.265 0.480 0.435 0.000 0.000 0.000 0.000 0.000 0.001 0.000

a Graphite electrode (vs SCE), Pt counterelectrode, 0.125 M phosphate buffer solution pH 7.4, mediator concentration 1 mM, T ) 303 K. b Scan rate 50 mV s-1. c Current ratios normalized to the highest current for each of the nitrite reductases. d Not detected.

Determination of Heterogeneous Rate Constants. Apparent heterogeneous rate constants kS were calculated from the peak separation in the cyclic voltammograms according to the method of Nicholson.36 The mediator voltammograms were measured in 5 mL of phosphate buffer containing 1 mM mediator with scan rates of 10, 25, 50, 100, 200, and 500 mV s-1. The peak potentials were determined in order to evaluate the apparent heterogeneous rate constants. Determination of Homogeneous Rate Constants. The homogenous rate constants k2 for the reaction between several mediators and the cytochrome c NiR were determined by chronoamperometry according to Ryan et al.35 The measurements were carried out for blank solutions containing 0.25 mM mediator and 1.0 mM nitrite and after adding increasing amounts of cytochrome c NiR: 1.40 × 10-10, 2.80 × 10-10, 4.18 × 10-10, 5.55 × 10-10, and 6.90 × 10-10 M. The background i vs t curve was subtracted from the experimental i vs t plots as described. In order to prevent disturbing effects from convection, the measurements were carried out over a 10 s time interval. Determination of the Enzyme Activity. The specific activity of cytochrome c nitrite reductase was assayed by enzyme-catalyzed conversion of nitrite to ammonia using Na2S2O4-reduced methyl viologen as the electron donor.37 Transformation of nitrite to ammonia was quenched after 5 min and ammonia was determined.38 One unit of enzyme activity was defined as that amount which caused the formation of 1 µmol of NH4+ min-1 at 310 K. Determination of the Activity of Immobilized Enzyme. The activity of immobilized enzyme was determined according to Jones (36) Nicholson, R. S. Anal. Chem. 1965, 37, 1351-5. (37) Liu, M.-C.; Peck, H. D., Jr. J. Biol. Chem. 1981, 256, 13159-64. (38) Boltz, D. F.; Taras, M. J. In Colorimetric Determination of Nonmetals. Chemical Analysis, 2nd ed.; Boltz, D. F., Howell, J. A., Eds. John Wiley & Sons Inc.: New York, Chichester, Brisbane, Toronto, 1978; pp 197-251.

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and Garland39 with several modifications. The enzyme-catalyzed reduction of nitrite was measured by the initial absorbance decrease of reduced methyl viologen based on a calibration curve for the reduction of 10 mM methyl viologen in phosphate buffer solution using defined concentrations of dithionite. The N2saturated phosphate buffer solution in the stoppered cuvette (1 cm) contained 10 mM methyl viologen and a piece (19.63 × 10-2 cm2) of the membrane with immobilized cytochrome c nitrite reductase. In order to obtain an absorbance of methyl viologen of A585 ≈ 1.4, dithionite was added from a stock solution. The reaction was started by adding anaerobically 1.0 mM NO2-, and the decrease in absorbance was measured at 585 nm every 10 s. Characterization of Electrodes. The biosensor response to nitrite was monitored amperometrically by following the steadystate currents in 0.125 M phosphate buffer (pH 7.4). RESULTS AND DISCUSSION Selection of Efficient Artificial Electron Donors. In order to select suitable electron donors for the nitrite reductases, a range of viologens (methyl, benzyl, and 1-methyl-1′-tetradecyl-4,4′ viologen), triarylmethane redox dyes (bromphenol blue and red), phenazines (NMP+, M-NMP+, Janus green B), phenoxazines (gallocyanine, safranin T, and phenosafranin), phenothiazines (azure A, B, and II), indigo dyes (indigo carmine, indigo trisulfonate), the coenzyme FADH2, and several other redox mediators (anthraquinone-2,6-disulfonic acid, N,N,N′,N′-TMPD) were investigated by cyclic voltammetry. The formal potentials (E°′ ) (Epa + Epc)/2), the difference between the peak potentials (∆Ep), and the ratio of anodic to cathodic peak current (|ia/ic|) are depicted in Table 1. The efficiency of the mediators acting as artificial electron donors for the NiR from P. denitrificans and S. deleyianum (39) Jones, R. W.; Garland, P. B. Biochem. J. 1977, 164, 199-211.

Table 2. Diffusion Coefficients and Heterogeneous Rate Constants of the Relevant Mediators As Determined by Chronoamperometry and Cyclic Voltammetrya mediator

diff coeff (10-6 cm2 s-1)

heter rate const (10-3 cm s-1)

phenosafranin M-NMP+ methyl viologen NMP+ FADH2 azure A

9.95 5.88 5.41 4.57 1.81 1.16

2.01 2.98 1.38 1.00 2.02 0.74

aSee

Figure 2. Cyclic voltammograms of 1.0 mM phenosafranin in 0.125 mM phosphate buffer at a graphite electrode (a), when 10 µL of nitrite reductase from S. deleyianum was entrapped at the electrode surface (b), and after addition of 10 mM nitrite to the solution (c). 0.125 M phosphate buffer solution, pH 7.4; T ) 298 K; ν ) 50 mV s-1.

was studied by amperometric steady-state measurements at a nitrite concentration of 1.0 mM. The polarization voltages used are listed in Table 1. The catalytic steady-state current measured under batch conditions was normalized to the highest current obtained with each mediator-NiR preparation. Triarylmethane redox dyes (bromphenol blue and red, bromphenol blue sodium salt), methyl viologen, phenazines (M-NMP+, NMP+), 1-methyl1′-tetradecylviologen, and FADH2 were suitable electron donors for cytochrome cd1 NiR. The cytochrome c NiR preferred phenazine dyes (phenosafranin, safranin T), methyl viologen, benzyl viologen, bromphenol blue, and FADH2. The standard potentials of the reduction of NO2- to NO (E0′ ) 0.109 V vs SCE) and NO2- to NH4+ (E0′ ) 0.099 V vs SCE) indicated thermodynamically that electron donors with formal potentials E0′ below 0 mV vs SCE were required. The cytochrome cd1 NiR was able to accept electrons from the donors studied that had a formal potential of at least -70 mV vs SCE (NMP+) and also from donors with much more negative potentials (methyl viologen, bromphenol blue). Unlike the one-electron-transfer NiR, tetraheme cytochrome c NiR preferred electron donors with formal potentials in the range -386 mV vs SCE (FADH2) to -739 mV vs SCE (bromphenol blue). With respect to these results, further studies were performed with methyl viologen and M-NMP+ as electron donors for cytochrome cd1 NiR as well as phenosafranin, safranin T, and methyl viologen for cytochrome c NiR. The catalytic behavior of NiR from S. deleyianum using the electron mediator phenosafranin as artificial electron donor is depicted in Figure 2. Phenosafranin (1.0 mM) showed a quasireversible cyclic voltammogram in phosphate buffer solution at a graphite working electrode (Figure 2a). The peaks of the anodic and cathodic current shifted slightly cathodically when 10 µL of cytochrome c NiR was retained at the graphite electrode behind a dialysis membrane. This may have been caused by adsorption of the enzyme (Figure 2b). The addition of 10 mM nitrite to the measuring solution caused a decrease in the anodic peak current

text for measuring conditions.

and a significant increase in the cathodic peak current indicating an efficient catalytic reaction. The same effect was observed for cytochrome cd1 NiR. Diffusion Coefficients and Heterogeneous Rate Constants. The diffusion coefficients (D0) for the oxidized forms of the dissolved species were used for the determination of the apparent heterogeneous rate constants kS by chronoamperometry33 (Table 2). The electrochemically active surface area of the graphite electrode was estimated to be 3.5 × 10-2 cm2, which agreed surprisingly well with the geometrical surface area of 3.14 × 10-2 cm2. The apparent heterogeneous rate constants for some of the most suitable electron donors for the cytochrome cd1 NiR (methyl viologen, M-NMP+, NMP+, FADH2) and for the cytochrome c NiR (phenosafranin, methyl viologen, FADH2) were determined from the peak separations of the corresponding voltammograms according to Nicholson36 (Figure 3). Hereby, the rate constants, identical diffusion coefficients for the oxidized and reduced forms of the mediators, and a transfer coefficient of 0.5 were assumed. From the data in Table 2 it should be noted that high values of diffusion coefficients and rapid heterogeneous electron transfer rates were obtained for those redox mediators found to be the most efficient as donors for the respective NiR. In contrast, the diffusion coefficient and the heterogeneous rate constant for the inappropriate electron donor azure A were significantly smaller. Presupposing a suitable negative redox potential for the redox mediator, it appears that both parameters are basic conditions for an efficient electron shuttle from the cathode to the reductase. Homogeneous Rate Constants. The reaction rates between selected artificial donors and the tetraheme cytochrome c NiR were determined by chronoamperometry as described by Ryan et al.35 Attempts to evaluate the rate constants from CV measurements40,41 yielded irreproducible results. At high scan rates (>200 mV s-1), large charging currents for the double-layer capacitance were observed, resulting in inaccurate determination of the peak currents. Therefore, chronoamperometry was applied as a more convenient method for determining the homogeneous rate constants.16,24,42 Figure 4A shows the background-substracted i-t data of the diffusion controlled currents with increasing NiR concentrations 1-5. Because of the catalytic effect, the current decreased (40) Nicholson, R. S.; Shain, I. Anal. Chem. 1964, 36, 706-23. (41) Cass, A. E. G.; Davis, G.; Hill, H. A. O.; Nancarrow, D. J. Biochim. Biophys. Acta 1985, 828, 51-57. (42) Hoogvliet, J. C.; Lievense, L. C.; van Dijk, C.; Veeger, C. Eur. J. Biochem. 1988, 174, 273-80.

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Figure 3. Cyclic voltammograms of a graphite electrode in 0.125 M phosphate buffer solution pH 7.4 containing 1.0 mM mediator, T ) 298 K, reference electrode SCE: mediators, phenosafranin (A), M-NMP+ (B), methyl viologen (C), NMP+ (D), FAD (E), azure A (F); scan rates, 10 (a), 25 (b), 50 (c), 100 (d), 200 (e), and 500 mV s-1 (f).

in the presence of increasing NiR concentrations less rapidly than in the case of simple diffusion of the mediator phenosafranin.The ratios of the catalytic and diffusion currents (ic/id) were plotted over the time interval 0.5-4 s to obtain linear plots (Figure 4B). These ratios were converted into values of the kinetic parameter λ42 and plotted vs time (Figure 4C). Initial slopes of these plots were obtained by linear regression analysis and plotted vs the NiR concentration (Figure 4D). The NiR concentration used in the calculation was one-sixth of the actual concentration, assuming that six molecules of reduced phenosafranin are needed for the reduction of nitrite to ammonia. The second order rate constant k2 was calculated from the slope of the straight line through the points.42 In this way, the homogeneous rate constants k2 for cytochrome c NiR and the artificial electron donors phenosafranin (Figure 4), methyl viologen, safranin T, and FADH2 were 812 Analytical Chemistry, Vol. 68, No. 5, March 1, 1996

estimated, resulting in the values 11.8 × 109, 10.1 × 109, 5.9 × 109, and 2.1 × 109 M-1 s-1, respectively. Taking into account the formal potentials of the donors ranging between -675 and -386 mV vs SCE, it is reasonable to assume that the different rate constants arise from the affinity of the enzyme for the donor22 and also from steric and electrostatic interactions.15 The rate constant for cytochrome c NiR and methyl viologen agrees roughly with the rate constants determined for the reduction of cytochrome c3 from Desulfovibrio vulgaris and D. desulfuricans by methyl viologen.16,43 Moreno et al.16 estimated the homogeneous rate constant for the reaction between methyl viologen and NiR from D. desulfuricans by cyclic voltammetry as 1.6 × 108 M-1 s-1 and by chronoamperometry as 2.2 × 108 M-1 s-1. The reason (43) van Leeuwen, J. W.; van Dijk, C.; Grande, H. J.; Veeger, C. Eur. J. Biochem. 1982, 127, 631-7.

Figure 4. Determination of the homogeneous rate constant k2 for nitrite reductase from S. deleyianum and the artificial donor phenosafranin by chronoamperometry. (A) Current-time plots: background current of the graphite working electrode, 0.125 M phosphate buffer pH 7.4, 0.26 mM phenosafranin, 10.3 mM KNO3- (0). Currents after adding increasing concentrations of NiR: 2.34 × 10-11 (1); 4.66 × 10-11 (2); 6.96 × 10-11 (3); 9.25 × 10-11 (4); 11.5 × 10-11M (5). (B) Plot of ic/id vs t for enzyme concentrations 1-5. (C) Plot of the kinetic parameter λ vs t for the NiR concentrations35 1-5. (D) Initial slope vs NiR concentration, obtained from λ vs t plots.

for the difference in measured rate constants between the NiR from D. desulfuricans and S. deleyianum could be related to differences in the structure of these enzymes.30 Sensor Parameters. For selecting the most suitable redox mediators (Table 1), the evaluation criteria were their mediating efficiency, quantified as amperometric steady-state current to the

corresponding NiR, and the polarization voltage required to reduce the electron donor. Although bromphenol blue was found to be the most efficient electron mediator, this compound needs a polarization voltage of about -800 mV vs Ag/AgCl, which would be accompanied by a strong interfering oxygen sensitivity of the potential sensor by both cathodic reduction of oxygen and the Analytical Chemistry, Vol. 68, No. 5, March 1, 1996

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Figure 5. Calibration curves of entrapped nitrite reductases at a graphite electrode using different electron donors: 0.125 M phosphate buffer solution, pH 7.4, T ) 303 K, stirring by bubbling N2 through the cell. Nitrite reductase from S. deleyianum and phenosafranin, E ) -600 mV (3), methyl viologen, E ) -750 mV (O) and safranin T, E ) -600 mV (]); nitrite reductase from P. denitrificans and M-NMP+, E ) -200 mV (4) and methyl viologen, E ) -750 mV (0) for high concentrations (A) and low concentrations (B). Counterelectrode, Pt wire, and polarization voltages vs SCE.

autoxidation of bromphenol blue. Such a sensor would be hard to handle under real conditions. A similar characteristic was found for methyl viologen, but for comparability with other works it was selected.15-17 Furthermore, phenosafranin and safranin T were used as the most acceptable compromise between the parameters sensitivity and polarization voltage. Calibration curves obtained with entrapped NiR (3 µL) from S. deleyianum and 1.0 mM mediator in 0.125 M phosphate buffer (pH 7.4) show a linear range up to 1 mM nitrite (Figure 5). The sensitivities of the electrodes using the mediators phenosafranin, methyl viologen, and safranin T were 274.5, 255.3, and 191.5 mA M-1 cm-2, respectively. Comparing the detection limits, the lowest value of 1 µM nitrite was obtained with both phenosafranin and methyl viologen. Cytochrome cd1 NiR was studied with the electron donor M-NMP+ at a working potential of -200 mV vs SCE. The amperometric cytochrome cd1 NiR/M-NMP+ system showed a linear response up to 750 µM nitrite with a sensitivity of 33 mA M-1 cm-2. The detection limit was 10 µM nitrite. The higher sensitivity obtained using the cytochrome c NiR compared with cytochrome cd1 NiR results from its capacity to transfer six electrons vs one electron in the case of cytochrome cd1 NiR, which produces a higher reduction current per nitrite molecule reduced. Therefore, the NiR from S. deleyianum was preferred for preparation of the nitrite sensor. Enzyme immobilization of the cytochrome c NiR (1070 units mg-1) was performed as described previously using 5, 10, and 20 µL enzyme solutions to prepare the enzyme immobilization layers. The enzyme activity of the different layers was determined to be 43.5, 48.6, and 58.1 milliunits cm-2, respectively. Calibration curves obtained with the membranes with different enzyme loadings using 1 mM dissolved phenosafranin in the measuring solution showed sensitivities of 427.7, 518.8, and 452.4 mA M-1 814 Analytical Chemistry, Vol. 68, No. 5, March 1, 1996

cm-2, respectively (Figure 6). From the corresponding calibration curves, apparent KM values of 140 (a), 205 (b), and 235 µM (c) were calculated for these sensors. The detection limit of about 1 µM nitrite was nearly identical for the different enzyme layers and was similar to that found for the entrapped enzyme. For practical applications it is advantegeous to use electron mediators that are fixed at the electrode surface. We have solved the problem by preparing hardly soluble forms of the mediators which were physically entrapped into a graphite-epoxy electrode material. Therefore, the solubility of mediator is a critical point with respect to the long-term stability of the working electrode, depending on mediator leaking at the one hand and the need of a certain solubility of the mediator enabling a homogeneous reaction with the redox-active center of the enzyme at the other hand. Phenosafranin and safranin T were precipitated in aqueous media by substitute their anions with Reineckate anions. As we found for phenazine Reineckates in SECM experiments,24 the mediator dissolves in aqueous media to a very small amount and forms a stable steady-state diffusion layer in close proximity to the electrode surface. Previous experiments for selecting suitable anions have shown that Reineckate anions are favorable substituents.24 The polarization voltage needed for both phenosafranin and safranin T was -600 mV vs SCE. The somewhat lower negative polarization voltage of these mediators compared with -750 mV for methyl viologen was an aspect in favor of these donors. The electrodes were combined with membranes with immobilized cytochrome c NiR (58.1 milliunits cm-2). Calibration curves of nitrite electrodes are shown in Figure 7. The sensors had a linear range up to at least 250 µM nitrite. The detection limit was again 1 µM nitrite. The sensitivities in the linear range were 446.5 mA M-1 cm-2 for the phenosafraninmodified electrode and 435.9 mA M-1 cm-2 for the safranin

Figure 6. Dependence of the sensor response on the membrane loading of nitrite reductase from S. deleyianum: 43.5 (0), 48.6 (O), and 58.1 milliunits cm-2 (4); 0.125 M phosphate buffer solution, pH 7.4, with 1 mM phenosafranin, T ) 303 K, stirring by bubbling N2 through the cell, E ) -600 mV vs SCE, for high concentrations (A) and low concentrations (B). Counterelectrode, Pt wire.

Figure 7. Calibration curves of mediator-modified enzyme sensors using nitrite reductase from S. deleyianum (58.1 milliunits cm-2): Phenosafranin-modified electrode (0), safranin T-modified electrode (O), 0.125 M phosphate buffer solution, pH 7.4, T ) 303 K, stirring by bubbling N2 through the cell, E ) -600 mV vs SCE, and counterelectrode, Pt wire.

T-modified electrode. The apparent KM values for nitrite of these sensors are 205 and 170 µM, respectively. The sensitivity and the apparent KM value for the phenosafranin-modified electrode were somewhat smaller than measured for an analogous immobilization (58.1 milliunits cm-2) in combination with dissolved mediator, which indicated that the system displayed only a small limitation from incorporation of the mediator-Reineckate form

in the electrode material. The 95% response time for 10 µM nitrite was 3 min. This arose from the thickness of the membrane of about 40 µm determined by an eddy-current measurement. A remarkable obstacle with respect to both NiR and mediator is the interference from oxygen, which decreases the sensitivity by about 50%. The detection limit increased from 1 to 3 µM in air-saturated measuring solutions, which may be due to the autoxidation of Analytical Chemistry, Vol. 68, No. 5, March 1, 1996

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enzyme and mediator and to oxygen radical-mediated changes of the active center of the enzyme. A decrease in activity by 50% of a phenosafranin-modified electrode with immobilized cytochrome c NiR was achieved after 17 h, when one measurement per hour was carried out at a nitrite concentration of 20 µM. The electrode was stored overnight in phosphate buffer at 277 K. Subsequent measurements with additional 0.1 mM phenosafranin in the measuring solution increased the current signals up to 120% compared with the measurement without the additional mediator. This effect may be due to a limitation of soluble mediator in the enzyme layer. However, the decrease in the signal with time is in the same range both for the mediator-modified electrode and for the mediator in the measuring solution, indicating a loss of sensor activity mainly by deactivation of the immobilized enzyme. The storage stability of immobilized cytochrome c NiR, which had been kept dry at about 281 K, was followed daily by combining a membrane from the same fabrication charge with a phenosafranin-modified electrode and measuring the catalytic current arising from 20 µM nitrite. The enzyme membrane retained about 85% activity after 8 days and about 45% of the initial activity after 14 days. CONCLUSIONS In this study we demonstrated for the first time that an immobilized nitrite reductase can be coupled via electron mediators to a redox electrode for the construction of an amperometric (44) EUsguideline for drinking water, 15.07.1980 (80/777/EWG, Publ. EU. 30.08.1980 Nr. L 229/11), 1980. (45) Verordnung u ¨ ber Trinkwasser und Wasser fu ¨ r Lebensmittelbetriebe (TrinkwasserverordnungsTrink V) vom 22.5.86 (BGBl. I S. 760) in der Fassung der Bekanntmachung vom 5.12.90 (BGBl: I S. 2612, 1991 S. 227) (BGBl. III 2126-1-7). (46) Jacobson, K. B.; Manos, R. E.; Wadinski, F. A. Biotechnol. Appl. Biochem. 1987, 9, 368-79.

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nitrite sensor. By use of the six-electron-transferring cytochrome c NiR from S. deleyianum in combination with a suitable artificial electron donor such as phenosafranin, selective and highly sensitive determination of nitrite in aqueous solution is achieved. The detection limit of the enzymic nitrite sensor is roughly half of the limit of this hazardous anion in drinking water according to EU guidelines44 and the decree of the F.R.G.45 Major disadvantages of the sensor are concerned with its limited stability, sensitivity to oxygen, and the relatively long response time of 3 min for the 95% value. The last aspect can be addressed by optimization of the thickness and composition of the immobilization matrix. A more complicated issue is the stabilization of the nitrite reductase. Current work in our laboratory concerns the effect of stabilizing additives such as sugars and polyelectrolytes on the immobilization matrix. Finally, the oxygen influence could either be handled by defined air saturation of the samples or by adding a protective enzyme layer consisting of an oxidase or the recently discovered oxyrase.46 ACKNOWLEDGMENT We are grateful to Dr. L. Haalck, Institut fu¨r Chemo- und Biosensorik, Mu¨nster, for isolating the NiR from P. denitrificans and AKZO-Faser AG, Wuppertal, for the generous gift of Cuprophan 80 M dialysis membrane. We thank R. Erben for skillful technical assistance and Dr. C. McNeil from the University of Newcastle upon Tyne (Great Britain) for a critical discussion of the manuscript. Received for review July 12, 1995. Accepted November 13, 1995.X AC950692N X

Abstract published in Advance ACS Abstracts, January 15, 1996.