Anal. Chem. 1995, 67, 2822-2827
Cyanide Determination Using an Amperometric Biosensor Based on Cytochrome Oxidase inhibition Aziz Amine,+ Mokarram Alafandy, and Jean-Michel Kauffmann* lnstitut de Pharmacie Campus Plaine, Universite Libre de Bruxelles, CP 205/6,7050 Bruxelles, Belgium
Marta Novak Pekli Department of Pharmaceutical Chemistry, Semmelweis University of Medecine, 1092 H-Budapest, Hungary
An amperometric reagentless biosensor, based on a
carbon paste electrode (CPE) modified by asolectin, cytochrome c, and cytochrome oxidase, is described for the sensitive determination of cyanide. The modified CP matrix mimics a biological membrane environment. The sensor, polarized at -0.15 V vs Ag/AgCl, generates the reduced form of cytochrome c, which in turn is oxidized by the enzyme cytochrome oxidase. The resulting current is related to the enzyme activity and is depressed by inhibitors of cytochrome oxidase such as cyanide. Concentrations of cyanide as low as 0.5 pM can be measured with half-maximal response at about 12 p M . The effects of pH, ionic strength, and temperature on this new cyanide biosensor are reported. The inhibition is reversible and reproducible (RSD = 4%), allowing cyanide determination for more than 2 months using the same probe. Possible use of this biosensor in flow systems is illustrated. Most of the enzyme-immobilized electrodes cited in the literature are developed for the determination of their substrates, and only a few reports are devoted to the determination of specific inhibitor^.'-^ This may be due to the facts that enzyme sensors based on inhibition require a rigorous control of substrate concentration and enzyme activity and that the inhibition should be re~ersible.~ In this paper, cyanide is determined by exploiting its noxious effect on cytochrome oxidase5-13 immobilized in the matrix of a lipid-cytochrome c-modified carbon paste electrode (CPE). ' Present address: Faculte des Sciences et Techniques "Mohammadia", Department of Biology, Mohammadia. Morocco. (1) Blum, L. J.; Coulet, P. R. Biosensor, principles and applications; Marcel Dekker: New York, 1992. ( 2 ) Griffiths, D.; Hall, G. Trends Biotechnol. 1993,11, 122. (3) Turner, A. P. F.: Kambe, I.: Wilson, G. S.Biosensor Fundamentals and Applications: Oxford University Press: Oxford, 1987. (4) Tran-Minh, C. Ion-Sel. Electrode Rev. 1985,7,41. (5) Keilin, D.; Hartree, E. F. Proc. R. Sot. London 1939,B127,167. (6) Yonetani, T.; Ray, G. S. J. Biol. Chem. 1965,240,3392. (7) Nicholls, P.; Van Buuren. J. H.; Van Gelder, B. F. Biochim. Biophys. Acta 1972.275,279. (8) Albery, W. A: Cass, A. E. G.; Shu, Z. X. Biosens. Bioelectron. 1990,5,397. (9) Albery, VI'. J.: Cass, A. E.: Hubbard. J. A. M.; Shu, Z. Biochem. Soc. Trans. 1986,14. 1212. (10) Hill, B. C.; Marmor, S.Biochem. J. 1991,279,355. (11) Mitchell, R.; Brown, S.; Mitchell, P.; Rich, P. R Biochim. Biophys.Acta 1992.
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Analytical Chemistry, Vol. 67, No. 17, September 1, 1995
Mixed enzyme CPEs are receiving considerable interest since they allow the preparation of fast-responding stable and reagentless biosensors.14--24 The development of a reagentless cyanide sensor is of considerable environmental interestLi Standard methods are based on the evolution of gaseous HCN from an acidified solution by distillation and purging with air. The HCN gas is collected by absorption in a suitable alkaline acceptor solution to form free CN- ions. Subsequently, cyanide can be determined by titrimetric, colorimetric, or potentiometric methodsSz5Problems encountered with these devices include cost and lack of selectivity, and often lengthy preparation times are required before sample analysis.25 Others methods use ion-selective electrodes, detecting cyanide at the micromolar level, but suffer greatly from interferences by sulfide, mercaptans, iodide, and chloride ions.26 Recently, enzyme electrodes which detect and quantify cyanide on the basis of its biological action, rather than its physical or chemical action, have been proposed.892 i 2 8 Ideally, a biosensor for cyanide measurement should incorporate cytochrome oxidase, Le., the specific biological component upon which cyanide acts to exert its lethal effect. Cytochrome oxidase is the terminal electron transport complex of the inner mitochondrial membrane; it requires phospholipids for a ~ t i v i t y . ~The ~ - ~ enzyme ~ contains four redox sites and (12) Berka, V.; L'ygodina, T.: Musatov, A,: Nicholls. P.; Konstantinov, A. A. FEES Lett. 1993,315, 237. (13) Nichols. P. Trends Biochem. Sci. 1983,8, 353. (14) Wang. J.; Wu. L.; Lu, Z.: Li, R.; Sanchez, J. Anal. Chim. Acta 1990.228, L31.
(15) Amine, A.; Kauffmann. J.-M.; Patriarche, G. P. Talantu 1991,38,107. (16) Amine, A.; Kauffmann, J:M. Bioelectrochem. Bioenerg. 1992.28,117. (17) Amine. A ; Kauffmann, J.-M.; Guilbault. G. G.: Bacha. S. Anal. Lett. 1993. 26, 1281. (18) Kulys. J.; Schuhmann. 'A'.; Schmidt, H. L. Anal. Lett. 1992,25,1011. (19) Bremle. G.; Persson, B.; Gorton, L. Electroanalysis 1991,3,77. (20) Yabuki. S.; Mizutani. F.: Katsura, T. Biosens. Bioelectron. 1992.7,695. (21) Bonakdar, M.; Vilchez. J. L.: Mottola, H. A. J. Electroanal. Chein. 1989. 266.47. ( 2 2 ) Smit, M H.: Rechnitz, G. A. Anal. Chem. 1992.64.245. (23) Amine. A.; Deni. J.: Kauffmann. J.-M. Bioelectrochem. Bioenerg. 1994,34, 123. (24) Amine, A.: Kauffmann. J.-M.; Palleschi, G. Anal. Chim. Acta 1993.273. 213. (25) Standard Methods for the Examination of Water and Wastewater, l i t h ed.; American Public Health Association: Washington. DC, 1989: Method 4500ch-, pp 4-20, (26) Ion-Selective Electrode Catalog; Orion Research Inc.: Boston, 1992. (27) Smit, M. H.: Cass. A. E. Anal. Chem. 1990.62,2429. (28) Smit. M. H.: Rechnitz. G. A. Anal. Chem. 1993,65,380. 0003-270019510367-2822$9.0010 0 1995 American Chemical Society
Cytochrome oxidase
Reagents. All reagents were of analytical grade, supplied by transfers electrons from reduced cytochrome c to molecular Sigma or Merck (Brussels, Belgium). Cytochrome oxidase (EC 0xygen.~,3~-~~ Its inhibition by cyanide is known to be noncom1.9.3.1.; 13 unitdmg), horse heart cytochrome c (oxidized form, petitive toward 02.13 A previously described cyanide amperometric b i o s e n ~ o r , ~ ~ ~C7752), tyrosinase (EC 1.14.18.1.; 13 900 unitdmg), and asolectin (L-phosphatidyl choline, type 11-S) extracted from soybean, con prepared by retaining cytochrome c and cytochrome oxidase with a dialysis membrane on the surface of a gold electrode, had a tainiig 18%of phosphatidyl choline along with other lipids (P5638), were from Sigma. The carbon paste was from Metrohm (EA 207c, limited operating lifetime, due probably to a lack of lipid environment in the sensor configuration. Thus, alternative enzymes have 76%graphite and 24%liquid paraflin). The supporting electrolyte been s u g g e ~ t e d . 2Peroxidasez7 ~~~~ and tyrosinasez8have been used, was phosphate buffer, and a solution of the desired pH was but these enzymes have a variety of substrate^,^^ and the halfprepared from a mixture of NaZHP04 and NaHzP04 solutions. maximal response for cyanide is highly dependent on substrate Stocks solutions of KCN, NaZS, and NaN3 were prepared daily. concentration.z7~z8Furthermore, the sensor based on these Solutions were prepared from reagent-grade chemicals using enzymes requires the addition of a mediator in s o l ~ t i o n . ~ ~ ~ ~ deionized ~ water. Caution: Since cyanide is a dangerous poison, In our previous work,z3rapid electron transfer of the redox all solution preparations were done in a fume cupboard. Experiprotein cytochrome c was obtained by incorporating negatively ments were performed in a well-aerated room. charged lipids (e.g., phosphatidyl serine, asolectin, or cardiolipin) Electrode Preparation. The modified CP electrode was into the CP electrode matrix. In the following report, we first prepared by thoroughly mixing in a mortar CP and the appropriate study the possible direct electroactivity of the enzyme cytochrome lipid (5% w/w) in the presence of a minimum amount of oxidase immobilized in the CPE in the presence of charged lipids. chloroform. After evaporation of the solvent, an appropriate Direct electron transfer was observed in cyclic voltammetry, but amount of cytochrome oxidase (0.5-5% w/w), alone or with its inhibition by cyanide was not detected. The ternary electrode cytochrome c (0.5-4%), was added and mixed with the lipid CP configuration (i.e., cytochrome oxidase, cytochrome c, and lipid) matrix. A portion of the resulting paste was packed into the well was, however, sensitive to cyanide. Scheme 1shows the sequence of the body of the BAS electrode (3 mm diameter, 2 mm depth). of cytochrome oxidase/cytochrome c reactions used in the After the surface was manually smoothed on clean paper, the biosensor configuration. enzyme electrode was tightly covered with a piece of dialysis membrane (Spectra pore) having a molecular weight cutoff of EXPERIMENTAL SECTION -3500. This prevented the swelling phenomenon of the mixed Apparatus. Cyclic voltammetry and amperometric measureprotein-lipid CPEz3and any leaching of cytochrome c out of the ments were performed with a CV 27 voltammograph (BAS, West probe into the solution. Lafayette, IN) connected to a Hewlett-Packard 709OAx-y recorder. Procedure. Before any cyclic voltammogram (CV) recording, All experiments were performed with a three-electrode cell the enzyme electrode was first conditioned over 30 min by cycling configuration containing the working electrode, a Ag/AgCl, KCl between +0.5 and -0.2 V vs Ag/Ag+ at a sweep rate of 5 mV s-l. saturated reference electrode, and a platinum wire as auxiliary For amperometric recordings, a constant potential (-0.15 V electrode. The pH of the solution was measured with a Tacussel vs Ag/Ag+) was applied to a freshly prepared enzyme-cytoMini 80 pH meter. All experiments were carried out at 23 f 1 chrome c-lipid CPE. The electrode was dipped into a 10 mL "C. A homemade cell was usedz4 for flow and batch injection stirred solution, and a stable residual current was obtained within analysis. Temperature studies were carried out in a double wall 30 min. This residual current corresponded to the electrochemical beaker, thermostatically controlled. reduction of the oxidized form of cytochrome c, catalyzed by enzymatic regeneration (Scheme 1). Scheme 2 illustrates the (29) Palmer, G. Pure Appl. Chem. 1987,59, 749. origin of the residual current. Step 1 corresponds to a residual (30) Kadenbach, B.; Jarausch, J.; Hartmann, R.; Merle. P. Anal. Biochem. 1983, 129, 517. current due to the electrochemical reduction of cytochrome c (31) Vik, S. B.; Capaldi, R. A. Biochemistry 1977,16, 5755. (oxidized form). Step 2 corresponds also to the electrochemical (32) Vik, S. B.; Capaldi, R. A Biochem. Biophys. Res. Commun. 1980,94, 348. reduction of cytochrome c but catalyzed by the enzyme cyto(33) Vik, S. B.; Georgevich, G.; Capaldi, R A Proc. Natl. Acad. Sci. U&4. 1981, 78, 1456. chrome oxidase (the catalytic effect resulted from the in situ (34) Fry,M.: Green, D. E. Biochem. Biophys. Res. Commun. 1980,93, 1238. enzymatic regeneration of cytochrome c in its oxidized form). Step (35) Robinson, N. C.; Strey, F.; Talbert. L. Biochemistry 1980,19, 3656. (36) Azzi, A. Biochim. Biophys. Acta 1980,594, 231. 3 corresponds to the inhibition of the enzyme in the presence of (37) Powell, G. L.; Abramovitch, D. A.; Kim, K FASEB]. 1988,2, 1638. a saturating amount of cyanide. The magnitude of the current in (38) Abramovitch, D. A.; Marsh, D.; Powell, G. L. Biochim. Biophys. Acta 1990, each step is related to the amounts of cytochrome c (step 1) and 1020, 34. (39) Nicholls, P. Biochem. J 1992,288, 1070. cytochrome c + enzyme (step 2). (40) Babcock, G. T.; Wikstrom. M. Nature 1992,356, 301. Following the initial stabilization period, aliquots of the inhibitor (41) Pan, L.-P.; Hazzard, J. T.; Lin. J.; Tollin, G.; Chan, S. I. ]. Am. Chem. SOC. (from the stock solution) were successively injected into the cell 1991,113,5908. (42) Enzyme Nomenclature; Academic Press, Inc.: San Diego, 1992. while the current was monitored. A calibration curve was obtained Analytical Chemistry, VoJ. 67, No. 17, September 1, 1995
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0
CPE
CPE Cyt. c Cyt. ox Saturated with inhibitor
Cyt. c
6
PE
step 2
v
by applying the standard addition method, with measurements at 95%of the steady state current. The measured current (I) is the difference between the current of unbound enzyme (residual current at step 2) and the current of inhibitor-bound enzyme. I, is the current at infinitely high inhibitor concentration and corresponds to maximal saturation of the enzyme by the inhibitor (step 3). K,, the apparent inhibition constant, is the inhibitor concentration required for half-maximal saturation (Zm,/2). This parameter corresponds to the dissociation constant of the inhibitor from the enzyme. The experimental data used in the Hill's plot were within the range 0.1-1OKi. The biosensor was stored dry at 4 "C between experiments. RESULTS AND DISCUSSION
Redox Behavior of Cytochrome Oxidase in the Lipid Carbon Paste Matrix. Direct electron transfer of small-sized redox proteins (e.g., cytochrome c, ferrodoxins, plastocyanine) has been rep0rted?~
