Anal. Chem. 1993, 65, 300-305
300
Toxin Detection Using a Tyrosinase-Coupled Oxygen Electrode Mark H,Smitt and Garry A. Rechnitz' Hawaii Biosensor Laboratory, Department of Chemistry, University of Hawaii, Honolulu, Hawaii 96822
therefore not limited by the concentration of the analyte, An enzyme-baud "eMrochemlcai canary" Is described for and low-level analysis is possible. Additionally, as resulting the detection of cyanide. Tho sensing system imitates sensors are based on the interaction of the analyte with the cyanide's site of toxicity In the mitochondria. The terminal m q u e n c o o f . k c b o n t r c u l d . r I n a ~ r ~ ~ k m l m l d < e d enzyme, the greater the inherent potency or toxicity of the modulator,the better the detection capabilitiesof the system. by mediator coupling of tyrosinase catalysis to an electroDue to its toxicity as a respiratory inhibitor, much research chemical system. An enzyme-coupied oxygen electrode k has been devoted to the analysis of ~ y a n i d e .These ~ methods created whlch Is senrltlve to selective pokonlng. Biocatalytlc of analysisare typicallybased on cyanide's chemicalproperties redudon of oxygen k profnotedby e a-Hy supplying rather than ita toxic properties. A biochemical understanding tyrosinase with electrons. Thus, ferrocyanide Is generatedat of the mechanism of action of cyanide at a molecular level a cathode and mediates the enzymatic reductlon of oxygen suggests an alternative method for its detection. to water. An enzymedependent reductive current can be Cyanide exerts ita noxious effect by binding to a specific monitored which le inhlbited by cyanlde in a concentratlonprotein in the mitochondria,cytochrome oxidase, and blocking dependent manner. Oxygen depletion in the reaction layer terminal electron transport to oxygen.&Cytochrome oxidase can bo minhnized by addreulng enzyme activity udng a and the catalytic mechanism for oxygen activation and potentialpukhrg routine. Enzymeacthrity is electrochemically reduction have undergone considerable investigation.6 TerInitiated and terminated and the sensor becomes capable of minal electron transfer to oxygen occurs at a bimetallic hemecontinuour monitoring. Cyanide pokonlng of the biologkai copper reaction center in this enzyme. This transfer of component is reverdbie, and it can be reused after rlndng. electrons culminates a series of electron-transfer reactions The resulting sensor detects cyanide based on its bloiogicai which are harnessed for energy. A schematic representation actlvlty rather than its physical or chemical properties. of the process disrupted by cyanide is shown in Figure la.
INTRODUCTION Like many drugs, toxins can exert their deleterious effects by specifically interacting with biological systems. The mechanisms of such injurious interactions are becoming understood as a result of fundamental discoveries in biochemistry. With this understanding has come the possibility of devisingselectivesensingsystemsthat discriminatepoisons through their toxic expression. A biosensor which duplicates the specificbiochemical system being poisoned would provide a certain means for the relevant detection of the respective toxin. The concept of using biological systems to detect poisons is not new and is epitomized by the use of canaries in coal mines. In this present report, we describe a system that mimics the canary, providing an electrochemical means for detecting respiratory toxins, of which we use cyanide as a prototype. The proposed sensor is an inhibition-basedsystem which detecta and determines cyanide on the basis of its biological action, not its physical or chemical properties. Recent biosensor research has attempted to exploit the specific functional properties of biological components by integrating them into sensing devices. Enzymes have been incorporated successfully into electrochemical biosensors because of their specific recognition and rapid turnover of Substrates.' Enzyme electrodes have also been proposed as sensors for the detection of biocatalytic inhibitors2 and activators.3 Such systems, which are based on biocatalytic modulation, incorporate the inherent amplificationimparted by the enzyme. The magnitude of the sensing signal is 'Present address: Hawaii Biotechnology Group, Inc., 99-193 Aiea Heights Drive, Aiea, Hawaii 96701. (1) Scheller, F.;Schubert, F. Biosensors. Techniques and Instrumentation in Analytical Chemistry; Elsevier: Amsterdam, 1992; Vol. 11. (2) Tran-Minh, C.Ion-Sel. Electrode Reu. 1985, 7 , 41-75. (3) Smit, M.H.; Rechnitz, G. A. Anal. Chem. 1990, 64, 2545-2549. 0003-2700/93/0365-03S0$04.00/0
Electrons are shuttled to cytochromeoxidase from an electron source D(red) (or cytochrome reductme) by a redox protein, cytochrome c. Once cytochrome oxidase receives electrons, oxygen is reduced to water. Although the precise mechanism of action of cyanide toxicity is still under investigation, it is generally agreed that it acta by binding to the metal centers of cytochromeoxidase? thus causingthe cessationof metabolic respiration. Ideally, a biosensor for toxin measurement should incorporate the specific biological component that the toxin acta on to exert its lethal effect. A respiratory toxin sensor using cytochrome oxidase has been described on the basis of such a principle, and cyanide's propensity to obstruct this transfer of electronshas been exploited as a bioelectrochemicalmeans for its sensing.8 However, the complex nature of cytochrome oxidase, its lipid requirements, and lack of stability, has made it difficult to incorporate this enzyme into a sensing device, and alternative enzymes have been suggested.9 In this report we employ current developments in mediated bioelectrocatalysis'o to construct an alternative bioelectrochemical sensor and mimic the final steps of mitochondrial respiration using a water-soluble enzyme, tyrosinase (EC 1.14.18.1, a polyphenol oxidase). One function of tyrosinase is to catalyze the four-electronreduction of molecular oxygen to water at the expense of substrates such as catechols. In these studies,we replace natural substrates with redox shuttles which carry electrons from a cathode to the active site of the enzyme. Such electrochemical promotion results in the bioelectrocatalytic reduction of oxygen in a manner which (4) Singh, H. B.; Wasi, N.; Mehra, M. C. Int. J. Enuiron. Anal. Chem. 1986,26, 115-136. (5) Nicholls, P. Trends Biochem. Sei. 1983,8, 353. (6) Babcock, G. T.; Wikstrdm, M. Nature 1992,356, 301-309. (7) Yoshikawa, S.;Caughey, W. S. J.Biol. Chem. 1990,265,7945-7958. (8)Albery, W. J.; Cass, A. E. G.; Hubbard, J. A. M.; Shu, 2.Biochem. SOC.Trans. 1986, 14, 1212-1213. (9) Smit, M.H.; Cass, A. E. G. Anal. Chem. 1990,62, 2429-2436. (10) Bartlett, P.N.;Tebbutt, P.; Whitaker, R. G. B o g . React. Kinet. 1991, 16, 55-155. 0 1993 Amerlcan Chemical Soclety
ANALYTICAL CHEMISTRY, VOL. 65, NO. 4, FEBRUARY 15, 1993
I CN
I
T
Comparlson of the terminal sequence of electron transport in aerobic respkatlon wtth the proposed sensor. (a) Mitochondrial electron transport: D correspondsto an electron donor (In this case cytochrome reductase): cytochrome c is an electron mediator; cytochrome oxldase accepts electrons and activates and reduces oxygen to water. (b) The electrons are donated from an electrodeto an electron mediator (M) such as ferricyanide; tyroslnase accepts electrons and reduces oxygen to water. The oxidetsre enzymes in both systems are sensitive to cyantde inhibltlqlr. Flgurr 1.
can be electrochemically monitored. A schematic representation of such a system is shown in Figure lb. In this scheme the electron mediator (M(ox))is reduced at the cathode and reoxidized by tyrosinase at the expense of oxygen. The rate at which M(ox) is catalytically regenerated is a function of enzyme viability. Thus, the resulting catalytic cathodic current at this enzyme-coupled oxygen electrode is sensitive to cyanide inhibition in a concentration-dependent manner.
EXPERIMENTAL SECTION Material and Methods. Tyrosinase(EC 1.14.18.1,2400unita/ mg) and glutaraldehyde (30%) were purchased from Sigma Chemical Co. and used without further purification. Ferricyanide and sodium cyanide were purchased from Aldrich. All water was distilled and deionized to 18Mi2 cm-l prior to use. Analytical grade sodium phosphate was used for the preparation of electrolyte, which was0.05 M phosphate buffer, pH 7.0. A threeelectrode setup was used in all experiments consisting of a glassy carbonworking electrode (3-mmdiameter, Bioanalytical Systems) a platinum wire counter electrode, and an Ag/AgCl reference electrode (against which all potentials are quoted). Cyclic voltammetric experiments for measuring the effect of enzymeconcentration on the bioelectrocatalysis of oxygen were performed in an 0.5-mL cell. Cyanide inhibition studies using both slow scan rate cyclic voltammetry and pulsed potential methods were conducted in a 10-mL cell. Electrochemical measurements were performed using a Bioanalytical Systems (BAS) CV-1B poten,tiostat or alternatively, a BAS lOOB potentiostat that was interfaced with an IBM 386 PC. For double potential pulse experiments, a reductive potential of 0 V was applied to the electrode followed by an oxidative pulse of the same width to 0.4 V. The current versus time data points were imported into the data analysis package "Igor" (WaveMetrics, Lake Oswego, OR) and analyzed using a Macintosh computer. Enzyme electrodes were constructed as follows: The glassy carbonelectrodewaspolished with O.05-pm alumina and sonicated for 5 min. The electrode was then activated by the method of Anjo et al. in 1 M sodium hydroxide by applying a potential of 1.5 V for 5 min." The electrode was air dried, and 20 pL of a solution containing tyrosinase (20 mg/mL) was applied. The enzyme was dried under a vacuum for about 15 min. A 10-pL aliquot of glutaraldehyde (1% in water) was then added to the dried enzyme layer and was allowed to react for 30 min. The (11) Anjo,D.M.;Kahr,M.;Khodabakhsh,M. M.;Nowinski,S.; Wanger, M. Anal. Chem. 1989,61, 2603-2608.
381
resulting cross-linkedenzymewas dried under a vacuum. Finally, the enzyme electrodewas coveredwith a dialysismembrane (Type "C" from Technicon) which was secured with an "0"ring and stored overnight at 4 "C in 0.05 M phosphate buffer, pH 7.0. Bioelectrocatalytic studies were done in a phosphate-buffered solution containing the electron mediator, ferricyanide. Concentrated ferricyanide solutions were made daily, on a weight basis, and added to the electrolyte buffer prior to immersion of the electrode. For inhibition studies, cyanide was added to the electrochemical cell with a Hamilton syringe so that the added volume did not exceed 1% of the total cell volume. The solution was then stirred for 10s, and a reading was taken following1min of equilibration. The reversibility of cyanide inhibition was determined by replacing the cyanide-containing solution with fresh buffer and mediator and stirring the solution for 1 min; this procedurewas repeated three times, before an electrochemical measurement was taken. Cyanide is a dangerous poison and appropriate precautions shouldbe taken to prevent any accidentalrelease; all preparations, additions, and disposals were performed in a fume cupboard. Fresh solutions were made up in phosphate buffer, pH 7.0, and stored in vials with little head space,to prevent the loss of cyanide from solution. Cyanide was disposed of by adding an excess of laundry bleach (sodium hypochlorite).
RESULTS AND DISCUSSION Tyrosinase was chosen as a subsitute enzyme for cytochrome oxidase for several reasons. It is water soluble, relatively stable, and commercially available in a preparation of high specific activity. Equally important is ita catalytic activity and susceptibility to cyanide poisoning. Tyrosinase catalyzes the ortho hydroxylation of monophenols to catechols (cresolase activity) and the oxidation of catechols to orthoquinones (catecholase activity), at the expense of molecular oxygen.12 Like cytochrome oxidase, tyrosinase is a metal-containing redox enzyme which activatesand reduces oxygen. Tyroeinase containsa pair of cupric ions that presumably act as the active site for electron exchange.13J4 These metal sites shuttle between the Cu2+and Cu+ states. The enzyme, in ita resting state,exists mainly as met-tyrosinase, where the copper sites are internally bridged through the protein in the Cu2+state. Electron transfer from a suitable substrate effects the reduction of the Cu2+to the Cu+ (deoxytyrosinase) state. The reduced copper can then bind molecular oxygen to form oxytyrosinase. Subsequent electron transfer from an additional electron donor results in the release of two molecules of water. The reaction scheme can be written as follows: D(red) + met-tyrosinase
-
D(ox) + deoxytyrosinase (1)
+ +
0, + deoxytyrosinase
oxytyrosinase
D(red) + oxytyrosinase 4H+ D(ox) met-tyrosinase
+ 2H,O
(2)
(3)
D(red) corresponds to a two-electron donor. For the catecholase process, the overall reaction is therefore the catalytic transfer of four electrons from substrates to one molecule of oxygen. This enzyme activity is inhibited by (12) Lerch, K. Advances in Pigment Cell Research. In Progress in Clinical and Biological Research; Bagnara, J. T., Ed.; Alan R. Lisa, Inc.: New York; 1988, Vol. 256, pp 85-100. (13) Jolley, R. L.; Evans, L. H.; Makino, N.; Mason, H. S. J. Biol. Chem. 1974, 249, 335-345. (14) Himmelwright, R. S.;Eickman, N. C.; LuBien, C. D.; Lerch, K.; Winkler, M. E.; Solomon, E. I. J.Am. Chem. SOC.1980,102,7339-7344.
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 4, FEBRUARY 15, 1993
cyanide in a manner that is competitive with oxygen.l5 This is probably due to the binding of cyanide to the coppers. A wide variety of substrates has been tested in our laboratory for use as electrochemically reversible electron donors. Apart from oxidizing naturally occurring orthoquinonessuch as dopamine, caffeicacid, and chlorogenicacid, tyrosinase was found to act on other catechol-containing substrates such as 1,2-dihydroxynaphthalene-4-sulfonate. However, most of these catechol substrates were found to inactivate the enzyme during catalytic turnover. More interesting, with regard to the development of biosensors, we have found that it is possible to supply the enzyme with reducing equivalents using one-electron shuttles such as ferrocenecarboxylic acid, ferrocyanide, and osmium bis(2,2’bipyridine). These inorganic redox species, in the presence of tyrosinase, promote the mediated bioelectrocatalysis of oxygen and do not inactivate the enzyme. Presumably, they initiate the catalytic cycle by reducing the copper centers of met-tyrosinase (eq 1)and propagate the cycle by acting as a substrate for oxytyrosinase (eq 3). Whether this electron transfer occurs directly or through some integral redox cofactor has not been determined. As such redox mediators exchange electrons with a solid electrode in a reversible manner, we were able to electrochemicallymonitor enzyme activity by the scheme shown in Figure lb. Cyclic Voltammetry Studies of Bioelectrocatalysis. Cyclic voltammetry has been demonstrated to be a useful technique to study the interaction of mediators with en~ymes.~6J7 In our studies, slow scan cyclic voltammetry was employed to select a suitable mediator for the promoted bioelectrocatalysis of oxygen. Several fundamental criteria had to be fulfilled by the mediator. Electron exchange between the enzyme and mediator in a manner that was electrochemically reversible was essential. Additionally, the redox potential of the mediator had to be sufficiently positive of the electrode potential at which oxygen was directly (nonenzymatically) reduced to hydrogen peroxide at the electrode surface; in this way the amperometric consumption of oxygen would be primarily due to enzyme catalysis. The electrode potential needed for direct electrolysis of oxygen was a function of the electrode material and resulted in our use of glassy carbon, which required high overpotentials (-0.2 V) for such reduction. Ferricyanide was therefore chosen as an electron mediator for tyrosinase as it fulfilled these criteria. In addition, ferricyanide’s redox potential is not affected by pH, and it exists in the bulk solution in the oxidized state. This final feature was important as we wanted enzyme catalysis to be electrochemically initiated. The effects of enzyme catalysis on the cyclic voltammetric profile for ferricyanide are shown in Figure 2. At a bare glassy carbon electrode, a poorly defined wave for ferricyanide alone was observed. Additionof enzyme resulted in a concentrationdependent increase in reductive current at electrode potentials close to the potential of ferricyanide reduction. The magnitude of the electrocatalytic increase in signal was in part dependent on the rate at which electrons are transferred from the electrode through the enzyme to oxygen. This catalytic current could be abolished by oxygen removal (nitrogen purging). The rate of oxygen reduction through this bioelectrocatalytic process is a function of the following principal (15)Duckworth, H. W.; Coleman, J. E. J.B i d . Chem. 1970,245,16131625. (16)Hill, H.A.0.;Sanghera,G.S. In Biosensors: A Practical Approach; Cass, A. E. G. Ed.; Oxford University Press: Oxford, U. K., 1990;Chapter 2. (17)Davis, G.In Biosensors: Fundamental and Applications;Turner, A. P . F., Karube, I., Wilson, G. S., Eds.; Oxford University Press: Oxford, U. K., 1987;pp 247-256.
OI
// /
-.i = I
10 Tvrosinasel
d 2?
//
\
/
/ I
14 mg/ml Tyrosinasel
1
I
I
I
200 250 300 3! Potential (mV) Flguro 2. CatalyHc currents as a function of enzyme concentration. Cyclic voltammetry was conducted on 100 I.IM ferrlcyankle using an unactivatedglassy carbon electrode, In 0.05 M phosphate buffer, pH 7.0. The sweep rate was 2 m V I Sstarting at 350 mV. A l-mg sample of enzyme was added repeatedly to a 0.5-mL cell In a cumulatlve manner between each sweep. 100
150
determinants: (1)the response of the mediator to an applied potential (the rate of substrate generation), (2) the bulk concentration of mediator, (3) the catalytic activity of the enzyme preparation, and (4)the concentration of oxygen. The increase in response with increasing enzyme concentrations, as depicted in Figure 2, demonstrated that conditions exist where the reductive current is determined by enzyme Concentration. It was under such enzyme-limitedconditions that we employed this enzyme mediator system as a poison detector. The enzyme was immobilized at the electrode surface so that reversible poisoning of the enzyme by cyanide could be studied. The catalytic reductive catalytic currents resulting during a potential scan of the enzyme-modified electrode in the presence of ferricyanide were attenuated by cyanide. The magnitude of inhibition of catalytic current was dependent on the concentration of cyanide added, allowing for cyanide determination following the formation of a calibration curve. Subsequent removal of cyanide by replacing the mediator solution resulted in a reinstatement of catalytic current. While cyclicvoltammetry waa a useful techniquefor studyingenzyme activity, data acquisition was typically slow, which resulted in our study of alternate electrochemical methods for measuring tyrosinase activity. Pulsed Potential Methods for Determining Enzyme Activity. Chronoamperometry has been shown to be of use in analyzing enzyme catalysis.18 The tyrosinase enzyme electrode was therefore analyzed using pulsed potential methods. As the mediator (ferricyanide) existed in bulk solution in the oxidized form, its reduction provided electrons to the enzyme which initiated and perpetuated the catalytic reduction of oxygen. This catalysis could be terminated by applying an oxidizing potential to the electrode, thereby removing substrate. Figure 3 shows a schematic diagram of this electrochemicalswitchingprocedure. Thus, the catalytic activity of the enzyme could be switched on and off electrochemically, through substrate promotion of bioelectrocatal(18)Cass, A.E.G.; Davis, G.; Hill, H. A. 0.; Nancarrow, D. J. Biochim. Biophys. Acta 1985,828, 51-57.
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 4, FEBRUARY 15, 1993
-
4
383
10
ON
OFF
Fe(CN):(ox)
Fe(CN)e.?d)
8
Fe(CN):(ox)
= L
h
'
z6 s X
Flgue 3. Schematic of electrochemicalswitching of enzyme catalysis. Biocatalysie is initiated by applying a potential of 0 V and terminated by applying a potential of +400 mV.
1.2 1.6 Time (sec)
c)
C
2.0
: 4
0'
2 5 0 0 ~ MNaCN
0 0.6 0.8 1 .o Time (sec) Flgure 5. Effect of cyanide on the current decay during a reductive pulse at a tyrosinase enzyme electrode: (a) decay during first second; (b) decay during second second of the same pulse. Solution and instrumental condltlons were as in Figure 4. Cyanide was added to a 10-mL cell, and the solution was stirred for 10 8. 0.0
0
I
I
I
I
I
I
I
50
100
150
200
250
300
350
Time (ms) Figure4. Profileof current decay duringa reductive pulse as a function of enzyme concentration. Cumulative addition of enzyme resulted in higher current densltles. Intermediateunlabeled decay curves corra spond to 2,4, and 6 mg/mL tyrosinase. The pulse was to 0 V (from 400 mV) in 100 HM ferricyanide, 0.05 M phosphate buffer, pH 7.0.
ysis. Additionally, the rate of enzyme catalysis could be adjusted by choosing appropriate intermediary reductive potentials. We have shown previously that the catalytic amplification of the sensing signal of such modulation-based sensors can be controlled through the proper selection of intermediate potentiala.3 The current resulting at a tyrosinase-modified electrode was monitored by following a reductive pulse in the presence of ferricyanide. Figure 4 shows the effect of enzyme concentration on transient currents during a reductive pulse. The difference between the noncatalytic current and the catalytic current represented oxygen reduction. Following 3-4 s, the catalytically enhanced faradaic current would eventually reach what appeared to be a constant magnitude. We attribute this apparent leveling of current to ratedetermining enzyme kinetics. The faradaic current was a function of the concentration of mediator, the concentration of oxygen, and the concentration of enzyme. As expected, reductive current density increased with increasing enzyme concentrations. This increase in cathodic current was what would be expected from an EC' reaction, or the catalytic regeneration of electroactive species,lg where C' is determined by the enzyme turnover rate. The inhibition of enzyme activity was analyzed by monitoring the current decay. Figure 5 shows typical current
0.2
0.4
profiles of ferricyanide reduction at a tyrosinase-modified electrode as a result of a potential pulse to 0 V, in the presence and absence of cyanide. Calibration curves could be constructed by measuring the current as a function of cyanide concentration. We found, however, that more reproducible results could be achieved by analyzing the integrated current, as we discuss below. Chronocoulometric Analysis of Enzyme Activity. When a reductive potential was applied at a tyrosinase electrode immersed in a buffered solution of ferricyanide, the total charge passed at the electrode could be related to the activity of the enzyme. The longer the period of current integration, the larger the absolute difference between the charge due to catalytic effects and the charge resulting from simple diffusion. Since the chronoamperometric response consisted of nonfaradaic as well as faradaic current, the viability of the enzyme was easier to monitor if the initial portion of the chronoamperometricdecay was ignored. Much of the early charge accumulation, which corresponded to a considerable fraction of the total current, was a result of double layer charging. This was especially true if protein was immobilized on the electrode surface. Figure6a shows a chronocoulometricresponse to a reductive pulse, at a tyrosinase electrode, in the presence of several concentrations of cyanide. The higher the concentration of cyanide, the lower the reductive charge passed at the electrode. The data represents the last 5 s of a 10-s pulse. Apparent steady-state currents which are possibly dictated by enzyme kinetics result at the enzyme electrode during this period, as are shown by the linearity of the charge accumulation. A calibration curve is shown in Figure 6b which is normalized by expressingthe response to cyanideas a percent inhibition of total charge. With this method of standardization, reproducible calibration curves between electrodes (19)Bard, A. J.; Faulkner, L. R.Electrochemical Methods: Fundamentals and Applications; John Wiley & Sons, Inc.: New York, 1980; p 456.
384
r
ANALYTICAL CHEMISTRY, VOL. 65, NO. 4, FEBRUARY 15, 1993
60
50
4
c
0 40.c .e
c c
z
30-
2010-
+ 0 -+
0
1
2 3 4 Time (sec)
5
could be achieved, since the fractional inhibition of enzyme is a function of the inherent affinity between cyanide and enzyme. We found that the sensitivity of the system to cyanide inhibition increased as the pulse time was lengthened. This was probably because cyanide is competitive with oxygen and oxygen is depleted as the pulse progresses. Therefore, while optimal sensitivity would entail the use of long potential pulsing routines, optimal sensor performance during continuous monitoringis also dictated by the finite concentration of oxygen. Optimization of Pulsing Scheme for Addressing Enzyme Activity. We are presently investigating methods of developing a sensor capable of continuous monitoring.This has involved optimizing both the concentrations of components, as well as the chronocoulometricmethod of analysis. The catalytic process being monitored is a modified pingpong reaction. Enzyme turnover is determined by the concentrations of both oxygen and mediator and their respective Km’s (see reaction eq 1-3). Since the oxygen concentration is dictated by ambient conditions, our initial efforts were to make the monitored enzyme catalysis strongly dependent on the interaction of mediator with enzyme. As the effectiveelectron-acceptingconcentrationof oxygen under air-saturated conditions is about 1 mM, we chose low concentrations of ferricyanide (100 rM) so that its (rather than oxygen’s) interaction with the enzyme was the primary determinant of biocatalysis. Apparent steady-state currents following an initial decay suggest this to have been achieved. It should be stressed however that this form of optimization is at the expense of decreased sensor sensitivity to cyanide, since cyanide is competitive with oxygen. The inevitable depletion of oxygen during prolonged reductive pulsing was also addressed. Studies were performed to investigate whether a pulsing routine could be adopted which would minimize problems associated with the electrolysis of oxygen. We found that shortening the pulse time
and allowing an interval between double potential pulses provided a suitable method for reproducibly addressing enzyme activity. Thus, a 2-5 pulse to 0 V, followed by a 2-5 pulse to +0.4 V, followed by an open circuit lag time of 1 min. resulted in reproducible catalytic currents during the reductive pulse. Charge accumulation during the second half of the reductive pulse was reproducible and showed no signs of diminishing during successive pulses. Therefore, this “bioelectrochemical breathing” provides a strategy for devising a system capableofconstant monitoring. The pulsing lengths and intervals are obviously dictated by the sensor configuration and would therefore depend on further sensor development. Reversibility of Cyanide Inhibition. The canary was a good example of a single-use sensor. Once poisoned it would have to be replaced. An electrochemicalcanary provides the possibility of creating a sensor that would not only give a varied response to different concentrations of cyanide but would also be reusable, since the binding of cyanide to tyrosinase is reversible. We investigated the recovery of the sensor following cyanide inhibition by exposing the enzyme to an electrolytic solution containingcyanide and ferricyanide and measuring the charge accumulationduringthe last second of a 2-5 pulse, as in Figure 5b. The electrode was rinsed by immersing it in a stirred, cyanide-free solution for 1 min. Following three transfers to fresh electrolyte and a final recovery period of 10 min, the enzyme activity was retested. We found that the recoverability of the sensor was a function of the concentration of cyanide and the exposure time. For example, challenging the electrode to 1 mM cyanide for 2 min would result in an initial inhibition of about 46% of the charge measured and a recovery of 90% of the charge in each of five cycles. Lower concentrations of cyanide would result in higher enzyme recoveries. Prolonged exposure (>lo min) of the enzyme to millimolar concentrations of cyanide would result in extremely poor recovery of the enzyme electrode (40-50 5% per cycle). We attribute this irreversible inactivation to the solubilization of the active site coppers, by cyanide.20
SUMMARY By imitating metabolic respiration, we have exploited cyanide’s toxic expression to create a biosensor. Tyrosinase replaces the in vivo target for cyanide poisoning, cytochrome oxidase, and is incorporated into an electrochemical environment. Using redox shuttles, electron transfer through tyrosinase to oxygen can be monitored. The introduction of cyanide to this system effectively shuts off the consumption of oxygen in a dose-dependent manner. Such inhibition is reversible, as cyanide dissociates from the enzyme’s catalytic site. As the sensor only consumes oxygen, it does not require the continual addition of reagents to maintain catalytic activity. Additionally, the enzyme can be t ~ n e on d and off by controlling the applied electrode potential. These latter features allow for the possibility of constant monitoringusing a potential pulsing strategy. These studies have demonstrated the feasibility of developing such a sensor but have required the use of a freely diffusing mediator. On-going studies are focused on the immobilization of mediators to polymers and other macromolecules in order to provide a true reagentless setting for measuring enzyme activity and ita poisoning. An earlier publication described an amperometric peroxidase-based sensor based on a similar concept.3 That sensor was developed around a two-electrode system which was sensitive to cyanide in submicromolar concentrations, and (20) Beltraminin,M.; Salvato,B.;Santamaria, M.; Lerch, K.Biochirn. Biophys. Acta 1990, 1040, 365-372.
ANALYTICAL CHEMISTRY, VOL. 85, NO. 4, FEBRUARY 15, 1993
50% of the bioelectrocatalyticactivity was inhibited by 2 pM cyanide. Our present tyrosinase system is less sensitive, as shown by Figure 6, and represents a compromisein an attempt to make a single-electrode ("probelike") sensor capable of continuous monitoring. Competition of oxygen with cyanide represented one factor limiting sensitivity. In addition, minimizing the dependence of the rate of bioelectrocatalysis on oxygen concentration affected our sensitivity. We found that increasing the mediator concentration to 5 mM (making the catalytic current more determined by oxygen concentration) resulted in an increase in sensitivity to cyanide but also resulted in rapid oxygen depletion. We are currently investigating data acquisition methods which can improve on our sensitivity. The specificity of the sensor depends on the susceptibility of the active site to toxin binding. Inhibition-based sensor sensitivity should increase with the toxicity of the poison. It is realized that tyrosinase is susceptible to a variety of inhibitors. For example, we have found that this system
985
responds very well to sodium azide. While further development of selectivity could involve the incorporation of selective membranes, we believe the true w e of this sensor to be as a warning device to the accidental (or purposeful) release of toxins. The poisons that would be sensed would therefore depend on the application or location of the sensor. It is hoped that sensors resulting from these and on-going studies wil target a broad spectrum of toxic enzyme inhibitors, making them applicablein a number of settingewhich require continuous monitoring.
ACKNOWLEDGMENT We gratefully acknowledge support from NIH Grant GM25308.
RECEIVEDfor review August November 23, 1992.
24,
1992.
Accepted
