Anal. Chem. 1994,66, 3158-3163
Chemically Amplified Voltammetric Enzyme Electrodes for Oxidizable Pharmaceuticals Thea J. Moore, G. Gina Nam,t Leonard C. Pipes,* and Louis A. Coury, Jr.' Department of Chemistry, Box 90346, Duke University, Durham, North Carolina 27708-0346
An electrocatalyticreaction occurringbetween several reduced flavin enzymes (glucose, lactate, and sarcosine oxidases) and the oxidized form of the analgesic acetaminophen is reported for the first time. Similar processes are characterized for reactions of glucose oxidase with norepinephrine and chlorpromazine. The utility of these reactions is demonstratedboth in solutionand for an enzyme immobilized on electrodesurfaces. Sensitivities and limits of detection for determinations of acetaminophenand norepinephrine at electrodesmodified with polymer-bound glucose oxidase are each improved by an order of magnitude over determinationsperformed at bare electrodes. Detection limits for acetaminophen and norepinephrine at modified electrodes of 100 and 300 nM, respectively, are demonstrated. Widespread availability of acetaminophen in over-thecounter analgesic preparations has led to an increase in accidental or suicidal ingestion of toxic amounts of t h e d r ~ g . ' - ~ On the basis of reports of the Rocky Mountain Poison Control Center at least one to two cases of acetaminophen poisoning occur daily in the United States, although this may represent an underestimation since it is believed that many cases go unreported.' Overdose is only achieved after 30-50 times the therapeutic dose (0.5 mg) has been ingested, and hence acetaminophen poisoning in adults and teens is frequently deliberate.6 Accidental poisoning occurs most often in children and is usually a result of chronic exposure to the drug rather than acute poisoning from a single doseas The most reliable method for diagnosing acetaminophen poisoning is to monitor changes in its concentration in blood serum over time. The amount of antidote used to treat the overdose is then based on the serum concentration of acetaminophen found, and treatment is most effective when initiated within 10 h after ingestion of the d r ~ g . ~Acute ,~ nausea and central nervous system toxicity are the common adverse side effects of the antidotes normally employed;9 thus 'Present address: Glaxo, Inc., 2.4124 Analytical Chemistry, 5 Moore Drive, Research Triangle Park, NC 27705. t Present address: Department of Chemistry, The University of California at Los Angeles, 405 Hilgard Avenue, Los Angeles, CA 90024-1569. (1) Arena, J. M.; Drew, R. H. Poisoning: Toxicology, Symptoms, Treatments; Thomas: Springfield, IL, 1986. (2) Bramwell, H.; Cass, A. E. G. Analyst 1990, 115, 185. (3) Price, C. P.; Hammond, P. M.; Scawen, M. D. Clin. Chem. 1983, 29, 358. (4) Munson, J. W.; Abidine, H. J. Pharm. Sci. 1978, 67, 1775. ( 5 ) Vaughan, P. A.; Scott, L. D. L.; McAleer, J. F. Anal. Chim. Acta 1991,248, 361. (6) Cotran, R. S.;Kumar, V.; Robbins, S . L.; Robbins' Pathological Basis of Disease; Sanders: Philadelphia, 1989. (7) Hammond, P. M.; Scawen, M. D.; Atkinson, T.;Campbell, R. S.; Price, C. P. Anal. Biochem. 1984, 143, 152. (8) Penna, A.; Buchanan, N. Brit. J. Clin. Pharmacol. 1991, 32, 143. (9) Dreisbrach, R. H.; Robertson, W. 0. Handbook of Poisoning: Prevention, Diagnosis and Treatment; Appleton and Lange: Norwalk, CT, 1987.
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rapid and accurate methods for diagnosing acetaminophen poisoning are required. Spectrophotometric, colorimetric, and electrochemical methods have each previously been developed for this p u r p ~ s e . ~ . ' ~ In this paper, we present the first report of a previously unknown reaction between the flavin enzymes glucose, lactate, and sarcosine oxidases and electrooxidized acetaminophen. We demonstrate that this reaction can be harnessed to engender a steady-state catalytic redox cycle, useful for chemical amplification of the oxidation current for acetaminophen, that results in both improved sensitivity and detection limit. This reactivity is exploited for the construction of a chemically amplified enzyme electrode, through immobilization of glucose oxidase in a polymer film. The advantage of this approach over all others previously reported for acetaminophen is that temporal discrimination against other faradaic (and nonfaradaic) signals is possible, since the charge monitored from the catalytic process uniquely displays a firstorder dependence on time. The reaction strategy is also extended to two other species of clinical interest: the vasoconstrictor hormone norepinephrine and the antipsychotic drug chlorpromazine.
EXPERIMENTAL SECTION Reagents. Acetamidophenol (acetaminophen), 4-aminophenol, sarcosine oxidase (SOD, from Bacillus), lactate oxidase (LOD, from Pediococcus), glucose oxidase (GOD; types X, VII, and VII-S, from Aspergillus niger), sarcosine, lactic acid, and /?-D-(+)glucose (97%anomerically pure) were purchased from Sigma Chemical Co. Chlorpromazine, norepinephrine, and hydroquinone were obtained from Aldrich Chemical Co. Analyte, enzyme, and substrate solutions were prepared in 0.5 M KC1/0.05 M phosphate buffer, pH = 7.00 (Fisher). Poly(viny1 alcohol) (hydrolyzed from poly(viny1 acetate); MW 124 000-1 86 000) andglutaricdialdehyde (50% (w/w) aqueous solution) were purchased from Aldrich. All compounds were used as received. Instrumentation. Cyclic voltammetric and chronocoulometric data were acquired with either a Cypress Systems CS1087 or a Bioanalytical Systems BAS- lOOB electrochemical analyzer. Both potentiostats were interfaced to laboratory 80386-based computers (Zeos) for data storageand processing. An HP-8452A photodiode array spectrophotometer was used to determine the concentration of active enzyme in prepared solutions. Electrodes and Cell Assembly. Small-volume (600 ML) electrochemical cells with openings for an external reference (10) Shearer, C. M.; Christenson, K.; Mukherji, A.; Papariello, G. J. J . Pharm. Sci. 1972.61, 1627.
0003-2700/94/03663158$04.50/ 0
0 1994 Amerlcan Chemical Society
electrode and gas inlet were used for the majority of experiments, while a conventional 10 mL, three-electrode cell was used for some of the modified electrode studies. Working electrodes were constructed from edge-plane pyrolytic graphite (Union Carbide), encased in epoxy either alone, or with a Pd ring auxiliary electrode in a coplanar arrangement." The reference electrode for all experiments was Ag/AgCl. The electrodes were polished with alumina (1-0.05 pm diameters) on Buehler Microcloth and then sonicated in H2O for 60 s in a Branson 1200 sonicating bath to remove alumina from electrode surfaces. Water used for all studies was purified by reverse osmosis (Barnstead ROpure-LP) and then ion exchange (Barnstead NANOpure). Preparation of Enzyme Solutions. Solutions of the flavin enzymes GOD, SOD, and LOD were prepared anaerobically by dilution of commercial preparations in septa-capped vials under Ar. The molar concentration of active enzyme in each preparation was determined from the difference in absorbance at 452 nm before and after in situ reduction of enzyme with substrate. For these calculations, the literature value for the differential molar absorptivity (Aa) at X = 452 nm for the flavin center common to all three enzymes of 1.3 X lo4 M-l cm-l was used.12 Preparation of Modified Electrodes. Poly(viny1 alcohol), was dissolved in deionized H20 to make a 6% (w/w) solution by heating at 120 OC in a convection oven until dissolved. Any water lost upon heating was replaced after the solution had cooled. The GOD/polymer deposition solutions were prepared from aqueous stock solutions to give final concentrations of 4% (w/w) polymer, 1.7% (w/w) glutaric dialdehyde, 0.017 N HCl, and 5% (w/v) GOD. Glutaric dialdehyde served as the cross-linking agent for the polymer and enzyme, and the acid functioned as a catalyst. Pyrolytic graphite working electrodes were coated with 10 pL of the deposition solution and allowed to dry at 25 "C overnight. Enzyme-modified electrodes were soaked in buffer for a minimum of 3 h before use to remove un-cross-linked materials. A solution of inactive GOD was prepared by thermal denaturation of the enzyme at 90 OC for 10 min and was added to deposition solutions in the place of active GOD for electrodes used in control experiments.
RESULTS AND DISCUSSION Solution Kinetic Studies. Catalytic currents are observed for the electrooxidation of acetaminophen in the presence of substrate-reduced glucose oxidase (GOD), lactate oxidase (LOD), or sarcosine oxidase (SOD). Although acetaminophen has been examined as a potential interferant for glucose electrodes,13 its catalytic behavior with flavin enzymes has apparently not been studied previously. The proposed mechanism for the electrocatalytic process is shown schematically in Figure 1 for the example of glucose oxidase as the reactive enzyme. Acetaminophen is known to undergo a two-electron, twoproton heterogeneous oxidation to form N-acetyl-p-quinon(1 1) Coury, L. A.; Oliver, B. N.; Egekeze, J. 0.;Sosnoff,C. S.;Brumfield, J. C.; Buck, R. P.;Murray, R. W. Anal. Chem. 1990, 62, 452. (12) Duke, F. R.; Weibel, M.; Page, D. S.; Bulgrin, V. G.; Luthy, J. J.Am. Chem. SOC.1969, 91, 3904. (13) Maidan, R.; Heller, A. Anal. Chem. 1992, 64, 2889.
/I cooH ~ O H HOCH HCOH
OH
Figure 1. Reaction scheme for an acetaminophen-mediated electrocatalytic process: (a) acetaminophen, (b) N-acetyl-pquinoneimine, (c) benzoquinone,(d) reduced flavin center of GOD, (e) oxidized flavin center of GOD, (f) Lbgiucose, (g) Sgluconolactone,and (h)bgluconate.
eimine.14 The quinoneimine is known to be fairly unstable, and it hydrolyzes to produce benzoquinone, the rate of hydrolysis being pH dependent.15 As will be shown, however, when a reduced flavin oxidase is present in solution with its primary substrate, the acetyl-quinoneimine accepts electrons from the reduced flavin center of the enzymeat a substantially greater rate than it hydrolyzes. Acetaminophen is thus regenerated by this reaction and can be subsequently reoxidized in a second heterogeneous electron transfer. When a saturating excess of primary substrate maintains the initial concentration of the reduced oxidase, a kinetically pseudo-first-order redox cycle is established between the enzyme, mediator, and electrode." The flow of electrons in the redox cycle is then mediated by the reduced and oxidized forms of acetaminophen, consequently current for the oxidation of the mediator is amplified in the chemical domain by repeat sensing at the electrode surface. Such a strategy has the advantage of increasing the signal without coamplification of the background response. Theory. Delahay and Stiehl16 first derived the mathematical expression describing the current for an electrode process that is coupled to a regenerative (catalytic) solution reaction. This equation is valid for catalytic currents observed over both the early transient and subsequent steady-state time domains and is given by
where kcat(in our case) is the bimolecular rate constant (cm3/ mol s) for the enzyme-mediator reaction, Dmdis the diffusion coefficient for the mediator (cm2/s), C*,,,cd and C*en, are the bulk concentrations of mediator and enzyme (mol/cm3), respectively, t is time (seconds), and the other parameters (14) Van Benschoten, J. J.; Lewis, J. Y.;Heineman, W . R. J. Chem. Educ. 1983, 60, 112. (15) Miner, D. J.; Rice, J. R.; Riggin, R. M.; Kissinger, P. T. Anal. Chem. 1981, 53,2258. (16) Delahay, P.; Stiehl, G. L. J . Am. Chem. SOC.1952, 7 4 , 3500.
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~~~~
~
~
Table 1. Comparlson of the Redox Potentlals, Half-Wave Potentlals, Blmoleoular Rate Constants, and Catalytlc Efflclencks for the Reactlon of GOD wHh Acetamlnophen and Its Llkely Hydrolyds Products mediator Ep,a' Ep,cb Eo' El/f kcaJ(M-' S-I)
acetaminophend 4-aminophenole hydroquinoneC
-400 94 127
53 0
74 64
409 74 175
1 . 1 x 105 L O X 105
1.5 x 105
i
E/(V vs Ag/AgCI)
Oxidation peak potential in millivolts for the mediator in the absence of GOD. Reduction peak potential in millivolts for the mediator in the absence of GOD. Half-wave potential of the catalytic voltammogram inmillivolts. [mediator] = 1.21 mM; [GOD]= 3.52pM;electrodearea = 0.096 cm2. [mediator] = 0.88 mM; [GOD]= 9.4 pM; electrode area = 0.096 cm2.
L
have their customary meanings.17 For the specific case where t = 100 s and kcat= lo8cm3/mol s, the error function term erf(kcatC*enzt)1/2exceeds 0.9999 and the exponential quotient s-Il2; thus, the current expression (now approaches describing the purely steady-state component of the response) may be simplified to
E/(V vs Ag/AgCI)
is, = nFAC*mcd(kcatDmedC*cnr)1/2 (2)
E/(V vs AgIAgCI)
In experimental terms, a voltammetric scan rate may thus be selected in a catalytic situation that is sufficiently slow to yield the limiting values of the erfand exp terms in eq 1, allowing for the straightforward extraction of kat from voltammograms. Equation 1 was originally derived for the pseudo-first-order kinetic case; thus its application in the present context is contingent upon the enzyme being a kinetically inexhaustible reactant. This is easily accomplished by saturating the enzyme with its primary substrate (e.g., P-D-glucose for GOD) to shift rate control to the enzymemediator reaction.11J8It should also be noted that thediffusion coefficient for the enzyme does not enter into the derivation of these equations, further facilitating the kinetic analysis. Comparison of Acetaminophen Kinetics with Hydrolysis Products. To properly interpret results, it is essential to verify the identity of the active mediator in the catalytic sequence. As was previously mentioned in the discussion of Figure 1, benzoquinone is a hydrolysis product of electrogenerated a~etylquinoneimine.~~ A somewhat less likely possibility is the hydrolysis of acetaminophen to p-aminophenol prior to electrooxidation. We have verified that the hydroquinone/ benzoquinone and p-aminophenol/quinoneiminecouples are capable of mediating electrocatalytic reactions with substratereduced GOD to yield steady-state voltammograms. The rate constants for the reactions of each of these couples with GOD are comparable to the rate constant for the reaction of acetaminophen/acetylquinoneiminewith GOD and are listed in Table 1. If benzoquinone and/or quinoneimine were present in solution with acetylquinoneimine, they could compete with the latter as electron acceptors for the reduced enzyme. For a mediator couple that undergoes a rapid heterogeneous oxidation, the half-wave potential ( E l p ) of the catalytic voltammogram will be approximately equal to Eo' for the L. R. Electrochemical Merhods: Fundamentals and Applications; Wiley: New York, 1980. (18) Coury, L. A.; Murray, R. W.; Johnson, J. L.; Rajagopalan, K. V. J . Phys. Chem. 1991, 95, 6034. (17) Bard, A. J.; Faulkner,
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L
Figure 2. Voltammograms for the oxidation of acetaminophen at a pyrolytic graphite electrode ( A = 0.073 cm2) in the absence (a, c, e) and presence (b, d, f) of substrate-reduced GOD (panel A), LOD (panel B), and SOD (panel C).
mediator in the absence of coupled solution kinetics. We note that the observed E112 for the acetaminophen catalytic wave in the presence of GOD is 409 mV, while the catalytic halfwave potentials for the p-aminophenol and hydroquinone reactions are 74 and 175 mV, respectively (Table 1). The fact that Ell2 for the acetaminophen reaction is shifted far positive of those for hydroquinone andp-aminophenol identifies the acetaminophen/acetylquinoneiminecouple as the most likely mediator. Furthermore, the persistence of steady-state waves upon repeat scanning suggests that the rate of hydrolysis of acetylquinoneimine is too slow to compete with its reduction by reduced GOD. Comparison with Other Flavin Enzymes. Acetaminophen mediates electrocatalytic processes for flavin enzymes other than GOD. Figure 2 presents catalytic voltammograms for substrate-reducedGOD (panel A), LOD (panel B), and SOD (panel C). In each case, catalytic currents are observed for the oxidation of acetaminophen in the presence of 3.52 ELM enzyme and an excess of substrate, and the associated kinetic data are listed in Table 2. The reaction between oxidized acetaminophen and LOD appears to be slightly more rapid than with GOD, and both are substantially faster than SOD. However, on a per-unit basis, thecommercial LOD preparation is nearly 500 times more costly than GOD, and hence GOD was used for the remainder of the studies reported below. In the case of SOD,steady-state catalysis was only achieved for relatively concentrated solutions of the enzyme (ca. 70 pM). Other Mediator-Analytes. Acetaminophen is not the only pharmaceutical compound of analytical interest capable of mediating electrocatalytic reactions with GOD. Catalytic currents are also observed for norepinephrine and chlorpromazine in the presence of substrate-reduced GOD (Figure 3).
Table 2. Klnetlc Data for the Reactlon of Oxldlzed Acetamlnophen wlth Three Flavln Oxldaws enzyme ip,a/(wV iC/(wVb k , t / W 1 s-'1
12 11 10 4.gd
glucose oxidasec lactate oxidasec sarcosine oxidasec sarcosine oxidase
1.1 x 105 1.4 x 105
32 37 11 4.6'
1.6 x 104
i, a = oxidation current for acetaminophen in the absence of enzyme; electrode area = 0.096 cm2 for all data. i, = oxidation current for acetaminophen in the presence of enzyme. [enzyme] = 3.52 pM; [acetaminophen] = 1.21mM.d [acetaminophen] = 0.375 mM. Steadystate current; [enzyme] = 70.6 pM; [acetaminophen] = 0.319 mM due to dilution from addition of enzyme.
// -40 cc 900 680
460
240
20
-200
Ie
E/(mV vs Ag/AgCI)
15 I
-25 -5
-45
I '
1000
775
550
325
100
E/(mV vs Ag/AgCI) Flgure 3. Voltammograms for the oxidation of norepinephrine (panel A) and chlorpromazlne (panel B) at a bare electrode (A = 0.096 cm2) in the absence (a, c) and presence (b, d) of substrate-reduced GOD.
For norepinephrine (Figure 3, panel A) the catalytic current is steady-state at scan rates from 20 to several hundred mV/s. At slower scan rates, electrooxidized norepinephrine undergoes an internal cyclization on the voltammetric time scale to form an indoline specieslgwhich is apparently not an efficient redox mediator for GOD. This results in loss of norepinephrine during the potential scan and gives rise to non-steady-state catalytic currents, diminished in absolute magnitude from those engendered at faster scan rates. In aqueous solutions, the phenothiazine compound chlorpromazine is electrochemically oxidized to chlorpromazine sulfoxide in two one-electron transfers. Chlorpromazine (Figure 3, panel B) undergoes the first oxidation step to form the chlorpromazine radical cation, exhibiting an anodic peak potential of 730 mV vs Ag/AgCl. The current observed at 900 mV in curve (c) of the figure is the onset of the second anodic wave, viz., the oxidation of the radical cation to the hydrolytically labile dication.*OJ A cathodic wave for the reduction of the chlorpromazine radical cation is not observed during the reverse scan for the solution conditions and time (19) Hawley, M. D.;Tatawawadi, S.V.;Piekarski,S.; Adams, R. N. J . Am. Chem. SOC.1961, 89, 447. (20) Cheng, Y . H.; Sackett, P. H.; McCreery, R. L. J . Am. Chem. SOC.1978,100,
962.
scale of these measurements, and homogeneous reactions of the radical cation are known to be catalyzed by phosphate (the buffer anion employed here).21 The catalytic response (Figure 3B,d) is large in magnitude but quite unusual in appearance and may be explained as follows: As the chlorpromazine radical cation (formed at potentials > 600 mV) accepts an electron from reduced GOD, anodic current increases as the reduced form is regenerated and then reoxidized. At potentials greater than about 750 mV, the catalytic current decreases as the radical cation dismutates or is further oxidized to the dication (which hydrolyzes to the sulfoxide).20*21Since the catalytic current diminishes at more positive potentials, we conclude that the reaction between chlorpromazine sulfoxide and reduced GOD is substantially slower than that of the radical species (as would be expected when comparing an atom transfer to a simple electron-transfer reaction). On the reverse scan of curve d, anodic current initially decreases but then starts to rise again at around 750 mV as the radical is no longer oxidized further and the catalytic reaction again begins to dominate the electrode process. Due to the complexity of the catalytic mechanism for chlorpromazine, rate constants cannot be obtained using eq 2. However, comparison of peak currents in the presence and absence of enzyme for a given set of solution conditions shows the reaction of the radical with GOD to be about twice as fast as that with SOD. This is in contrast to the order-of-magnitude difference observed for the acetaminophen reaction. Although theorigin of this effect is unclear, we note that chlorpromazine is more hydrophobic than acetaminophen; thus the reaction sites for the mediators may differ in their hydrophobicities among different flavin enzymes, leading to different trends in observed reactivity. Enzyme-Modified Electrodes. The results of the above solution experiments were next used to design enzyme-modified electrodes and to study the effects on catalytic currents when the enzyme is confined inside a polymer layer. Glucose oxidase was immobilized on pyrolytic graphite disks by coating the electrode surfaces with aqueous solutions of poly(viny1 alcohol) polymer, GOD, and a cross-linking agent. Figure 4 shows voltammograms for acetaminophen at such an electrode (a) in 0.5 M KCl/phosphate buffer and (b) in a solution also containing 0.8 M /3-D-glucose. As is shown, steady-state catalytic currents similar to those observed for freely diffusing enzyme are observed for immobilized GOD in the presence of saturating levels of substrate. A distinct advantage of a steady-state response for analytical work is that integration of the current response leads to a signal for the catalytic process that increases linearly with time: sisdt = Q = nFAC*,~(D,~C*,,,k)'/21
(3)
By contrast, background charge due to double-layer capacitance reaches a constant value quite quickly, whereas charge due to other diffusing electroactive species not involved in the (21) Mayausky, J. S.;Cheng, H. Y.; Sackett, P. H.; McCrecry, R. L. In Aduances in Chemistry Series, No. 201 Electrochemical and Spectrochemical Studies of Eiological Redox Components; Kadish, K. M., Ed.; American Chemical Society: Washington, DC, 1982.
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Tabk 3. comprrbon dAhe 8a” CHWl C8kuWod Ddrotkn Unlbforth.oxldaumdAW” md Norat B.nandModwkd Ekctrodn sensitivity/ working electrode (mC M-’cm-2) detn limit/(M)
1000
760
520
280
40
-200
Acetaminophen bare ( A = 0.086 cmz) 9.8 X polymer/GODib( A = 0.088 cmz) 4.4 X 1.2 X polymer/GOD,C ( A = 0.089 cm2) Norepinephrine bare ( A = 0.086 cm2) 1.1 x polymer/GOD,c ( A = 0.089 cm2) 3.9 X
104 104 106
1x10-6 4x104 1 x 10-7
104
2x104 3 X 1V7
lo5
Electrode areas listed in parentheses. * Deposition solution prepared with inactive GOD. Deposition solution prepared with active GOD.
E/(mV vs Ag/AgCI) Flgur. 4. Voltammograms for acetaminophen oxidation at (a) an enzyme electrode (bare area = 0.080 cm2) in 0.5 M KWphOephate buffer, and (b) a solution of 0.8 M @+glucose, 0.5 M KCl/phphate buffer. Inset: Calibration plots for the oxidation of acetaminophen in a solution of 0.8 M @oglucose,0.5 M KCi/pho@late buffer at (A) an electrode (A = 0.088 cm2) morHfled with a polymer Rm containing thermel)y denatured GOD, (6) a bare electrode ( A = 0.086 cm2), and (C) an electrode(A = 0.089 cm9 modifled with a film containingactlve W. The y axis represents (analyfe charge background charge), normalized for the electrode area measured prior to modification.
-
catalytic reaction obeys a t‘JZdependence according to the integrated Cottrell equation: Q = ~~FACL,,,,(D,,~/T)‘~~
(4)
Thus, in the limit of long time (ignoring convection effects), the signal-to-background charge ratio increases without bound for backgrounds arising from both nonfaradaic and noncatalytic faradaic processes. Natural convection does not appear to influence the catalytic reaction within polymer coated electrodes since nonlinearities (viz., negative deviations) in plots of signal vs time were never obseryed, even for long integration times (up to 300 s). Sensitivity and detection limit studies were conducted by integrating currents at plateau potentials for 300 s (inset of Figure 4). Charge observed in the absence of analyte (Qb) was subtracted from the analyte data (Q),and this difference was normalized by the area of the working electrode (determined by chronocoulometry prior to enzyme immobilization), to yield the net charge, (Q- &)/A. Calibration plots were generated for acetaminophen at (B) a bare electrode, (C) an active enzyme-modified electrode, and (A) an electrode containing only thermally denatured GOD in the polymer film. The electrode modified with inactive GOD served as a control to account for any changes in accessible electrode area due to anchoring of the polymer film. The lack of enzymatic activity of the enzyme preparation following thermal denaturation was easily confirmed spectrophotometrically since the absorbance maximum for the flavin center of denatured GOD is blue shifted to 448 nm and the absorbance intensity does not decrease at ,A, when substrate is added. There is roughly a 10-fold improvement in both the sensitivity and detection limit (viz., 100nM) for acetaminophen and norepinephrine (300 nM) at active enzyme electrodes (Table 3) relative to bareelectrodes. In thesestudies, detection limit was defined as the experimental analyte concentration that corresponds toa signal that equals three times thestandard 3162
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0‘ 0
I
20
40
60
Time/(days)
-
Flgwe 1. Net charge responses (viz., [& b ] / a r e a ) for enzyme electrodes to 28.6 pM acetaminophen in 0.8 M @+glucose, 0.5 M KCi/phosphate buffer. The data represented by Riled ckcles are for an enzyme electrode ( A = 0.088 Cm? that had been stored h 0.5 M KCi/phosphate buffer at 4 OC when not in use. AH other data are for enzyme electrodes that were stored dry at 4 OC for (A) 1 week, (E)2 weeks, and (C) 3 weeks before testing the electrode response. Each electrode was soaked in 0.5 M KCi/phosphate at 4 OC for 12-15 h before testing and the respecthre areas of the u n “ d electrodes were (A) 0.089 cm2, (E)0.088 cm2, and (C) 0.086 c”.
deviation of signals which are independent of concentration (Le,, well below the detection limit). A similar improvement is not seen for chlorpromazine at modified electrodes, perhaps due to its larger size and/or increased hydrophobicity, either of which could impede its permeation into the swollen polymer film. This explanation is supported by a comparison of the voltammetric response times for acetaminophen, norepinephrine, and chlorpromazine at modified electrodes, where response time is defined as the time required for the magnitude of the catalytic current to reach a constant value during repetitive scanning after immersion of the electrode into an analyte solution. For acetaminophen and norepinephrine the response time is consistently less than 3 min, while chlorpromazine exhibits response times in excess of 23 min. The long-term stability of the enzyme films was evaluated for modified electrodes stored under different conditions (Figure 5). The lifetime of an enzyme electrode which had been stored in a solution of 0.5 M KCl/phosphate buffer at 4 OC when not in use was monitored over a 54 day period. The average value of the initial net charge observed for the oxidation of 28.6 pM acetaminophen at a modified electrode stored under “wet” conditions was reduced to 77% after 3 weeks of use. After 6 weeks, the relative response was 54% of the mean value observed for the first week of operation and at the end of the 54 day test period the response was down to 42%. Similarly, the responses of electrodes that had been stored
dry in the refrigerator for periods of (A) 1, (B) 2, and (C) 3 weeks did not differ significantly from those of the modified electrode stored in 0.5 M KCl/phosphate buffer. This presumably indicates that loss of GOD from the film through soaking is not responsible for the diminishing response over several weeks but is more likely due to slow denaturation of the enzyme in the polymer layer. By comparison, for glucose oxidase immobilized on a platinum electrode in a poly(pyrro1e) film, the current response is reportedly reduced to 10% after 7 days,**which was attributed to the leaching of active GOD from the polymer matrix.
CONCLUSION A rapid catalytic reaction sequence develops between the reduced flavin center of glucose oxidase and electrooxidized acetaminophen, resulting in amplification of the signal observed in the chemical domain. The flavin-containing enzymes lactate oxidase and sarcosine oxidase were also evaluated in terms of their reactivities under similar conditions, and SOD was found to react an order of magnitude more slowly than either GOD or LOD. Such analyte-mediated electrocatalytic processes are potentially applicable to the determination of compounds other than acetaminophen, and hence the feasibility of this scheme was demonstrated for the cases of chlorpromazine and norepinephrine reacting with substrate-reduced GOD. One distinct advantage of the enzymatic amplification strategy for enzyme electrode work is the temporal discrimination between analyte and background (22) Umafla, M.; Walker, J. Anal. Chem. 1986, 58, 2979. (23) Coury, L. A.; Heineman, W. R. J . Electroanal. Chem. 1988, 256, 327.
signals afforded by integration of currents to yield charges. The background charge normally saturates in less than a few hundred milliseconds at carbon electrode^,^^ and the charge due to noncatalytic faradaic processes is proportional to r1/2. By comparison, steady-statecurrent yields a charge that grows linearly and without bound in t . Enzyme reactivity is not diminished by confinement of the protein in a polymer matrix, and catalytic currents generated during the oxidation of all analytes studied at enzyme electrodes closely resemble those observed at bare electrodes with freely diffusing enzyme. For chlorpromazine however, the time required for the catalytic current to reach a constant value is considerably longer at a modified electrode, evidently due to its more sluggish permeation through the polymer film. This demonstrates the potential for selectivity based on the size and hydrophobicity of the analyte/mediator through control of the polymer chemistry employed.
ACKNOWLEDGMENT This material is based upon work supported in part by the North Carolina Biotechnology Center. Acknowledgment is also made to the Duke University Research Council for additional suport. G.G.N. and L.C.P. were recipients of Duke Undergraduate Research Grants in 1991 and 1992, respectively. Received for review February 24, 1994. Accepted June 1, 1994.' Abstract published in Advance ACS Abstracrs, August 1, 1994.
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