Anal. Chem. 1995, 67, 1896- 1902
Enzymatically Amplified Voltammetric Sensor for Microliter Sample Volumes of Salicylate Thea J. Moore,t Melissa J. Joseph,t Barry W. Allen,* and Louis A. Coury, Jr.*st Department of Chemistry, Sox 90346, Duke University, Durham, North Carolina 27708-0346, and Department of Anesthesiology, Box 3094, Duke University Medical Center, Durham, North Carolina 27710
A new voltammefric sensing strategy for salicylate employing two enzymes and applicable to microliter sample volumes is demonstrated. The method involves the use of the enzyme salicylate hydrowlase to convert salicylate to catechol,which is oxidized at a carbon electrode. The product of this oxidation reaction, o-quinone, is then reduced by a second enzyme, glucose oxidase, to regenerate catechol. Reoxidation of catechol results in a signal that is amplified due to repeated cycling of catechol molecules between the oxidizedand reduced states. This chemistry is implemented in two configurations. (i) A paper disk into which both enzymes have been absorbed is mounted on a coplanar three-electrode assembly for aqueous experiments. Determination of salicylate in a nonprescriptiondermatological product is demonstrated. (ii) A small solution volume confined directly on the coplanar electrodes is used for determination of salicylate in whole blood. The advantages of the use of two enzymes and of monitoring steady-state catalytic currents are discussed. Acetylsalicylic acid (aspirin), sodium salicylate, and related derviatives are generally regarded as safe for use by adults in the treatment of pain, inflammation, and fever.' For aspirin alone, the reported consumption in the United States was $2.7 billion in 1990.2 Such widespread use of these over-the-counter drugs has resulted in problems from overdose, principally observed in three clinical situations. (i) Therapeutic intoxication may be found in persons suffering from chronic idammatory diseases, who take these drugs habitually.' (i) Suicidal overdose from salicylates has been idenmed as a recurring problem, particularly among teenagers and older women.' (ii) The incidence of accidental ingestion of aspirin (especially flavored formulations) by children less than 5 years of age has been ~ t u d i e d . ~ Upon absorption, aspirin is rapidly hydrolyzed (half-life = 15 min) to salicylic acid, which circulates in the blood as sali~ylate.~ Serum levels of salicylate typically reach a maximum 2 h after ingestion. Blood concentrationsof salicylate in excess of 2.17 mM (300 pg mL-l) are considered toxic, while the effective concentra* Corresponding author e-mail address:
[email protected]. Department of Chemistry. + Department of Anesthesiology. (1) Rainsford, K D. Aspirin and the Salicylates; Buttenvorths: London, U.K., 1984; pp 56-58 and 245-248. (2) Feinman, S. E. Beneficial and Toxic EffectsofAspin'n; CRC Press: Boca Raton, FL, 1994; pp 23-30. (3) Done, A. K. Pediatrics (Suppl.) 1978,62, 890-897. (4) Thiessen, J. J. In Acetylsalicylic Acid: New Usesfor an Old Dmg Barnett, H. J. M., Hirsh, J., Mustard, J. F., Eds.; Raven Press: New York, 1992; pp 49+
61.
1896 Analytical Chemistry, Vol. 67, No. 11, June 1, 7995
tion range for a therapeutic dose is 1.09-2.17 mM (150-300 pg mL-') .5 Because of the relatively small concentration difference between therapeutic and toxic dosages, a rapid and specitic method for determining salicylate levels in the blood is desirable for the diagnosis of salicylate intoxication. The Trinder test is the classical clinical method for determination of blood salicylate.' This colorimetric technique is based on the formation and detection of a purple Fe3+-salicylic acid complex with concomitant denaturation of proteins by mercuric chlorides6 Although the Trinder test is rapid and inexpensive to perform, it is subject to interferences from Fe3+complexes with aliphatic enolic or phenolic compounds and other drugs which may be present in the blood or urine.7 Chromatographic and spectroscopic techniques can be less susceptible to interferences from endogenous species. However, as has been previously discussed, many of these techniques require time-consuming sample pretreatment steps (e.g., extraction, derivatization, or preconcentration) that would prohibit their use in emergency situations.6 Several detection schemes employing the flavin enzyme salicylate hydroxylase (SH) have been r e p ~ r t e d . ~These - ~ methods share the approach of exploiting the speciticityof the SH reaction, viz., the irreversible decarboxylation and subsequent hydroxylation of salicylate to form catechol: salicylate
+ P-NADH + 0, + ZH+ 2 catechol
+ P-NAD' + H,O + C02
Enzyme-based spectrophotometric methods are typically used to determine salicylate concentration by either measuring the absorbance of the blue compound formed by the reaction of catechol with Caminophenol or monitoring the change in absorbance at 310 nm due to the consumption of NADH.8 Electrochemical measurements of the consumption of reactants or the formation of products in the above reaction include the following: (i) a modsed oxygen electrode to measure the reduction current for oxygen under steady-state conditions? (ii) a potentiometric sensor to determine the carbon dioxide evolved,10and (iii) an amperometric biosensor to measure catechoL8 (5) Tietz, N. W. Textbook ofclinical Chemisty; Saunders: Philadelphia, PA 1986 p 1856. (6) Trinder, P. l3iochem.J. 1954,57, 301-303. (7) Rahni, M. A. N.; Guilbault, G. G.; De Oliveria, G. N. Anal. Chim.Acta 1986, 181, 219-225. (8) Frew, J. E.; Bayliff, S. W.; Gibbs, P. N. B.; Green, M. J. Anal. Chim. Acta 1989,224, 39-46. (9) Neumayr, M.; Friedrich, 0.;Sontag, G.; Pittner, F. Anal. Chim. Acta 1993, 273, 469-475. (10) Fonong, T.; Rechnitz, G. A Anal. Chim. Acta 1984,158, 357-362.
0003-2700/95/0367-1896$9.00/0 0 1995 American Chemical Society
The latter approach of detecting catechol rather than salicylate has several advantages8 Although salicylate is electroactive and the peak current for its oxidation increases linearly with concentration, the sensitivity of amperometric measurements is higher when the species being detected is electrochemically reversible or quasi-reversible (e.g., catechol). Also, at the modest potentials (relative to salicylate) where catechol oxidation is observed, background currents from common electroactive blood components (e.g., ascorbate, uric acid, acetoacetate) are less severe? Detection at a decreased operating potential thus has the effect of increasing the selectivity of the measurement. Finally, film formation at the electrode surface due to a buildup of products and their subsequent oligomerization is less severe for the oxidation of catechol" than for salicylate.12 We recently developed an enzyme-based electrochemical method that produces a chemically amplified signal for the oxidation of catechol.'3 In this paper, we build upon our earlier work to determine salicylatein a nonprescription drug preparation and in spiked, whole blood samples. The strategy employed involves coupling the salicylate hydroxylase reaction to an electrocatalytic redox cycle that regenerates catechol after its electrochemical oxidation. Amplification ensues via a solution reaction involving the reduced form of glucose oxidase (GOD); thus, steady-state catalytic currents are observed for the repeated oxidation of catech01.'~ These currents have the benefit of being independent of applied potential a few hundred millivolts beyond E"' as well as being independent of time. The latter helps distinguish between coincident redox proce~ses'~ since i = to for the amplified process, i = t-'I2 for nonamplified redox reactions, and i = for non-faradaic signals. Two implementations of this chemistry for sensing purposes will be demonstrated. The first utilizes a filter paper reagent disk mounted on a coplanar, three-electrode assembly. This arrangement is similar to the "gel-cell" and the microlithographically defined microcell (cell-on-achip)previously described by Murray and c o - ~ o r k e r s . ' ~In - ~our ~ case, the GOD/SH reagent disk is placed on the tip of an inverted electrode assembly, and microliter quantities of analyte solutions also containing NADH and glucose are deposited onto the disk. The second implementation involves placing droplets of whole blood that have been mixed with enzyme solutions directly onto the electrode surface. In each case, a voltammetric signal is monitored that is related to the concentration of salicylate in the sample. Most conventional biosensors are designed to be used in solution experiments with immersed electrodes, typically necessitating 1mL or more in sample volume for an analysis. The use of the miniature cells described here for sensor construction reduces the volume and consequently the total amount of sample (11) Ryan, M. D.; Yueh, A; Chen, W.-Y.J. Electrochem. SOC.1980,127, 14891495. (12) Kafil, J. B.; Last, T. A J. Chromatogr. 1985,348, 397-405. (13) Moore, T. J.; Nam, G. G.; Pipes, L. C.; Coury, L.AAnal. Chem. 1994,66, 3158-3163. (14) Oliver, B. N.; Egekeze, J. 0.;Murray, R W . j . A m . Chem. SOC.1988, 120, 2321-2322. (15) Oliver, B. N.; Coury, L. A; Egekeze, J. 0.;Sosnoff, C. S.; Zhang, Y.; Murray, R W.; Keller, C.; UmaAa, M. X. Electrochemical Reactions, Enzyme Electrocatalysis, and Enzyme Immunoassay Reactions in Hydrogels. In Biosensor Technology Fundamentals and Applications; Buck, R P.,HaUield, W. E., UmaAa, M., Bowden, E. F., Eds.; Elsevier: New York, 1990 pp 117135. (16) Morita, M.; Longmire, M. L.; Murray, R W. Anal. Chem. 1988,60,27702775.
required for an electrochemical experiment. This work also embodies more involved enzyme chemistry than previously achieved at coplanar, three-electrode microcells. Despite this complexity, salicylate determinations are easy to perform, are reproducible, and yield catalytic currents comparable to those observed for large-volume solution experiments. The singledetermination protocols used permit polishing of the working electrode between experiments to remove components of the sample matrix that may gradually foul the electrode (e.g., electrooxidized NADH"). The advantages of using filter paper over other polymeric matrices for enzyme immobilization include the following: fi) the lack of a need for cross-linking agents, which may damage some enzymes; (ii) the reproducibility of the geometry of filter paper disks; (ii) the extremely low metal content of filter paper, which avoids enzyme inhibition; (iv) the observation that buffer-soaked filter paper is less sensitive to variations in ambient humidity than many hydrophilic polymers; (v) the fact that coenzymes (such as NADH), which are difticult to immobilize in an active form in polymer films, can be easily absorbed into filter paper; and (vi) the low cost of filter paper. EXPERIMENTAL SECTION Reagents. Salicylic acid (2-hydroxybenzoicacid), ,&nicotinamide adenine dinucleotide, reduced form @-NADH), salicylate hydroxylase (SH, from Pseudomonas; EC 1.14.13.1), glucose oxidase (GOD, type VIES, from Aspergillus niger), and P-D(+)-glucose (97% anomerically pure) were purchased from Sigma Chemical Co. Some lots of SH were also obtained from GDS Technology, Inc. (Elkhart,IN). The catechol derivatives were purchased from either Aldrich or Sigma. All reagents were used as received from the manufacturer with the exception of 3-methylcatechol,which was recrystallized twice from hot toluene. Kinetic studies of reactions between electrooxidized catechol mediators and substrate-reduced GOD were conducted in solutions of 0.5 M KCV0.05 M phosphate buffer, pH 7.00 (Fisher Scientific). Except for the kinetic studies, all analyte, enzyme, cofactor, and substrate solutions were prepared in 0.1 M phos phate (Baker) buffer, pH 7.13. The enzyme solutions were prepared by dissolving a lyophilized powder containing the active enzyme (GOD or SH) in either 0.5 M KCVphosphate buffer or 0.1 M phosphate buffer to make solutions that were typically 5.2% (w/v) in SH and 23.0% (w/v) in GOD. The molar concentration of active GOD employed in kinetic studies was determined spectrophotometrically,as described previou~ly.'~ Water used for solution preparation was purified by reverse osmosis (Barnstead ROpure-LP) and then by ion exchange (Barnstead NANOpure). Instrumentation, Electrodes, and Cell Assembly. Cyclic voltammetric data were acquired with either a Cypress Systems CS1087 or BASlOOB electrochemical analyzer, each interfaced to a laboratory 80386based computer (Zeos) for data storage and processing. Coplanar electrode assemblies were comprised of an edge-plane pyrolytic graphite (Union Carbide) working electrode (A = 0.088 cm2),Ag wire (Aldrich) pseudoreference electrode, and Pd (Aldrich) ring auxiliary electrode, all encased in epoxy. A small volume electrochemical cell (600 pL) capable of accommodating an external reference electrode and gas inlet tube for anaerobic experiments was employed for some solution experiments. The kinetic studies were the only experiments conducted (17) Jaegfeldt, H. 1.Electroanal. Chem. 1980, 110, 295-302.
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1897
under anaerobic conditions, and they utilized an external Ag/AgCl reference electrode. Between experiments, the electrode assembly was polished with alumina (1-0.05 pm diameter) on Buehler Microcloth and then sonicated in deionized HzO for 60 s in a Branson 1200 sonicating bath to remove alumina from the electrode surfaces. Methodsfor Salicylate Determinations. Blank filter paper disks of uniform size were cut from Whatman filter paper, grade 42 (Ashless, 0.2 mm thickness), with a standard office hole punch. Salicylate was determined directly by placing an untreated filter paper disk on top of a coplanar electrode assembly, depositing 30 p L of salicylate in phosphate buffer onto the filter paper, and measuring the oxidative peak current (ip,Jfor salicylate. SH-only reagent disks were prepared by depositing 5 pL of SH onto blank filter paper disks and allowing them to dry in air. Indirect determination of salicylate was achieved by monitoring after 2 min for catechol produced in situ by depositing 30 pL of a salicylate/b-NADH solution onto an ‘SH reagent disk positioned on top of a coplanar electrode assembly. An incubation period of 2 min in air was sufficient to ensure reproducible conversion of salicylate to catechol (i.e., currents were independent of length of incubation beyond this time). GOD/SH reagent disks were prepared by treating filter paper disks with 5 p L of SH/O.l M phosphate buffer solution and 5 jiL of GOD/O.l M buffer solution. Reagent disks were allowed to dry in air between treatments. For the indirect determination of salicylate by monitoring a chemically amplified catechol signal, 20 pL of a salicylate/@-NADHsolution was deposited onto a GOD/ SH reagent disk and allowed to react for 2 min before adding 10 pL of P-D(+)-glucose. The current at 600 mV was measured from the resulting voltammograms for quantitation purposes.
Determination of Salicylate in a Nonprescription Drug. A 19.8 mg sample of a commercial 17% by weight salicylate solution (Compound W Whitehall Laboratories) was added to 3.00 mL of phosphate buffer @H 7.0) and sonicated for 5.0 min in a Branson 1200 sonicator. Various volumes (5,10,15, or 20 pL) of the supernatant were diluted with 100 pL of 6.11 mM P-NADH plus 200 pL of buffer. After brief agitation, 10pL of each solution was delivered onto separate SH/GOD disks. The solutions were incubated for 300 s in air before the addition of 30 p L of a 3.75 M ~-D-ghCOSe.Droplets were mixed by repeatedly drawing and withdrawing solution into the tip of an Eppendorf pipet for 60 s, after which each voltammetric scan was initiated. Assay of Whole Blood Samples. Experiments involving whole blood were conducted by placing droplets of solutions directly onto the surface of coplanar electrodes. For this work, a short length of plastic tubing (Le., a simple drinking straw) was fitted over the end of the inverted coplanar electrodes to form a reservoir with a capacity of approximately 200 pL. In a typical experiment, 100 pL of whole, human blood (from an anonymous donor at the Duke University Medical Center) was spiked with varying amounts of salicylate and placed into a 1.5 mL capacity poly@ropylene)microcentrifuge tube. Next, 50 pL of SH solution (40 units/mL) was added, followed by 200 pL of 6.11 mM NADH in pH 7 (0.1 M) phosphate buffer. The components were mixed in a laboratory vortexing apparatus (Vortex-Genie, Scientific Instruments, Inc.) for 10 s and then allowed to incubate for 300 s while exposed to the air. Finally, 50 pL of 21.7% (wt/vol) GOD and 50 pL of 4.8 M P-D-glucosewere added to the centrifuge tube and vortexed for 10 s. A 100pL aliquot of this solution was taken 1898 Analytical Chemistry, Vol. 67, No. 7 7 , June 7, 7995
COO
Figure I. Schematic representation of the chemical and electrochemical processes involved in the chemical amplification of salicylate after its enzymatic conversion to catechol.
via Eppendorf pipet and delivered into the reservoir above the coplanar electrodes, and the voltammetric scan was initiated. Wanting: Human blood should be treated as a potentially toxic substance. Contraction of hepatitis and/or HIV from whole blood samples represents a serious risk. While conducting the experiments described here, personnel wore full face shields, lab coats, and latex gloves at all times. All pipet tips, centrifuge tubes, used gloves, polishing pads for electrodes, and paper tissues employed were placed in “biohazard containers and then incinerated after completion of this work. Glassware, electrodes, Nalgene wash bottles, and lab bench tops were scrubbed after use with aqueous hypochlorite (bleach) solutions. RESULTS AND DISCUSSION
Characterizationof the Reaction between GOD and Oxidized Catechols. In a recent p~blication,’~ we reported that the current observed for the oxidation of norepinephrine (a catechol compound) was enhanced considerably in the presence of GOD and a saturating excess of its primary substrate, P-D-glucose. This signal amplification was attributed to repeat sensing of the catechol at the electrode surface due to redox cycling. During this process, the c-quinone formed oxidatively at the electrode surface was rapidly reduced to norepinephrine by a solution reaction with substrate-reduced GOD. This enzymatic amplificationwas shown to result in greater sensitivity and improved limits of detection for norepinephrine. To assess the feasibility of indirect determination of salicylate via recycling of catechol by GOD (Figure l), the reactions of substrate-reduced GOD with various oxidized catechols were examined. The suitability of GOD as a reductant appears to be general for o-quinone compounds as steady-state catalytic voltammograms were observed for each of the catechols in Table 1, despite their varying substitution patterns and charges. Limiting currents were experimentally verified to be independent of scan rate over a 20 to several hundred mV s-l range. In such cases, the steady-state currents are then described byI8
where C*med and C,* are the concentrations of the mediator (catechol) and enzyme (GOD), respectively (units, mol ~ m - ~ ) , (18) Bard, A. J.; Faulkner, L. R Electrochemical Methods: Fundamentals and Applications; Wiley: New York, 1980.
Table 1. Bimolecular Rate Constants for the Reaction of Catechol Mediators wlth Substrate Reduced Glucose Oxldase
catechol neutral (1) khlorocatechol (2) catechol (3) DOPEGC (4) 7,8dihydroxy-&methoxycoumarin (5) 3-methoxycatechol (6)3-methylcatechol (7) Cte7t-butylcatechol (8) Cmethylcatechol cationic (9) DHBAd (10) dopamine anionic (11) DOPACC (12) hydrocaffeic acid (13) caffeic acid
kcat (M-l
s-l)O
9 f 1 10.7 f 0.8 5.4 f 0.9
0.52 f 0.03 2.4 f 0.4 3 i 1 2.4 f 0.8 7 f 2 1.8 f 0.3 3.4 f 0.7
0.38 =k 0.08 0.4 f 0.1 0.7f 0.1
nb 40
8 7 5 3 8
1100 800 500 200 -100
E/(mV vs Ag)
3%z3
7 5 18
x -10 -20
5 7
500 300
13 5 5
Mean f [ t w ( n ) 1 / 2where ] tm is the t-test value at 90%confidence level and s is the standard deviation. Number of replicate measurements. 3,4-Dihydroxyphenylethyleneglycol. d 3,dDihydroxybenzylamine. e 3,CDihydroxyphenylaceticacid.
100 -100 300
E/(mV vs Aa)
c
40
5-20
3 -80 -140 6 0 0 400 200
0
-200
E/(mV vs Ag) is the diffusion coefficient of the mediator (cm2 s-I); k,,, is the bimolecular rate constant for the enzyme-mediator reaction (cm3mol-' SI); and the other parameters have their customary definitions.ls Equation 1was originally derived for pseudo-firstorder catalytic processes;'* however, it has been shown to apply to enzyme reactions when the reduced form of the enzyme is kinetically inexhaustible by virtue of being saturated with s u b strate. '9 In accord with eq 1, the limiting currents for the catechol reactions were linearly proportional to the mediator concentration and square root of the enzyme concentration. The bimolecular rate constants for the reaction of reduced GOD with 13 substituted, electrooxidized catechols (o-quinones) were determined, and these values are presented in Table 1. As is evident, reaction rates were highest for neutral and cationic catechols and were consistently lower for anionic catechols. This may indicate that the region of interaction of the o-quinones with the enzyme is anionic, although it is not clear if the various species examined even share a common enzymic reaction site. Steric bulk also appears to be a factor since the smallest species (catechol) gives the largest k,,, and the larger, bicyclic coumarin reacted at a considerably slower rate, despite the fact that both compounds are uncharged. Effect of m e n on Catechol Reactions. In bacteria, GOD catalyses the oxidation of glucose to b-gluconolactone. During this process, the oxidized flavin in the enzyme is regenerated by reducing 02 ,to H~02.2~ In the electrocatalytic reaction sequence described above, &quinone replaced 0 2 as the electron acceptor for reduced GOD. Consequently, the kinetic studies discussed in the preceding section were conducted under anaerobic conditions to eliminate interference from oxygen. Since oxygen is a necessary reactant in the SH-catalyzed conversion of salicylate to catechol however, it is important to establish that its presence
&ed
(19) 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, 452-458. (20) Blanchard, J. S.; Wong, K. IC Isotope Effects on Enzymecatalyzed Redox Reactions. In Enzyme Mechanismfrom Isotope Effects;Cook, P. F., Ed.; CRC Press: Boca Raton, FL, 1991; pp 350-351.
Figure 2. Solution experiments performed in a small volume electrochemical cell (ca. 600 pL) using a coplanar electrode assembly. All scan rates were 10 mV/s. (Panel A) Cyclic voltammograms demonstrating the effect of successive scans; 0.196 mM salicylate in 0.1 M phosphate buffer. Lower curve is first scan. (Panel B) Voltammogram of catechol produced in situ by the hydroxylation of 0.196 mM salicylate in the presence of salicylate hydroxylase (0.52% (w/v)), NADH (0.203 mM), and dissolved oxygen (air-saturatedbuffer). (Panel C) In the presence of 2.3% (w/v) GOD and 0.6 M P-D(+)glucose, catalytic currents are observed in successive scans for oxidation of catechol generated by the reaction of 0.196 mM salicylate with salicylate hydroxylase. Lower curve is first scan.
will have no deleterious effect on the GOD/@quinone reaction when the oxidative catalylic cycle is coupled to the reactions of SH. To address this concern, additional kinetic experiments were conducted under aerobic and anaerobic conditions. The bimolecular rate constant for the reaction of substrate-reduced GOD with electrooxidized Cmethylcatechol in an oxygen-saturated solution was indistinguishablefrom the rate constant obtained for the same reaction in a deaerated solution. This result may indicate that the electron transfer from reduced GOD to oxygen is slower than the electron transfer to electrooxidized catechol. Altema tively, GOD may rapidly reduce 02 to H202 without significantly altering the concentration of b-D-glucose. The latter must be present in excess to saturate GOD and ensure pseudo-first-order reaction conditions (steady-statecurrents). The H202 formed by such a reaction should be of little consequence for the oxidative regeneration cycle (Figure 1) since the chemical oxidation of catechol by hydrogen peroxide proceeds at an appreciable rate only at alkaline pH (>9) and in the presence of a catalyst (e.g., c o (H20)62+).2' Voltammetry of Salicylate. Electrochemical oxidation of salicylate in aqueous phosphate buffer is irreversible at graphite electrodes (Figure 2, panel A). The anodic prepeak observed on the initial scan has been reported elsewherez2and was absent on (21) Alexiev, A A; Angelova, M. G. Mikrochim. Acta 1980,2, 187-194.
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subsequent scans. Furthermore, the peak current for the process at 750 mV was reduced to 66% of its original value on the second scan and continued to decrease with successive scans, presumably due to electrode fouling.12 This immediate loss in electrode response illustrates the problems inherent in attempting to quantitate salicylate by its direct oxidation. Solution Reactions of SH. Electrode contamination due to coupling of the reaction products from catechol oxidation is minimal in comparison to salicylate.ll Catechol is rapidly generated in situ by the reactions of SH with salicylate, NADH, and dissolved oxygen (Figure 2, panel B). The quasi-reversible process observed at -50 mV corresponds to the oxidation of catechol, while the anodic process seen at 500 mV is the oxidation of excess NADH. It is interesting to note that catalytic reactions between NADH and o-quinones have been studied previou~ly.~~ In that work, the rate constant for the bimolecular reaction between Cmethyl-oquinone and NADH was found to be kat = 3010 f 50 M-l s-1,23 which is relatively slow compared to the reaction with GOD (cf. Table 1). The chemical reversibility observed for catechol in Figure 2B suggests that if a similar reaction occurs between NADH and oquinone, its rate is too slow to be observed for the solution conditions and time scales employed in our studies. Catalytic currents were only observed in our experiments when GOD and /3-D-glucose were added to a solution of enzymatically generated catechol (Figure 2, panel C). The half-wave potential of the catalytic voltammogram shifted to a more positive potential on the second scan. This is attributed to fouling of the electrode surface during oxidation of residual NADH, the products of which are known to adsorb strongly to carbon electrode^.'^ Although the magnitude of the limiting current was reduced by 4% on the second scan, quantitation was not affected since this change in response was within the precision of the experiment. Both the shift in half-wave potential and the decrease in signal may be avoided however by simply selecting a less positive switching potential. Little to no current was observed for catechol oxidation when GOD and ,6-D-glucose were introduced to the reaction mixture before the conversion of salicylate to catechol by SH had time to proceed. This indicates that the velocity of reaction between GOD and @-glucose was considerably faster than the reactions of salicylate hydroxylase. The oxygen required to oxidize the flavin center of salicylate hydroxylase during formation of catechol appears to have been consumed instead by GOD, which reduced 02 to HzOz during the oxidation of @-glucose. Since the concentration of oxygen in air-saturated water is only about M,24the sequence of the addition of reagents in the scheme shown in Figure 1appears critical to avoid the parasitic consumption of oxygen by GOD. This requirement held true for reactions conducted with reagent disks (see below), despite the greater flux of atmospheric oxygen to solution for the large surface-to-volume ratio operative in such experiments. Studies with Reagent Disks. The mechanics of performing salicylate determinations on GOD/SH or SH reagent disks are illustrated in Figure 3, panel A. Catalytic voltammograms generated using disks resembled the steady-state voltammograms observed for solution experiments conducted in small-volume (22) Fung, Y.-S.; Luk, S.-F.Analyst 1989,114, 943-945. (23) Carlson, B. W., Miller, L. L. 1.Am. Chem. SOC.1985,107, 479-485. (24) Brett, C. M. A; Brett, M. 0. Electrochemisty: Principles, Methods, and Applications; Oxford New York, 1993; p 140.
1900 Analytical Chemisfry, Vol. 67,No. 7 7, June 1, 1995
B 20 r
-
-40
-60 -80' ' ' 600 400 200
'
0
~
-200
E/(mV vs Ag)
Figure 3. (Panel A) Diagram illustrating the mechanics of performing a salicylate determination on the tip of a coplanar electrode assembly with a GOD/SH reagent disk (ref, reference; WE, working; aux, auxiliary electrodes). (Panel B) Catalytic voltammogram observed for catechol after depositing 20 pL of a 90.6 pM salicylate/l.56 mM NADH solution plus 1OpL of 1 M P-D(+)-glucose solution onto a GOD/ SH reagent disk that had been treated with 5 pL of a 5.2% (w/v) SH solution and 5 p L of a 23.0% (w/v) GOD/phosphate buffer solution.
electrochemical cells (Figure 3, panel B), in terms of both halfwave potentials and limiting currents. This result is somewhat surprising given that diffusion rates might be expected to be slower in filter paper compared to aqueous solutions. The grade of filter paper employed as a solid support for SH and GOD is a porous material, designed to retain 2.5 pm particles. Since both enzymes have dimensions that are considerably smaller than the pore size of the filter paper, diffusive movement of the enzymes through pores should be rapid. When the relatively large volume of the analyte solution (-20 pL) was placed onto the surface of a disk, the solution permeated the paper and wet the electrode surface to form a thin solution layer between the filter paper and the electrode assembly. The total volume of the analyte droplet was not contained by the filter paper, consequently much of the solution formed a meniscus above the reagent disk. Thus, three solution regions exist in these experiments: (i) the droplet above the paper disk (which is exposed to air), (i) the region inside the disk itself (including the pores), and (iii) the thin layer of solution between the disk and the relatively rough graphite electrode. The observation of steady-state catalytic currents indicates that the rate of the electrode reaction was controlled by the bimolecular catalytic step and not by mass transport rates for any of the reactants. If the catalytic reaction layer were to extend beyond the thin layer of solution beneath the disk, biphasic kinetics would be expected due to the different transport rates for catechol through water and through the hydrated filter paper. The most likely explanation for the resemblance between reagent disk and solution voltammograms is that the reaction layer did not extend into the paper disk. It is also expected that the enzyme concentration in the solution layer adjacent to the electrode was constant due to equilibration occurring during the SH incubation step. JusMcation for this hypothesis may be demonstrated by calculation of the catalytic reaction layer thickness (u) as defined byz5
Assuming that the enzyme activity was unchanged by absorp tion into the paper and that the values for k,, (1 x lo9 cm3 mol-' (25) Delahay, P. New Instrumental Methods in Electrochemisty;Interscience: New York, 1954 R. E. Krieger Publishers: Huntington, NY, 1980 pp 100-103.
300
25
3 W
I
I
20
I
!
I
15 10
5
0
e
0
0.0 0.2 0.4
0.6 0.8 1.0
[Salicylic Acid]/(mM) Figure 4. Calibration plots for nonamplified analyte determinations. (0) Direct oxidation of salicylate in a solution of 0.1 M phosphate buffer on untreated filter paper disks (limit of detection = 26pM). (0) Plot for catechol oxidation arising from salicylate hydroxylation in a solution of 1.56 mM P-NADH on filter paper disks treated with 5 p L of a 5.2% (w/v) salicylate hydroxylase/O.l M phosphate buffer solution values for salicylate and (LOD = 23 pM). The y-axis represents ip,a catechol, respectively.
and &ed (6.4 x cm2/s) were the same as those in aqueous solution, p is calculated to be 2 pm for a typical GOD concentration of 170 pM. This value is reasonable for the thickness of a solution layer next to the electrode, particularly considering the roughness of the graphite surface. (Electrode areas measured electrochemically even after 100-500 ms were about 110%of the values obtained by visual measurement of the electrode diameter, indicating appreciable surface roughness.) Quantitation with Reagent Disks. In order to critically assess the merits of enzymatic amplification reactions for reagent disks, catechol was first determined without amplification by GOD. The calibration plot for catechol generated by the in situ decarboxylation and subsequent hydroxylation of salicylate on SH reagent disks was found to be linear in salicylate concentrations between 23 and 350 pM (Figure 4, solid symbols). At higher concentrations of salicylate, current for the oxidation of catechol reached a maximum value, perhaps due to incomplete conversion of salicylate due to oxygen depletion. A linear calibration plot was also obtained for the direct oxidation of salicylate on blank filter paper disks (Figure 4,open symbols). By comparing the slopes of the linear regions of the calibration plots in Figure 4, the sensitivity of an electrochemical salicylate determination is seen to be greater when salicylate is first converted to catechol, consistent with earlier work? In contrast, however, the detection limits for catechol and salicylate were similar: 23 and 26 pM, respectively. (Detection limit is defined here as the analyte concentration calculated to equal three times the standard deviation of signals obtained in solutions for which current is not a function of analyte concentration.) Enzymatic amplification of the catechol signal was found to further improve the sensitivity for salicylate detection using reagent disks. When salicylate determinations were performed on GOD/SH filter paper disks, a l@foldimprovement in sensitivity over SH-only disks was observed (cf. Figures 4 and 5). The reproducibility of these measurements was addressed by performing three to six replicate determinations for each salicylate concentration. Each of the points plotted in Figure 5 is thus the mean obtained from these replicates, and the error bars shown represent one sample standard deviation unit about the mean. The linear dynamic range observed in these analyses was similar to that for catechol in the absence of GOD (Figure 4). Also, the
0.00
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[salicylate]/(mM) Figure 5. Calibration plot for salicylate. Points represent the catalytic current for catechol, measured at 600 mV. Each point represents the mean current generated for the addition of 20 ,uL of varying amounts of salicylate dissolved in a 1.56 PNADH solution plus 10 p L of 0.1 M p-D(+)-glucose to GODISH reagent disks prepared with 5 pL of 5.2% (w/v) SH and 23.0% (w/v) GOD in phosphate buffer. Observed limit of detection = 10 pM.
lo
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s-1)
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E/( mV vs Ag) Figure 6. Catalytic voltammogram for a commercial pharmaceutical preparation ([salicylate] = 0.113 mM) using SH/GOD reagent disk. Scan rate was 10 mV/s, with reagent disk composition as in Figure 5. (Inset) Calibration plot for duplicate salicylate determinations with enzyme disks. R = 0.989.
limit of detection for the amplified reaction sequencewas improved by about 50% (10 pM) compared to the nonamplified case. Due to the small volumes employed, this represents an absolute detection limit of 32 ng for a 20 p L analyte droplet. No attempt was made to optimii the experiment for lower concentrations, however, since the target applications (see below) involve millimolar concentrations. Application I: Determination of Salicylate in a Nonprescription Drug. Topical solutions and ointments containing salicylic acid are widely used to treat benign skin lesions of viral originz6 (warts) and to treat more serious afflictions, such as ps0riasis.2~One commerically available preparation, stated by the manufacturer to be 17%by weight salicylic acid, was assayed using the reagent disk procedure discussed above. Inactive ingredients in the product included collodion, ethyl cellulose, and Polysorbate 80; hence, a sonochemical extraction procedure was developed to release the salicylate into an aqueous buffer. Four different dilutions of the extract were prepared as described in the Experimental Section. F i r e 6 shows a typical voltammogram obtained at a scan rate of 10 mV s-l. The current did not retrace itself on the reverse potential sweep due to fouling of the electrode from oxidation of excess NADH (ca. E = 750 (26) Physicians’ Desk Reference for Nonprescription Drugs; Medical Economics Company: Oradell, NJ, 1985. (27) Pet, J.; Strmefiova, M.; PalenEArovP, E.; Pullmann, R; Funiakod, S.; Vi,Sfiovsks;,P.; Buchanec, J.; LazarovA, 2. Cutis 1992,50,307-309.
Analytical Chemistry, Vol. 67, No. 11, June 1, 1995
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A -12 B C n -29 1 D
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E / (mV vs Ag) Figure 7. Catalytic voltammetry in whole blood samples. [Salicylate] = 0, 1.29, 1.94, 3.89, and 5.84 mM for curves A-E, respectively. Scan rate = 10 mV/s. See Experimental Section for details of solution conditions. (Inset) Calibration plot for salicylate determined in whole blood. Point at x = 3.89 mM is the mean value from triplicate measurements, with error bars representing one sample standard deviation about mean. R = 0.970.
mV). Duplicate determinations of the current at 600 mV on the forward scan were conducted for each of the solutions, and electrodes were repolished between each experiment. The mean current values obtained are shown in the inset of Figure 6 plotted versus the salicylate concentration calculated from the manufacturer‘s specifkation. The correlation coefficient for the regression line in the inset was 0.989. ApplicationII: Determinationof salicylate in Whole Blood Samples. Initial attempts to use reagent disks for blood samples proved unsatisfactory due to the inability of the blood droplets to reproducibly wet and permeate the enzyme-loaded disks. Subsequent experiments involvingvortex mixing of blood directly with the enzyme reagents were found to be more reliable. Figure 7 shows five voltammograms obtained in whole blood samples, spiked with varying amounts of salicylate and mixed with SH, GOD, NADH, and glucose. Curve A corresponds to a sample to which no salicylate had been added. Only the irreversible oxidation of NADH was observed, appearing at around 750 mV. For “low”salicylate concentrations ( < 2 mM) ,both the catalytic current due to catechol (at 400 mV) and the oxidation current for NADH (ca. 750 mv) were seen (curves B and C). For higher salicylate concentrations, large catalytic waves were observed. The calibration plot for these data (Figure 7, inset) shows a larger linear dynamic range but with diminished sensitivity compared to the reagent disk experiments for salicylate dissolved in buffer (28) KlimeS, J.; Sochor, J.; Zahradnifek, M.; Sedlafek, J. J. Chromatogr. 1992, 584,221-228.
1902 Analytical Chemisfry, Vol. 67,No. 7 1, June 7, 7995
(Figure 5). One possible explanation for this is the reaction of the o-quinone formed at the electrode surface with endogenous thiols. This would be expected to supress currents at lower salicylate concentrations but have less of an effect at higher concentrations (which are the critical levels for diagnosing an overdose). It is also noted that salicylate binds to erythrocytes?* thereby reducing the fraction of fi-eely diffusing salicylate in blood. This could also result in a diminished sensitivityfor measurements in blood. Finally, the precision of the measurements was assessed by conducting triplicate measurements at [salicylate] = 3.89 mM. The mean catalytic current was found to be 37.3 PA with a standard deviation of 3.9 PA, demonstrating that responses observed in blood for therapeutic levels of salicylate (B and C) are readily distinguishable from toxic concentrations (D and E). CONCLUSIONS The reported strategy improves the sensitivity and detection limit for catechol-based salicylate determinations in buffer solutions through chemical amplification of currents. Although salicylate can be determined directly with voltammetric methods, its oxidation is chemically irreversible. Greater sensitivity (roughly an order of magnitude increase) is observed instead by detecting and recycling catechol. Furthermore, the conversion of salicylate to catechol minimizes the decrease in the electrode response with time, since electrode fouling due to film formation on the electrode surface by the oxidation products is less severe for catechol than for salicylate. Despite the complex enzyme chemistry involved with a dual enzyme sensing sequence, reagent disk experiments generate reproducible currents that resemble those observed in solution experiments with immersed electrodes. Salicylate determinations using reagent disks only require small amounts of sample, are simple and rapid to perform, and are inexpensive to implement. The use of the two-enzyme system in whole blood samples gives rise to catalytic signals that are easily distinguishable from those due to NADH oxidation. Catalytic signals are observed over the range of salicylate concentrationsrelevant for diagnosis of salicylate overdose. ACKNOWLEDGMENT This material is based on work supported in part by the North Carolina Biotechnology Center through Grants 9113-ARG-0103and 9313-ARG-0101. M.J.J. was a recipient of a Duke Undergraduate Research Grant in 1990. We also are grateful to Claude Piantadosi, M.D., Director of the Duke Hyperbaric Medicine Center, for assistance in obtaining the human blood samples. Received for review January 30, 1995. Accepted March 16, 1995.@ AC950101T @
Abstract published in Advance ACS Abstracts, May 1, 1995.
