Anal. Chem. 1991, 63, 2993-2994
2993
Organic-Phase Enzymatic Assays with Ultramicroelectrodes Joseph Wang,* Li-Huey Wu, and Lucio Angnes' Department of Chemistry, New Mexico State University, Las Cruces, New Mexico 88003
INTRODUCTION There is a growing biotechnological interest in the use of organic solvents as media for enzymatic reactions (1-3). Such organic-phase enzymology offers a number of potential advantages compared to the common use of enzymes in aqueous solutions. These include increased solubility of hydrophobic substrates, enhanced thermostability, alteration of substrate specificity, shifts of thermodynamic equilibria, or reduction of side reactions. Enzymebased assays can ais0 greatly benefit from the use of nonaqueous solutions (4). In particular, recent work by Turner (5,6) and others (7,8) has illustrated that interesting biosensing possibilities accrue from organic-phase enzymology. Improved detection of phenolic compounds, cholesterol, or hydrogen peroxide was thus obtained by operating amperometric or thermal transducers in chloroform, toluene, or diethyl ether. While biocatalysis in organic media is still in its infancy, it should lead to many interesting analytical applications and opportunities. This paper describes the utility of ultramicroelectrodes for enzymatic assays in nonaqueous solvents. Unlike the traditional use of biocatalysts in aqueous solutions, amperometric monitoring of enzymatic reactions in organic media may suffer from severe ohmic effects (as the applied/detection potential is largely lost in the bulk solution). The ohmic-drop problem becomes extremely Severe since nonpolar organic solvents offer higher biocatalytic rates and are thus preferred for organicphase enzymology (9). The very small currents drawn at ultramicroelectrodes result in a dramatic minimization of ohmic (iR) losses (10, 11) and thus hold great promise for enzymebased amperometric measurements in resistive organic media. The feasibility of such measurements is demonstrated in the following sections. EXPERIMENTAL SECTION Apparatus. Voltammetric and amperometric measurements were performed with the Bioanalytical Systems (BAS) Model lOOA and EG&G PAR Model 174A electrochemical analyzers, respectively. The former was used also for measuring the uncompensated cell resistance. Experiments were carried out in a l@mL electrochemical cell (BAS, Model VC-2). The working electrode, reference electrode, (Ag/AgCI; BAS Model RE-1) and platinumwire auxiliary electrode joined the cell through holes in its Teflon cover. A magnetic stirrer and a stirring bar (1.0 cm long) provided the convective transport during amperometric measurements. The preparation of the carbon-fiber microcylinder electrodes (7-wm diameter, 5-mm length) has been described in detail previously (12).A glassy-carbon disk (3-mm diameter; Model MF 2012, BAS) was used for comparison purposes. Reagents. Horseradish peroxidase (HRP, EC 1.11.1.7, 90 units/mg) and tyrosinase (EC 1.14.18.1, 3870 units/mg) were obtained from Sigma. Ethanol (200 Proof, Quantum Chemical) and acetonitrile (EM Science Co.), with maximum water contents of 0.1 and 0.3%,were used as received. tertButy1 hydroperoxide, 2-butanone peroxide, o-phenylenediamine,1-[(1-hydroperoxy)cyclohexyl)dioxyl]cyclohexanol, and tetrabutyhonium bromide (TBAB) were purchased from Aldrich. Procedure. Experiments were performed at room temperature, with the enzyme present in the organic solvent in a form of undissolved suspension. (In the case of tyrosinase, the enzyme particles clumped together and stuck to the fiber electrode.) Permanent address: Instituto de Qumica da USP, Siio Paulo, Brazil. 0003-2700/91/0363-2993$02.50/0
Substrate concentrations were measured through voltammetric (squarewave)or amperometric monitoring of the reaction product. The latter were performed after applying the desired potential and allowing the background transient current to decay.
RESULTS AND DISCUSSION Various comparative experiments (to conventional-size electrodes) have been performed to illustrate the substantial improvements associated with the minimization of resistance effects at ultramicroelectrodes during organic-phase enzymatic assays. Model enzymes, horseradish peroxidase (HRP)and tyrosinase, known for their stable biocatalytic reactivity in various organic solvents (13,5), were thus employed for the quantitation of peroxide and phenolic substrates, respectively. (It should be pointed out that these enzymes retain a thin aqueous f i ,essential for their catalytic-activeconformation, in organic media.) Figure 1 compares square-wave voltammograms for increasing concentrations of tert-butyl hydroperoxide (1-5 mM (b-f)) in ethanol, as recorded at conventional-size glassycarbon (A) and carbon-fiber (B) electrodes. In the absence of deliberately added electrolyte, the large electrode is prone to severe ohmic distortions and no meaningful results are observed. In contrast, a well-defined cathodic response, associated with the reduction of the enzymatically oxidized mediator, is observed at the microelectrode. This current increases in proportion to the peroxide concentration. The consequences of such minimization of resistance effects upon amperometric biosensing are illustrated below. Figure 2 compares the dynamic amperometric response of the carbon-fiber microelectrode to successive additions of 1-[(1-(hydroperoxy)cyclohexyl)-dioxy]cyclohexanol(A), tertbutyl hydroperoxide (B), and p-chlorophenol (C) in the absence (a) and presence (b) of the corresponding enzyme (HRP (A, B) and tyrosinase (C)). Ethanol and acetonitrile solutions were employed, with no deliberately added electrolyte (15% v/v water was essential in the latter case). Despite the resistive media, the microelectrode responds very rapidly to these dynamic changes in the substrate concentration, producing steady-state signals within 0.5-1 min. A linear concentration dependence is observed in the first case. The favorable signal-to-noise characteristics permit convenient quantitation of millimolar and submillimolar concentrations. No response is observed in analogous measurements without the enzyme (a). Figure 3 shows calibration plots for 2-butanone peroxide, over the (1-12) X lo4 M range, under different experimental conditions. As expected, no response is observed in the absence of the enzyme (a-c). Essentially the same plots, with a nearly linear response, are observed in ethanol solutions in the absence (e) and presence (d) of electrolyte (0.1 M TBAB). Higher sensitivity coupled to a curved concentration dependence characterize the electrolyte-free acetonitrile solution (f). The different concentration profiles (ethanol vs acetonitrile) reflect solvent-induced kinetic alterations of the enzyme catalysis. Dramatic changes in the biocatalytic efficiencies have been reported in different organic media (9). The microelectrode/organic-phaseenzymology concept can be very useful for in situ investigations of such solvent effects. All solvents contain impurities, including traces of electrolyte. The level of electrolyte in the solutions employed in the present work was assessed through measurements of the 0 199 1 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 24, DECEMBER 15, 1991 I
I
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-0.5
I
0
POTENTIAL (VI
I
I
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2
0.5
10
CONCENTRATION, mY
Flgure 1. Square-wave voltammograms obtained at the conventional glassyearbon (A) and carbon-fiber (B) electrodes upon increasing the tert-bulyl hydroperox[de concentration in 1 mM steps (b-9. Also shown is the blank voltammogram (a). Solution: pure ethanol with 0.06 mg/mL HRP, 1 X loa3 M .o-phenylenediamine, and no added electrolyte. Voltammetric waveform has 15-Hz frequency, 25-mV amplitude, and 4-mV step.
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I
I
Figure 3. Dependence of the amperometrlc response upon the concentration of P-butanone peroxide In the absence (a-c) and presence (d-f) of HRP (0.08 mg/mL): (a, b, d, e) pure ethanol; (c, f) pure acetonitrile; (a, c, e, f) no electrolyte; (b, d) 0.1 M TBAB. Conditions: applied potential, -0.3 V; stirring rate, 250 rpm; 1 X M ophenyienedlamine.
Table I. Uncompensated Cell Resistance with the Solutions Employed in This Work"
solvent
pure
pure + 1 mM o-phenylenediamine
ethanol acetonitrile
2.7 MQ 1.2 MQ
2.1 Ma 700 KQ
pure + 1 mM TBAB 400 KQ 270 KQ
"As measured with the BAS l00A analyzer, using the iR star command.
a a
3 0
iL/c 2 min
i'
a
TIME
Flgve 2. Amperometrlc response to succBss/ve additions of 1 X lo4 M 1-[( l-(hydroperoxy)cycl~xyl)diixy]cyclohexanol(A), 1 X 10-3 M tert-bvtyl hydroperoxide (B), and 7 X lo4 M pGhlorophenol(C) in the absence (a) and presence (b) of the enzyme (HRP (A, B), tyrosinase (C); 0.08 and 0.1 mg/mL I-RP and tyrosinase,respectively). Conditions: solutions (A, B) pure ethanol and acetonitrile containing 1 X M o-phenylenediamine, (C) acetonitrlle/water (85:15 v/v); applied potentials, -0.4 V (A, B) and -0.3 V (C); stirring rate, 250 rpm.
cell resistance (Table I). These data clearly illustrate the very high resistivity of the media employed (even when compared to values measured in the presence of only 1m M TBAB). In addition, even the acetonitrle/water mixture (of Figure 2C) yielded a resistance of 600 KQ, compared to 26 KQ in the presence of 1 mM TBAB. In conclusion, the above results demonstrate that the application of ultramicroelectrodes for monitoring enzymatic reactions in organic media is very promising. Rapid and sensitive assays can thus be obtained in organic solutions containing no (deliberately added) supporting electrolyte. The bioanalytical use of organic-phaseenzymology is still at a very early stage. The unique coupling of ultramicroelectrodeswith biocatalysis in organic media should thus lead to a major expansion in the scope of enzymatic assays. Applicability to other enzymes, water-insoluble substrates, or solvent systems
can be easily envisioned. While enzyme suspensions were employed in the present work, similar advantages can be obtained for enzyme microelectrodes (based on adsorption/ attachment of the enzyme and mediator). Indeed, operation in organic solvent can simplify the immobilization procedure (5). Besides their analytical utility, microelectrodes may provide valuable insights in future studies and development of biocatalysis in organic media.
LITERATURE CITED Kllbanov, A. M. Chemtech 1986, June, 354-359. Volkin, D. B.; Staubll, A.; Langer, R.; Kllbanov, A. M. Bbtechnol. Bioeng. 1991, 37, 843. Halllng, P. J. Blotechnol. Bioeng. 1990, 36, 691. Kazandllan, R. 2.; Dordlck, J. S.; Kllbanov, A. M. Blotechnol. BioenQ. 1988, 28, 417. Hall, 0. F.; Best, D. J.; Turner, A. P. F. Anal. Chim. Acta 1968, 213, 113.
Hall,. G. F.; Turner, A. P. F. Bbsensors '90, First World Congress on Bbsensors, Singapore, May, 2-4, 1990; Elsevier Semlnars: Oxford, U.K., 1990. Connor, M. P.; Wana. J.; Kubiak. W.; Smvth. M. Anal. Chlm. Acta 1990, 229, 139. Danlelsson, B.; Flygore, L.; Velev, T. Anal. Lett. 1989, 22, 1417. Laane, C.; Boeren, S.;Vos, K.; Veeger, C. Bbtechnol. Bioeng. 1987, 30. 81. Bond, A. M.; Fleischmenn, M.; Robinson, J. J . Electfaenel. Chem.
1984, 168, 299. Wang, J., Ed. Mlcrcelectfodes;VCH Publishers: New York, 1990. Wang, J.; Tuzhl. P.; Zadell, J. Anal. Chem. 1987, 59, 2119. Dordlck, J. S.; Marietta, M. A.; Klibanov. A. M. Bbtechnol. Bioeng. 1987, 30, 31.
RECEIVED for review June 3,1991. Accepted September 25, 1991. This work was supported by the U.S.Environmental Protection Agency (Grant No. CR-817936-010). L.A. acknowledges a fellowship from Fundac5o de Amparo 6 Pesquisa do Estado de SBo Paulo (FAPESP), Brazil.
