Anal. Chem. 1993, 65, 513-518
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Affinity Biosensors Based on Preconcentration/Voltammetric Analysis. Detection of Phenothiazine Drugs at Langmuir-Blodgett Films of Tyrosine Hydroxylase Joseph Wang' and Yuehe Lin Department of Chemistry, New Mexico State University, Las Cruces, New Mexico 88003
Arkadi V. Eremenko and Ilya N. Kurochkin Research Center of Molecular Diagnostics & Therapy, Simpheropolysky Boulevard 8, 113149 Moscow, Russia
Maya F. Mineyeva Institute of Pharmacology, Academy of Medical Sciences of Russia, 125315 Moscow, Russia
A new route for operatlng afflnlty biosensor8 based on the vdtammetrk monltorlng of the accumulated guest (analyte) k dercrlbed. Hlgh unrltlvlty and sdectlvlty accrue from the and the coupling of the spoclflc receptor blndlng pr-88 InherontsedtMtyof theprocancentratkn/pottammetrk scheme. The redox (measurement) process results In dkroclatlon of the receptor-gued complex, thus allowlng multlpk analytkal determlnatlons. The receptor layer a b serves a8 an effective barrkr that exdudes Intetferlngrp.ch8. The new concept of preconcentratlon/voltammotrlcafflnlty Mownsors k Ilhistrated In connectlon wlth the detection of phonothlazlne drug6 udng Langmulr-Blodgett f l h of thelr receptor, the enzyme tyroslne hydroxylase. The effect of varlow exp&nental varlablw upon the sensor performance Is dercrlbed.
INTRODUCTION A relatively new area in the biosensor field is the coupling of receptors to transducers to form molecular recognition elementa.112 Different signal transductions can be used to follow the interaction between the host receptor and the guest target analyte. Most commonly,the specific binding triggers specific changes, such as ion transport across a membrane or a sudden change of the membrane potential.3 Intact chemoreceptors, isolated receptors, or synthetic hosta have thus been employed for the detection of different stimulants. Receptor binding, unlike antigen-antibody interactions, is usually specific to classes of substances rather than individual ones. The present article describes a new and novel voltammetric approach to follow the host-guest interaction. In this approach, the receptor binding process serves as an in situ preconcentration step of the target analyte. Subsequently, the accumulated analyte is being quantified (and removed) by applying a suitable voltammetric waveform. Such unique use of receptor electrodes as preconcentrating surfaces couples the high selectivity of the receptor recognition event with the inherent sensitivity of preconcentration/voltammetric schemea. As such, this concept representa a powerful addition to the field of preconcentrating modified voltammetric electrodes (1)Thompson, M.; Krull, U. J. Anal. Chem. 1991,63,393A. (2)Buch, R. M.; Rechnitz, G. A. Anal. Chem. 1989,61,533A. (3)Odashima, K.; Sugawara, M.; Umezawa, Y. Trends And. Chem. 1991,IO, 207.
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(commonlyrelying on electrostatic and coordination effect# and holds great promise for many practical biosensing applications. The new approach is illustrated in the following sections within the framework of voltammetric measurements of phenothiazine neuroleptic drugs and Langmuir-Blodgett (LB) films of their receptor, the enzymetyrosine hydroxylase. It could be extended to the biosensing of numerous bioanalytee by the appropriate choice of the receptor layer.
EXPERIMENTAL SECTION Apparatus. A 10-mL voltammetric cell (Model VC-2, Bioanalytical Systems (BAS))was used. The cell was joined to the gold working electrode (2-mm diameter, Model MF2014, BAS), reference electrode (Ag-AgC1, Model RE-1, BAS), and platinum wire auxiliary electrode through holes in its Teflon cover. Cyclic voltammetrywas performedwith a CV-27voltammograph(BAS) and recorded with a BAS Model RXY recorder. Differentialpulse-voltammetricdata were recorded with an EG&G Princeton Applied Research (PAR)Model 264A voltammetric analyzer and an BAS Model RXY recorder. A homemade LB system was used in connection with a Wilhelmy f i i balance. Reagents. All solutions were prepared with double-distilled water. Ascorbic acid (Baker), promethazine, uric acid, chlorpromazine, trimipramine, imipramine, perphenazine, and thioridazine (Sigma)were used as received. The supporting electrolyte was a 0.1 M phosphate buffer (pH 7.4). Tyrosine hydroxylase (TH, EC 1.14.16.2) from rat hypothalamus (male albino mongrel rata, 180-200 g) was obtained by affinity chromatography on diodine-tyronine sepharose 4B, as was described earlier.5 Preparation of the Sensor. LB films from TH were prepared (at room temperature)by spreading0.5 mL of the enzyme solution (1mg/mL in phosphate buffer, pH 7.0) on the liquid subphase of the same buffer (30 mL) in the LB trough (working area, 100 cm2). After 60 min the TH monolayer was compressed by moving the mobile barrier at 10 cm2/min. Figure 1 shows the surface pressure-molecular area (PA) isotherm for TH using the above conditions. Acondensed monolayer is formed at surfacepressures up to 25 mN/m, with a collapse (destruction) of the monomolecular film at higher pressures. All subsequent work employed a constant surface pressure of 20 mN/m. The compressed film was transferred onto the surface of a polished gold electrode using the Langmuir-Schafer method.e According to this scheme, (4) Wang, J. In Electroanalytical Chemistry, Bard, A. J., Ed.; Marcel Dekker: New York, 1989, Vol. 16, pp 1-88. (5)Mineyeva, M. F.; Kudrin, V. S.; Shemanov, A. Y. In Drug Dependence and Emotional Behavior, Valdman, A. V., Ed.; Consultant Bureau: New York, 1986, pp 303-325. (6) Eremenko, A. V.; Mineyeva, M. F.; Chernov, S. F.; Kurochkin, 1. N.; Kuznetaova, Valdman, A. V. Bull. Exp. Biol. Med. (Engl. Transl.) 1991,103,850.
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 5, MARCH 1, 1993
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the gold support touched the TH film in parallel to the surface of the liquid subphaseand was lifted together with the adsorbed film. The buildup of multilayer8of TH involved a 10-8air drying after the uptake of each enzyme layer. Multilayer structures, consisting of 5-20 TH monolayers, were used. Procedure. The electrochemicalsensingprocedure relied on the preconcentration/voltammetric scheme. The affinity sensor was placed in the 10-mLsolution of the phenothiazine drug. The preconcentration proceeded at open circuit, while stirring the drug solution (at 300 rpm) for 5 min. Following this period, the stirring was stopped and the initial potential was applied. After a 15-8rest period a differential-pulsescan in the positive direction was initiated, with the simultaneous recording of the voltammogram. The electrode was cleaned in the same solution by applying a potantial of 0.75 V for 1min, while the solution was stirred. Such a constant potential facilitatedthe removal of drugs from the TH layer, as was indicated by the subsequent differential-pulsevoltammogram (recordedin same solution or in the blank solution). The electrode was then ready for use in the next measurement cycle. Experiments were performed at room temperature. After each experiment, the electrode was kept in phosphate buffer solution at 4 "C.
RESULTS AND DISCUSSION The new concept of preconcentrationlvoltammetricaffiity biosensors will be illustrated in connection with the detection of phenothiazineneuroleptics. The preconcentrating surfaces have been created by depositing LB films of the enzyme tyrosine hydroxylase (TH) on a gold electrode. TH is a neurospecific enzyme, that plays an important role in psychotic diseases.5 Neuroleptic drugs are ligands of TH, and their strong binding to the enzyme eliminates the substrate inhibition.5 We have demonstrated recently that thin f i e of TH can be deposited onto a gold surface using the Langmuir-Schafer method.6 This LB technology allows convenient creation and deposition of oriented protein films with a high density of receptor molecules. The selective and strong TH-neuroleptic interaction is coupled in the following sections with the inherent sensitivity of preconcentrationl voltammetric procedures to offer an attractive biosensing of
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Cyclic voltammograms for several compounds obtained, at
the bare (A) and TKcoeted (E) goid electrodes, after 0 and 5 mln of stining (solid and dotted lines, respectively). Analytes: (a) 1 X lo4 M chlorpromazine; (b) 1 X lo4 M thlorldarlne; (c) 1 X lo4 M
trhnlpramlne;(d) 2 X 10-4 M ascorbic a c e (e) 2 X lo4 M uric acid. Conditions: scan rate, 20 mV/s; supporth~electrolyte, 0.05 M phosphate buffer (pH 7.4); TH coatlng (E), 20 monolayers. phenothiazine compounds. Additional gains in the selectivity accrue from the exclusion of interfering species and the characteristic redox behavior (E,) of the target drug. Other nonspecific preconcentrationlvoltammetric schemes for measuring phenothiazine drugs, based on adsorptive and hydrophobic accumulationsat ordinary and lipid-coatedelectrodes, have been reported.7~8 Figure 2 displays cyclic voltammograms obtained with the plain (A) and TH-coated (B) electrodes that had been immersed for 5 min in stirred solutions of several compounds of pharmaceutical and biological significance. The corresponding response without a stirring period is also shown (as solid lines). Note that none of these oxidizable compounds accumulateonto the bare gold surface. In contrast, the affinity sensor exhibits larger peaks for the chlorpromazine (a) and thioridazine (b) neuroleptice, indicating their effective uptake by the immobilized receptor. The well-developed voltammograms indicate that the redox activity of the drugs is retained upon binding to the receptor. (Notice also the enhanced signals at the TH electrode even without an accumulation period.) The other compounds (which are not ligands of TH), including the tricyclic antidepressant trimipramine (c), ascorbic acid (d), and uric acid (e), are not ''collectedWby the receptor fii. Indeed, comparison to the response of the plain electrode indicates that the TH layer forma an effective barrier for the transport of these compounds. Hence, the data of Figure 2 illustrate that the TH binding process serves to enhance both the sensitivity and selectivityof the voltammetric sensing. Analogous adsorptive and hydrophobic preconcentration schemes are not specific and cannot discriminate against the interfacial accumulation of other nonpolar compounds, such as tricyclic antidepressants. The LB technology offers the advantage of tailoring the sensitivity of affinity sensors via control of the number of deposited fiis. Figure 3 (dotted line) shows cyclic voltammograms for 1x lo-' M perphenazine, recorded following (7) Wang, J.; Freiha, B. A.; Deshmukh, B. K. Anal. Chen. 1985,14, 457. (8) Khodari, M.; Kauffmann, J. M.; Patriarche, G. J.; Ghandour, M. A. Electroanalysis 1989, I , 501.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 5, MARCH 1, 1993
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5 min of stirring, using sensors with different numbers of TH layers (0-20, a-e). The corresponding response without accumulation is shown as a solid line. Significant current enhancements are observed upon increasing the number of enzyme films (i.e. receptor binding sites). For example, a 5-fold increase in the sensitivity is observed at the 20-layer sensor. Irreversible binding often prevents the reuse of affinity biosensors. An important aspect of the new voltammetric affinity sensor is its suitability for multiple analytical determinations. Such renewable character results from the fact that the stability of the association is strongly dependent on the redox state of the guest analyte. The redox process (Le. the measurement step) thus results in dissociation of the receptordrug complex, with the immobilized receptor remaining intact, so that the sensor is ready to be used (with no memory effect). Accordingly, the preconcentrationl voltammetric operation of the affiiity biosensor can be represented by the following reaction scheme: R
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where R and A are the receptor and analyte, respectively. The ability to reproducibly renew the binding capacity is illustrated in Figure 4. The six preconcentrationlvoltammetric cycles for 1 X 10-5 M perphenazine result in reproducible peak currents (RSD = 2.3 % ;mean = 2.88 p& range = 2.81-2.89 FA). The small peaks, observed on the second sweep (dotted lines), correspond to the oxidation of the solution-phasedrug. No peaks were observedwhen the second sweep was carried out in the blank solution (not shown), indicating a complete removal of the analyte from the surface. Such "cleaning" was facilitated by holding the electrode at the final potential for 1min. The data of Figure 4 indicate also that the background current is not affected by the multiple drug determinations and that there is no drift in the
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TIME (min ) Figure 5. Dependence of the peak current on the preconcentratkm time for 2 X M chlorpromazine (a) and perphenazlne (b). Condbns: scan rate, 10 mV/s. Other condmons were as In Flgure 28.
binding activity. Indeed, the same sensor functioned in the normal fashion for 4-5 days with no apparent loss in the binding activity. There was only a 10% decrease in the activity over a period of 20 days, with a faster decrease during longer periods. Apparently, an analytically useful response is observed as long as an "analyte-free"surface is regenerated and the LB layer is mechanically stable. The LB technology offers also a reproducible casting of the receptor layer (with film-to-film reproducibility of ca. 5%). Figure 5 shows the dependence of the peak current on the accumulation time for chlorpromazine (a) and perphenazine (b). As can be seen from these data, the incorporation of both drugs proceeds rapidly. As the accumulation time increases, the response rises rapidly at first and then more slowly. Apparently, the binding reaches equilibrium within 8min. Profiles of similar shape (reflecting the kinetics of the binding process), but with different current responses, are expected for different numbers of TH monolayers. We have shown that voltammetry can be used to follow receptordrug interactions. In order for the receptor binding
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 5, MARCH 1, 1993
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Figure 6. (A) Voltammograms obtained after increasing the perphenazlne (a) and chlorpromazlne (b) concentration in 2 X M steps. Dotted lines represent the response of the blank soiutlon. (B) Dependence of the peak current on the concentratlon of promethazine (a), perphenazlne (b), thlorldazine (c)and chlorpromazine(d). Conditions were as in Figure 4.
process to possess significant analytical utility, it must exhibit a well-defined concentrationdependence. Figure 6A displays differential-pulse voltammograms obtained after successive standard additions of perphenazine (a) and chlorpromazine (b), each effecting a 2 X 104 M increase in concentration. The coupling of in situ preconcentration and a sensitive differential-pulse waveform offers convenient quantitation of micromolar concentrations. Also shown in Figure 6B are the resulting calibration plots for several phenothiazine drugs. For promethazine (a) and perphenazine (b),the peak current increases linearly with concentration over the entire range examined (sensitivities: 1-01and 2.35 pA/pM, respectively). Thioridazine (c) and chlorpromazine(d) exhibit a curvature in the response at concentrations higher than 8 X lo+ M (slopes of the linear portion, 3.15 and 3.99 pA/pM, respectively). Apparently, the trend in sensitivity reflecta the affinity order chlorpromazine > thioridazine > perphenazine > promethazine. The receptor binding event offers a high degree of selectivity. In particular, the voltammetric sensing of the accumulated drug can be performed in the presence of macro solution constituents with similar redox properties. For example, Figure 7 illustrates the quantitation of perphenazine in solutions containing an excess of ascorbic acid (a) and uric acid (b). When the measurement is performed at the plain
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Figure 7. Cyclic voltammograms for 2 X lo-' M ascorbic ecM (a) and uric acM (b) in the absence and presence of 1 X lo4 M perphenazlne (dotted and solid Ilnes,respectiVely)at bare (A) and TKcoeted (6)gold electrodes. Conditions: scan rate, 10 mV/s. Other COnditlonS were as In Figure 2. gold electrode (A), the phenothiazine peak is obscured by the overlapping acid peaks and its quantitation is not feasible. In contrast, the TH film (B)excludes these acids from the surface, while enhancing the phenothiazine response, hence allowing a convenient quantitation of the drug. Analogous adsorptive stripping measurements of phenothiazine drugs require a medium-exchange step to discriminate against solution-phase electroactive species.' In conclusion, we have demonstrated a new electrochemical transduction scheme for monitoringreceptor-ligand binding events, based on voltammetric measurements of the bound analyte. The sensitivity and selectivity of the voltammetric sensing scheme are both enhanced by using the receptor accumulationapproach. Additional advantages accrue from the reproducible LB creation of the receptor layers, the exclusion of interfering species, and self-cleaning capability. Such performance characteristics have obvious implications on voltammetric bioassays of body fluids. In addition to the bioanalytical utility, the new affinity biosensor should be a useful tool for direct (in situ) probing of receptor-drug interactions. This work further illustrates the prospect of applying the Langmuir technology to the design of biosensors. While the new concept is presented in terms of biosensing of phenothiazine drugs, it could be extended to many other bioanalytes through the judicious choice of the receptor.
RECEIVED for review July 28, 1992. Accepted November 24, 1992.
