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Anal. Chem. 1990, 62,826-829
Highly Stable PhospholipidKholesterol Electrode Coatings for Amperometric Monitoring of Hydrophobic Substances in Flowing Streams Joseph Wang* and Ziling Lu Department of Chemistry, New Mexico State University, Las Cruces, New Mexico 88003
Composite electrode coatings based on a mixture of phosphatidykhollne and chdesteroi offer remarkable mechanical stabiltty under vlgorous hydrodynamic condttbns. The greatly improved stabllity (over singledomain phosphoiipld layers) is attained without compromising the attractive permselective response of iipld electrodes. The enhanced stability is attributed to changes in the fWy/packting associated with the presence of cholesterol. Due to its high stabllity, simpiiclty, and fast and permselective response, the coated electrode seems well-sulted for flow measvrements of hydrophobic compounds. Parameters affecting the film permeability and the amperometric response are explored in the presence of numerous solutes of biological and phamaceuticai significance. Prevention of surface fouling (in the presence of surfactants) and appikablilty to selective assays of urine samples are illustrated. Such controlled access to the surface, based on solute polarity, greatly enhances the power of electrochemical flow detectors.
INTRO DUCT1 0N Amperometric detection for flowing streams has proven to be a viable technique in many analytical problems demanding high sensitivity and selectivity ( I , 2). Further improvement in the performance of electrochemical detectors is highly desired to meet new challenges posed by clinical and environmental samples. One promising avenue to achieve such improvements is to design surface microstructures that meet specific detection needs (3,4 ) . Permselective coatings represent one direction by which modified electrodes can benefit flow analysis. This is accomplished by rejection from the surface of undesired, interfering species while allowing the transport of the analyte. The size or charge exclusion discriminative properties of polymeric coatings such as cellulose acetate ( 5 , 6), perfluorinated (7, 8 ) or polyester (9) ionomers, or poly(viny1pyridine) (10) have been exploited successfully for flow measurements. Hence, high specificity toward small solutes or oppositely charged ones has been achieved in both flow injection and liquid chromatography operations. The prevention of surface fouling has been documented in the presence of various surfactants. New membrane barriers, based on different discrimination mechanisms, should provide new levels of selectivity to amperometric detectors. This paper describes the properties, advantages, and utility of a glassy carbon thin-layer detector coated with a mixed lipid layer. Much research effort has been devoted recently to the development of electrode surfaces derivatized with hydrophobic materials (11-14). It has been shown that lipid coatings can prevent polar electroactive compounds from reaching the electrode surface but remain highly permeable to hydrophobic substances. Substantial improvements in the selectivity toward hydrophobic compounds have thus been demonstrated. Enhanced sensitivity has also been reported in connection with
the hydrophobic accumulation into the lipid layer. Such properties, that are very attractive for flow analysis, have been illustrated by using cast films of palmitic or stearic acids (13, 14) and particularly phosphatidylcholine (11, 12), for voltammetry under batch (static) conditions. A major obstacle to the development of lipid-based flow detectors has been the poor mechanical stability of the lipid layer. Such films rapidly lose their integrity under vigorous hydrodynamic conditions, which characterize amperometric detection. Several studies reported on difficulties in keeping lipid layers intact even under static conditions (11,IZ). Unlike short-term batch experiments, the long-term stability of the attached layer is a key factor for the performance of flow detectors. We have observed that a composite coating material, consisting of a mixture of phosphatidylcholine (PC) and cholesterol (CH), offers significantly higher mechanical stability than films of PC alone. It is well-known that doping of phospholipid membranes with steroids offers a useful means for internal modification of membrane parameters such as molecular packing or fluidity (15-18). In particular, the reduced water penetration and condensed packing thus obtained greatly enhance the mechanical strength. The present study illustrates that cast PC/CH coatings exhibits remarkable stability, while maintaining attractive permselective properties based on polarity. We wish to report on these observations, as well as on the features and analytical utility of PC/CHcoated amperometric detectors, in the following sections.
EXPERIMENTAL SECTION Apparatus. The flow injection system consisted of the carrier reservoir, a Rheodyne Model 7010 injection valve (10-pL sample loop), interconnecting Teflon tubings, and a glassy carbon thinlayer detector (Model TL-5, Bioanalytical Systems (BAS)). All potentials are reported vs a Ag/AgCl reference electrode (Model RE-1, BAS). Flow of the carrier solutions was maintained by gravity. Some experiments used a rotating disk electrode (Model DDI 15, Pine Instruments). Experiments were conducted with an EG&G PAR Model 364 polarographic analyzer in connection with a Houston Omniscribe chart recorder. Reagents. All solutions were prepared with double-distilled water. Ascorbic acid (Baker),cholesterol,L-a-phosphatidylcholine (Type XI-E), acetaminophen, cysteine, promethazine, uric acid, chlorpromazine, trimipramine, tyrosine, desipramine, and perphenazine (Sigma) were used without further purification. The supporting electrolyte/carrier solution was a 0.05 M phosphate buffer (pH 7.4). The urine sample was obtained from a healthy volunteer and diluted with the supporting electrolyte solution. Procedure. Before its modification the glassy carbon surface was hand-polished with a 0.05-pm alumina slurry, rinsed with double-distilled water and sonicated in a water bath for 2 min. The "mixed" PC/CH solution was prepared by adding the desired amount of CH (usually 12 or 18 mg) to 1mL of chloroform solution containing 10 mg of PC. The electrode was coated by placing (with a micropipet) 5 pL of the mixed PC/CH solution (to cover the active disk and its surroundings)on the electrode and allowing the solvent to evaporate. The resulting coating had thickness of about 2 wm. Ten microliters was employed for coverage of the rotating disk electrode. The solvent was allowed to evaporate in air for 5 min. Analogous experiments were peformed with
0003-2700/90/0362-0826$02SO/OG 1990 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 62, NO. 8, APRIL 15, 1990
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Figure 2. Current-time response for 2.5 X lo4 M acetaminophen (A) and 5 X M uric acid (B) at the lipid-coated and bare electrodes (solid and dotted lines, respectively): rotation speed, 1600 rpm. The composite layer contained 18 mg/mL CH. Other conditions are given in Figure 1.
TIME Figure 3. Detection peaks for ascorbic acid (a), tyrosine (b), promethazine (c), and trimipramine (d) at bare and PC/CH-coated electrodes (bottom and top, respectively). Conditions, are given in Figure
16.
(c), and trimipramine (d) at bare and PC/CH-coated electrodes (bottom and top, respectively). Unlike the nearly complete elimination of the ascorbic acid and tyrosine signals, large peaks are observed, at the coated electrode, for the hydrophobic drugs. A fast response to dynamic changes in the concentration of such substances is also observed. For example, the peak width (at 0.6Cm,) of trimipramine is 5.1 s, as compared to 4.6 s at the bare electrode. This rapid response indicates effective "wash out" of hydrophobic compounds from the lipid layer, as desired for use in flow systems. Figure 4 shows the dependence of the film permeability for 10 solutes of biological and pharmaceutical significance. The ratio between the current at the film-coated electrode over that at the bare one, im/ib, is used as a measure of the permeability. The permeability profile of the PC/CH film (A) is in excellent agreement with changes of the solute polarity. For example, effective discrimination (im/ib C 0.05) is observed against the polar species tyrosine, uric and ascorbic
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ANALYTICAL CHEMISTRY, VOL. 62. NO. 8, APRIL 15, 1990 I
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Permeability of PC/CH (A) and CH (6) films for different solutes: ascorbic acid (l),tyrosine (2), uric acid (3),acetaminophen (4), cysteine (5), desipramine (6), perphenazine (7), trimipramine (8), promethazine (9),and chlorpromazine(10). Conditions are as in Figure 16, except that an applied potential of +0.9 V was used for 1, 5, 6, and 8. Figure 4.
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COMPOUND Flgure 5. Permeabiliiy of PC/CH film containing dflerent levels of CH: 18 (a), 12 (b), and 6 (c) mg/mL. Solutes used were ascorbic acid (l), tyrosine (2), promethazine (3), and trimipramine (4). Conditions are
given in Figure 16.
acids, cysteine, and acetaminophen. In contrast, the composite film permits passage of the hydrophobic antidepressant and phenothiazine drugs ( i m / i b > 0.2). For most of these compounds the film coverage results with only modest attenuations of the response. The ability to measure selectively such drugs in the presence of polar interferences, indicated from this profile, will be illustrated in the following section. In addition to hydrophobicity, the permeability may be affected by electrostatic interactions (associated with the charge of the lipid layer). Also shown in Figure 4 is the corresponding permeability profile at the CH-coated electrode (B). This single-domain coating blocks access of d l 10 solutes ( i m / i b < 0.3), exhibiting only a slight preference toward hydrophobic substances. An analogous profile for the single-domain PC film is not shown because of the poor mechanical stability. This coating, however, exhibited higher permeability for all 10 compounds, with i m / i b ranging from 0.4 to 0.95 (as was indicated also from analogous cyclic voltammetric experiments). The quantity of cholesterol present in the composite film can affect its permselective response and mechanical properties. Figure 5 shows the dependence of the f i i permeability on the composition of the mixed PC/CH coating. The three compositions tested exhibit the desired permselective properties. Increased permeability is observed for the four solutes upon decreasing the CH content. As a result, only 92% rejection of the polar tyrosine and ascorbic acid species is observed by using the 6 mg/mL CH coating. Such behavior is attributed, in part, to changes in the film thickness. The 12 mg/mL CH film offers the best compromise between effective
I
TIME Flgure 6. Injection peaks at bare (top) and PC/CH-coated (bottom) electrodes: (A) (a) 5 X lo4 M Chlorpromazine, (b) same as part a but after addition of 5 X lo4 M uric acid; (6)(a) 1 X lo4 M promethazine, (b) same as part a but after addiiion of 1 X lo4 M cysteine; (C) (a) diluted (1:40) urine, (b) same as part a but after addition of 1 X lo-' M trimipramine. Applied potential (6,C) was +0.9 V. Other conditions are given in Figure 16. exclusion of polar compounds, sensitive detection of hydrophobic ones, and good mechanical stability. Similar to polymer-coated detectors (6, IO), the response of the lipid electrode yielded a negligible dependence on the flow rate. For example, the limiting current for 5 X lo4 M promethazine remained stable over the 0.7-5.0 mL/min range. Analogous rotating disk experiments (at rotating speeds ranging from 400 to 2500 rpm) exhibited a similar independence upon the convection rate. Such attractive behavior indicates that transport through the film becomes the major contributor to the total diffusional transport. The permeability profiles of Figures 4 and 5 and the different mechanical stabilities indicate that the composite PC/CH coating exhibits properties superior to those of the two components alone. Such improvements are attributed to changes in fluidity/ packing associated with the incorporation of sterols into phospholipid membranes. Such effects have been widely reported in the literature (15-18). For example, nuclear magnetic resonance studies with mixed phospholipid/CH systems indicated a strong interaction between the fatty acid chains of the phospholipid and CH (18). X-ray diffraction and capacitance measurements illustrated that the presence of CH decreases the depth of water penetration into phospholipid layers (15). Others reported on the condensation of a PC monolayer upon addition of CH (16). Such effects may account for the remarkable stabilization of the lipid layer observed in the present work. The additional separation step, performed in situ, at the electrode surface, can benefit flow measurements in many practical situations. For example, Figure 6 illustrates the potential of the permselective transport characteristics of PC/CH-coated glassy carbon detectors for selective amperometric detection in flow injection systems. With the bare electrode (top) it is not feasible to detect chlorpromazine (A) or promethazine (B) selectivity in the presence of uric acid or cysteine, respectively, because of the additive (oxidation) response. In contrast, the PC/CH-coated electrode (bottom) effectively excludes the polar uric acid and cysteine from reaching the surface; as a result, the phenothiazine drugs are selectively detected (compare peaks a and b). The analytical advantage and practical utility that accrue from such rejection of polar compounds are best illustrated in Figure 6C for the selective detection of trimipramine in a diluted urine sample. A nearly complete elimination of the contribution of endogeneous oxidizable constituents is observed at the coated electrode (a). As a result, quantitation of the hydrophobic
ANALYTICAL CHEMISTRY, VOL. 62, NO. 8, APRIL 15, 1990
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Figure 7. Current-time response for 2.5 X M promethazine at the lipid-coated (a) and bare (b) electrodes. Arrows indicate addition of 50 ppm gelatin. Other conditions are given in Figure 2.
drug in urine is possible (with no sample treatment) under flow-injection conditions (b). These flow injection data indicate that lipid coatings can add a new dimension of selectivity to amperometric detectors based on solute polarity. In addition to flow injection measurements, such discriminative properties may offer significant improvements for liquid chromatography detectors. Coverage of such detectors with a permeable lipid layer would permit "isolation" of hydrophobic solutes from coeluting ones. Complex chromatograms may thus be simplified without lowering the operating potential. Obviously the scope of such applications will be limited by the compatibility of the film integrity with the chromatographic mobile phase. We investigated also the utility of the PC/CH film as a protective layer, aimed a t minimizing electrode poisoning effects. Figure 7 shows current-time rotating disk data for promethzaine at PC/CH coated (a) and bare (b) electrodes. A rapid and significant loss of the activity of the bare electrode is observed in the presence of the analyte ( t = 0 to 6.5 min) or organic surfactant ( t = 6.5 to 12 min). The lipid electrode, in contrast, offers excellent resistance to surface fouling due to accumulation of reaction products (e.g. of oxidation of phenothiazine compounds) or adsorption of gelatin. Notice also the rapid response to the addition of the hydrophobic drug ( t = 0). The PC/CH-coated amperometric detector exhibits a well-defined concentration dependence and reproducible data. For example, a calibration plot constructed from repetitive injections of promethazine solutions of increasing concentration (2-14) X M exhibited a linear response over the entire range (slope, 9.9 nA/pM; intercept, 4.4 nA; correlation coefficient, 0.998; conditions as in Figure 1B). A detection limit of 1.4 X lo4 M promethazine (Le. 4.4 ng in the 1 0 - ~ L injected volume) was estimated based on the signal-to-noise characteristics ( S I N = 3) of the 2 X M promethazine peak. Analogous measurements of 2 X M chlorpromazine
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and trimipramine yielded detection limits of 1.7 x IO4 and 2.7 x lo4 M, respectively. Hence, the inherent sensitivity of amperometric detectors is not compromised by coverage with lipid layers (when hydrophobic substances are concerned). A series of 24 successive injections of a 2.5 x M promethazine solution was used to evaluate the precision of the response (conditions as in Figure 1B). The mean peak current found was 3.9 wA, with a range of 3.7-4.1 FA and a relative standard deviation of 3 70.Such precision compares favorably with that obtained for phenothiazine compounds at bare electrodes. In conclusion, the experiments described above illustrate that doping of PC film with CH imparts remarkable mechanical stability into phospholipid coatings. The high stability, under vigorous hydrodynamic conditions, allows use of lipid-coated electrodes for monitoring flowing streams. Such coverage of amperometric detectors with hydrophobic materials offers a new avenue for controlling the access to the surface, and hence greatly benefits flow measurements. The additional separation step, performed in situ at the detector surface, reduces further the attention necessary for sample pretreatment. Changes in packing/fluidity, associated with the incorporation of CH, particularly the decreased water penetration, appear to be responsible for the improved stability. Other steroids or lipid mixtrures may be suitable for this task. Additional advantages may be achieved by coupling the hydrophobic layer with other permselective films. These and related coatings may benefit other electrochemical and nonelectrochemical sensing schemes. Studies in these directions are in progress. Registry No. Carbon, 7440-44-0; cholesterol, 57-88-5; L-aphosphatidylcholine, 60-87-7; chlorpromazine, 50-53-3; trimipramine, 739-71-9; desipramine, 50-47-5; perphenazine, 58-39-9; ascrobic acid, 50-81-7;acetaminophen, 103-90-2;cysteine, 52-90-4; uric acid, 69-93-2; tyrosine, 60-18-4.
LITERATURE CITED (1) Stulik, K.; PacLkova. V. Nectroanalyfical Measurements in Flowing Liquids ; Ellis Horwood: Chichester, England, 1987. (2) Kissinger, P. T. Anal. Chem. 1977, 49, 447A. (3) Dong, S.;Wang, Y. Electroanalysis 1989, 1 , 97. (4) Zak, J.; Kuwana, T. J . Electroanal. Chem. 1983, 150, 645. (5) Sittampalam, G.; Wilson, G. S . Anal. Chem. 1983, 55, 1608. (6) Wang, J.; Hutchins, L. D. Anal. Chem. 1985, 5 7 , 1536. (7) Wang, J.; Tuzhi, P.; Golden, T. Anal. Chim. Acta 1987, 197, 129. (8) Kaaret, T. W.; Evans, D. H. Anal. Chem. 1988, 6 0 , 657. (9) Wang, J.; Golden, T. Anal. Chem. 1989, 61, 1397. (10) Wang, J.; Golden, T.; Tuzhi, P. Anal. Chem. 1987, 5 9 , 740. (11) Garcia, 0. J.; Quintela, P. A.; Kaifer, A. E. Anal. Chem. 1989, 6 1 , 979. (12) Chastel, 0.;Kauffmann, J. M.; Patriarche, G. J.; Christian, G. D. Anal. Chem. 1989, 61, 171. (13) Tanaka, K.; Tamamushi, R. J . Electroanal. Chem. Interfacial Elecfrochem. 1987, 236, 305. (14) Uchida, I.; Ishino, A.; Matsue, T.; Itaya, K. J . Electfoanal. Chem. Interfacial Electrochem. 1989, 266, 455. (15) Simon, S. A,; McIntosh, T. J.; Latorre, R. Science 1982, 216, 65. (16) Benz, R.; Cros, D. Biochim. Biophys. Acta 1978, 506, 265. (17) Yeagle, P. L.; Hutton, W. C.; Huang, C.; Martin, R. B. Biochemistfy 1977, 16, 4344. (18) Darke, A.; Finer, E. G.; Flook, A. G.: Phillips, M. C. J . Mol. Biol. 1972, 6 3 , 265.
RECEIVED for review November 2, 1989. Accepted January 16, 1990. This work was supported by the donors of the Petroleum Research Fund, administered by the American Chemical Society, and by the National Institutes of Health (Grant No. GM 30913-06).
