Amperometric Detection with

Anal. Chem. 1999, 71, 3901-3904

Integrated Electrophoresis Chips/Amperometric Detection with Sputtered Gold Working Electrodes Joseph Wang,* Baomin Tian, and Eskil Sahlin

Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces, New Mexico 88003

An on-chip electrochemical detector for micromachined capillary electrophoresis (CE) systems, based on sputtering a gold working electrode directly onto the capillary outlet, is described. The new on-chip detector preparation requires no microfabrication or alignment procedures nor a decoupling mechanism. The attractive performance of the integrated electrophoresis chips/amperometric detection was demonstrated for the anodic detection of neurotransmitters. The response for dopamine was linear from 20 to 200 µM, with a LOD of 1.0 µM and a sensitivity of 52 pA/µM. Such intimate coupling of capillary electrophoresis chips and electrochemical detection facilitates the realization of complete integrated microanalytical devices. Microfabricated fluidic devices, integrating the sample-handling processes and the measurement step onto microchip platforms, are of considerable recent interest.1,2, Particular attention has been given to micromachined capillary electrophoresis (CE) chips because of their fast and efficient separation capabilities.2,3 Currently, such electrophoresis chips rely primarily on laser-based fluorescence to achieve a sensitive detection. Yet, such detection requires a bulky and complex supporting instrumentation and is limited to compounds that fluoresce or are amenable to derivatization with a fluorophore. Surprisingly, little attention has been given to the integration of electrophoresis chips with electrochemical detection,4 despite the inherent miniaturization, remarkable sensitivity, compatibility with advanced microfabrication, and minimal cost and power requirements of electrochemical devices. Electrochemical detection has already proven to be extremely valuable for conventional capillary electrophoresis applications.5,6 Several detector designs, based primarily on end-capillary or offcapillary configurations, have been used in connection to various CE applications. Recently, Woolley et al.4 reported on capillary electrophoresis chips with integrated electrochemical detection, based on the photolithographic placement of the working electrode just outside the exit of the electrophoresis channel. * Corresponding author. Tel.: (505) 646-2140. Fax: (505) 646-6033. E-mail: [email protected]. (1) Kovacs, G. T.; Petersen, K.; Albin, M. Anal. Chem. 1996, 68, 407A. (2) Manz, A.; Harrison, J. D.; Verpoorte, E.; Widmer, H. M. In Advances in Chromatography; Brown, P., Grushka, E., Eds.; Marcel Dekker: New York, 1993; Vol. 33, p 1. (3) Effenhauser, C. S.; Manz, A.; Widmer, H. M. Anal. Chem. 1993, 65, 2637. (4) Woolley, A. T.; Lao, K.; Glazer, A. N.; Mathies, R. A. Anal. Chem. 1998, 70, 684. 10.1021/ac9904720 CCC: $18.00 Published on Web 08/04/1999

© 1999 American Chemical Society

Figure 1. Capillary electrophoretic system. (A) Glass microchip, (B) separation channel, (C) injection channel, (D) pipet tip for buffer reservoir, (E) pipet tip for sample reservoir, (F) pipet tip for sample waste reservoir, (G) Plexiglass body, (H) buffer reservoir, (I) sample reservoir, (J) sample waste reservoir, (K) detection reservoir, (L) Au working electrode, (M) working electrode contact, (N) insulator layer, (O) channel outlet, (P) counter electrode, (Q) reference electrode, (R) contacts to high-voltage electrodes. Chip and chip holder are separated for clarity. Dimensions are not in scale.

In this paper, we describe a simple and attractive alternative route for the on-chip integration of amperometric detection based on the sputtering of the working electrode directly onto the capillary outlet (Figure 1). Such a flexible avenue eliminates the need for photolithographic electrode microfabrication or for a careful capillary/electrode alignment, places the detector directly at the end of the separation capillary (with a negligible interference from the separation electric field), and is compatible with a wide range of metal electrode surfaces. An analogous deposition of metal film electrodes was reported recently in connection to conventional capillary electrophoresis separations.7 The attractive analytical performance of the new on-chip electrophoresis/ electrochemistry microsystem is reported in the following sections. EXPERIMENTAL SECTION Apparatus. A laboratory-built high-voltage power supply, with an adjustable voltage range between 0 and +3000 V, was used for the electrophoretic separations. Amperometric detection was performed with an Electrochemical Analyzer 621 (CH Instruments, Cordova, TN) which was connected to a Pentium 166 MHz (5) Curry, P.; Engstron-Silverman, C.; Ewing, A. G. Electroanalysis 1991, 3, 587. (6) Holland, L. A.; Lunte, S. M. Anal. Commun. 1998, 35, 1H. (7) Voegel, P. D.; Zhou, W.; Baldwin, R. P. Anal. Chem. 1997, 69, 951.

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L-3,4-dihydroxyphenylalanine (L-dopa)

Figure 2. Microscopic image of the sputtered gold electrode at the capillary outlet. Magnification, ×125.

computer with 32 MB RAM. To minimize the noise level, the cooling fan of the analyzer was disconnected. Data were collected without filtration. The glass microchannel chips, fabricated by combining photolithographic, wet-chemical etching and thermal bonding techniques, were purchased from Alberta Microelectronic Corp. (AMC, model MC-BF4-001, Edmonton, Canada). The chip was modified by AMC by eliminating the original waste reservoir for accommodating the end-capillary electrochemical detector. The layout of the glass chips is shown in Figure 1, along with a schematic of the homemade Plexiglas chip holder. The chips consisted of a l20 × 87 mm glass plate with a separation channel and a sample-injection channel. The separation channel (between the buffer and detection reservoirs) was 77 mm in length, while the injection channel (leading from the sample reservoir hole to the sample waste reservoir) was 10 mm in length. The intersection of the separation and sample injection channels was located 5 mm from the buffer reservoir, halfway between the sample and sample waste reservoirs, yielding a separation channel with an effective length of 72 mm. The channels had a maximum depth of 20 µm and a width of 50 µm (at their top). Pipet tips were placed in the holes of the buffer, sample, and sample waste reservoirs, as shown in Figure 1. A thin gold film, sputtered around the outlet of the separation channel, served as the working electrode of the amperometric detection (Figure 2). Sputtering on the chip surface was performed with a Denton Vacuum Desk-II, using an argon pressure of 50 mTorr and a current of 40 mA for 7 min, and resulted in a film thickness of about 200 nm. The channel outlet was not protected during the sputtering. Electrical contact of the gold electrode was achieved by gluing a lead with silver epoxy and covering it with an insulator layer (Ercon ink R-488C1), exposing a gold disk area of 0.78 mm2 (Figure 1L). The Plexiglas holder of the CE microchip is also shown in Figure 1. The holder consisted of a buffer reservoir, a sample reservoir, a sample waste reservoir, and a detection reservoir. A platinum wire was inserted into each of the reservoirs and could be connected to the high-voltage power supply. The detection reservoir also contained an Ag/AgCl wire reference electrode and an additional platinum wire as the counter electrode for the amperometric detection. The Ag/AgCl reference electrode was prepared by oxidizing a silver wire in 0.1 M hydrochloric acid. Microscopic images were obtained with an Olympus Video Microscope (MTV-3), connected to a Mitsubishi Color Video Printer (model CP-10U). Chemicals. 3,4-Dihydroxyphenethylamine (dopamine), 1-[3′,4′dihydroxyphenyl]-2-isopropylaminoethanol (isoproterenol), and 3902 Analytical Chemistry, Vol. 71, No. 17, September 1, 1999

were obtained from Sigma. 2-(4-Morpholino)ethanesulfonic acid hydrate (MES hydrate) was obtained from Lancaster. The hydrochloric acid was of reagent grade and was obtained from J. T. Baker. The electrophoresis buffer consisted of a MES buffer (25 mM, pH 5.1 or 6.5) and was filtered through a 0.45 µm filter (Gelman Acrodisc) prior to use. Stock solutions of the various analytes (10 mM) were prepared daily in 25 mM MES buffer (pH 6.5) in the case of dopamine and isoproterenol, and in 0.1 M hydrochloric acid for L-dopa, and were diluted in the electrophoresis buffer prior to use. Electrophoresis Procedures. All capillary zone electrophoretic separations were carried out in uncoated channels. For reducing the peak tailing, the channels were treated by filling them overnight with a 1% hydrochloric acid solution. Prior to use, the channels were flushed with deionized water. For a separation, the buffer, sample, and sample waste reservoirs in the chip holder, and the corresponding pipet tips on the microchannel chip, were filled with 250 µL of buffer, 200 µL of sample, and 200 µL of buffer solutions, respectively. The chip was then placed in the chip holder with the pipet tips pointing downward into the reservoirs in the chip holder. Finally, the detection waste reservoir was filled with buffer solution, and the reservoirs were connected to the high-voltage power supply. The sample loading was carried out in two ways. In both cases, the injection channel was filled initially (at the start of a series of repetitive runs) with the sample solution electrokinetically. A separation potential of 2000 V was then applied to the buffer reservoir (with the detection reservoir grounded) until a flat amperometric baseline was achieved. Subsequently, a potential of +500 V was applied for 1 s to the sample reservoir (with the detection reservoir grounded while the buffer and sample waste reservoirs were floating). Alternately, a potential of +2000 V was applied for 5 s to the sample reservoir (with the sample waste reservoir grounded and buffer and detection reservoirs floating). Because of the sample dispersion into the separation channel (during its loading),8 it is difficult to estimate the exact injection volume. Unless mentioned otherwise, the separation was performed by applying +2000 V to the buffer reservoir with the detection reservoir grounded. (Safety Considerations: To avoid electrical shock, the high-voltage power supply should be handled with extreme care.) Electrochemical Detection. Unless stated otherwise, a potential of +0.70 V (vs Ag/AgCl wire) was applied to the working electrode during the electrophoretic separations. The injections were performed after a baseline stabilization. The electropherograms were sampled with a time resolution of 0.1 s.

RESULTS AND DISCUSSION A primary advantage of the on-chip deposition of the electrochemical detector (compared with the design of ref 4) is the simplicity of the design and construction. The rapid sputtering protocol results in an extremely thin (∼200 nm) metal electrode film at the capillary outlet (Figure 2). Such on-chip metal deposition leads to an effective end-column detection and obviates the need for a separate electrode microfabrication effort or for a decoupling device. Such self-isolation from the high separation (8) Jacobson, S. C.; Hergenroder, R.; Koutny, L. B.; Warmack, R. J.; Ramsey, J. M. Anal. Chem. 1994, 66, 1107.

Figure 3. Electrophoretic separation of (A) dopamine, (B) isoproterenol, and (C) L-dopa on CE microchips with amperometric detection. Conditions: sample containing 100 µM dopamine, isoproterenol, and L-dopa; electrophoresis buffer, 25 mM MES buffer (pH 5.1); separation potential, 2000 V; sample loading by applying +500 V to the sample reservoir for 1 s (with the detection reservoir grounded); amperometric detection at +0.70 V.

Figure 5. Hydrodynamic voltammograms for (A) dopamine and (B) isoproterenol. Conditions: 100 µM dopamine and isoproterenol; 25 mM MES buffer (pH 6.5); sample loading, 2000 V for 5 s to the sample reservoir (with the sample waste reservoir grounded); other conditions as in Figure 3.

Figure 6. Electropherogram for (a) 40, (b) 80, (c) 120, (d) 160, and (e) 200 µM (A) dopamine and (B) isoproterenol. Conditions: 25 mM MES buffer (pH 6.5); sample loading, 2000 V for 5 s to the sample reservoir (with the sample waste reservoir grounded); other conditions as in Figure 3.

Figure 4. Influence of the separation voltage on the electropherograms. Separation performed at (a) 1000 V, (b) 1500 V, (c) 2000 V, (d) 2500 V, and (e) 3000 V. Conditions: 100 µM (A) dopamine and (B) isoproterenol, 25 mM MES buffer (pH 6.5); other conditions as in Figure 3.

potential is attributed to the dramatic drop of the potential across the capillary (to a negligible value at its outlet)6 and to the relatively large detection reservoir.9 The analytical performance of the on-chip CE/electrochemistry microsystem was characterized for the determination of catechols. Figure 3 displays an electropherogram obtained for an equimolar (100 µM) mixture of dopamine (A), isoproterenol (B), and L-dopa (C), recorded at an electric field strength of 260 V/cm (2000 V applied between the buffer and detector reservoirs). Well-defined and resolved peaks are observed for the three compounds. The assignment of the various peaks is based on single runs of each compound. Such defined response, coupled with the very low noise level, offers convenient quantitation of micromolar levels of catecholamine compounds. The very low noise level (50 pA peakto-peak) also indicates an effective isolation from the high separation potential (despite the absence of a decoupling mechanism). Such isolation is examined in Figure 4 which illustrates the effect of the separation potential upon the amperometric response and separation efficiency. As expected, increasing the separation

potential from 1000 to 3000 V (in 500 V increments, a-e) dramatically decreases the retention time for both dopamine (A) and isoproterenol (B), from 111 to 36 s and from 125 to 40 s, respectively. The peak width (at half-height) decreases from around 2.5 s at 1000 V to ∼1.0 s at 3000 V. The separation efficiency, represented by the plate number, corresponds to 13 000-14 600 for dopamine and 7400-10 100 for isoproterenol. The high electric fields result in larger amperometric signals (expected for the sharper peaks) and in similar background noise levels. Relatively flat baselines are also observed for the various separation potentials, with no dramatic change in the baseline slope between 1000 and 3000 V. An initial baseline decay/slope was observed over the first 10 s of the 3000 V separation (not shown). Most subsequent work thus employed a potential of 2000 V. No further attempt has been made to optimize the separation. Figure 5 depicts typical hydrodynamic voltammograms for the oxidation of 100 µM dopamine (A) and isoproterenol (B). The curves were developed pointwise by making 100 mV changes in the applied potential. Both compounds display similar currentpotential profiles, with defined waves, starting around +0.1 V and leveling off above +0.7 V. The half-wave potentials are +0.30 V (A) and +0.37 V (B). All subsequent amperometric work employed a potential of +0.7 V. Figure 6 displays electropherograms obtained at the CE chips (operated with a separation potential of 2000 V) for mixtures containing increasing levels of dopamine (A) and isoproterenol (B) in 40 µM steps (a-e). Well-defined and resolved peaks are observed for these micromolar concentrations. This series was a Analytical Chemistry, Vol. 71, No. 17, September 1, 1999

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part of a 10-point calibration experiment over the 20-200 µM range. The resulting calibration plots were linear over the entire range, with sensitivities of 52 (dopamine) and 44 (isoproternol) pA/µM and correlation coefficients of 0.995 and 0.991, respectively. The limit of detections (LOD) was determined to be 1.0 µM dopamine and 1.3 µM isoproterenol (S/N ) 2). A slightly higher LOD (3.7 µM dopamine) was reported for an on-chip microfabricated electrochemical detector,4 while similar LODs (0.6-1.2 µM dopamine) were obtained for the deposition of the working electrode onto conventional silica capillary tips.7 A detection limit of 0.4 µM dopamine was reported for conventional end-column amperometric detection based on placing a carbonfiber electrode at the end of a fused silica capillary.10 A series of 10 repetitive injection/separation cycles (of a 100 µM dopamine/ isoproterenol mixture) was used for estimating the precision. Mean peak currents of 8.82 and 8.70 nA were obtained for dopamine and isoproterenol, respectively, with relative standard deviations of 7.7 and 8.6%. CONCLUSIONS The data presented here clearly demonstrate that metallic working electrodes can be deposited directly on-chip to produce (9) Chen, M. C.; Huang, H. J. Anal. Chem. 1995, 67, 4010. (10) Sloss, S.; Ewing, A. G. Anal. Chem. 1993, 67, 577.

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effective end-column electrochemical detectors for micromachined CE systems. The on-chip integrated amperometric detection obviates the need for large optical systems, common to laser-based fluorescence detectors. Such a simple detector preparation route requires no microfabrication or alignment procedures, allows the sputtering of a wide range of metal films, and obviates the need for a decoupling mechanism. Current efforts in this laboratory are aimed at miniaturization of the other components (particularly the potentiostatic and computer circuitry) and on-chip integration of them, as well as sputtering the reference and counter electrodes, in a move toward the production of complete miniaturized devices. Because of their inherent miniaturization, low-power requirements, and attractive performance, this and other4 integrated electrochemical detectors are expected to increasingly find a role in microchip separations and other analytical microsystems. ACKNOWLEDGMENT This work was supported in part by Sandia NL. E.S. acknowledges financial support from The Swedish Foundation for International Cooperation in Research and Higher Education (STINT). Received for review May 4, 1999. Accepted June 22, 1999. AC9904720