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Microchip Capillary Electrophoresis with an Integrated Indium Tin Oxide Electrode-Based Electrochemiluminescence Detector Haibo Qiu, Jilin Yan, Xiuhua Sun, Jifeng Liu, Weidong Cao, Xiurong Yang,* and Erkang Wang*
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, China
This paper describes an indium tin oxide (ITO) electrodebased Ru(bpy)32+ electrochemiluminecence (ECL) detector for a microchip capillary electrophoresis (CE). The microchip CE-ECL system described in this article consists of a poly(dimethylsiloxane) (PDMS) layer containing separation and injection channels and an electrode plate with an ITO electrode fabricated by a photolithographic method. The PDMS layer was reversibly bound to the ITO electrode plate, which greatly simplified the alignment of the separation channel with the working electrode and enhanced the photon-capturing efficiency. In our study, the high separation electric field had no significant influence on the ECL detector, and decouplers for isolating the separation electric field were not needed in the microchip CE-ECL system. The ITO electrodes employed in the experiments displayed good durability and stability in the analytical procedures. Proline was selected to perform the microchip device with a limit of detection of 1.2 µM (S/N ) 3) and a linear range from 5 to 600 µM. Over the past decade, there has been considerable of interest in a miniaturized total analysis system (µ-TAS).1,2 Particularly, capillary electrophoresis (CE) in the microchip format has attracted great attention in the separation field. The advantages of microchip CE include high performance, shorter analysis time, portability, disposability, and consumption of minute sample and reagent.3,4 Although these virtues of microchip CE have been documented, UV detection, a standard detection mode in conventional CE,5,6 is not easily applied to microchip CE because of its lack of sensitivity. To satisfy these urgent needs, a wide variety of different detection modes have been employed to microchip CE.7-10 At present, laser-induced fluorescence, due to its high * Corresponding author. E-mail:
[email protected]. Phone: +86-431-5262003. Fax: +86-431-5689711. (1) Manz, A.; Graber, N.; Widmer, H. M. Sens. Actuators, B 1990, 1, 244-248. (2) Harrison, D. J.; Fluri, K.; Seiler, K.; Fan, Z.; Effenhauser, C. S.; Manz, A. Science 1993, 261, 895-897. (3) Reyes, D. R.; Iossifidis, D.; Auroux, P. A.; Manz, A. Anal. Chem. 2002, 74, 2623-2636. (4) Auroux, P. A.; Iossifidis, D.; Reyes, D. R.; Manz, A. Anal. Chem. 2002, 74, 2637-2652. (5) Culbertson, C. T.; Jorgenson, J. W. Anal. Chem. 1998, 70, 2629-2638. (6) Moring, S. E.; Reel, R. T. Anal. Chem. 1993, 65, 3454-3459. (7) Chiem, N.; Harrison, D. J. Anal. Chem. 1997, 69, 373-378. (8) Li, J.; Kelly, J. F.; Chernushevich, I.; Harrisom, D. J.; Thibault, P. Anal. Chem. 2000, 72, 599-609. 10.1021/ac034500x CCC: $25.00 Published on Web 09/18/2003
© 2003 American Chemical Society
sensitivity and ease of coupling to microchip, is particularly popular and commercially available. However, most of the analytes are not of high fluorescence efficiency or cannot even be made to fluoresce unfavorable derivatization of the samples with suitable fluorophores becomes necessary. Furthermore, only a small number of wavelengths can be selected for excitation. Recently, mass spectrometric (MS) detection has been initially employed to microchip CE. Although rich chemical information can be provided and micromolar limit of detection can be readily obtained, the high cost and large size of the instrument limit the use of MS coupling with miniaturized systems. Electrochemical (EC) detection10-16 is receiving increased attention because of its high sensitivity, simplicity, low cost, and ease of miniaturization. But EC detection is easily affected by the high separation electric field, and the interference results in a larger background current and a shifted redox potential. Palladium17 and platinum18 decouplers were fabricated by some groups to decouple the circuit of EC detection from the separation electric field to some extent. In addition, the contamination of the working electrode also influences the reproducibility and stability of the detection. Electrochemiluminescence (ECL) based on tris(2,2′-bipyridyl)ruthenium(II) first reported by Tokel and Bard19 has received considerable attention in chemical analysis because of its inherent sensitivity, selectivity, and wide linear range in the utility in conventional flowing systems such as HPLC,20-23 flow injection (9) Liu, B.; Ozaki, M.; Utsumi, Y.; Hattori, T.; Terabe, S. Anal. Chem. 2003, 75, 36-41. (10) Wolley, A. T.; Lao, K.; Glazer, A. N.; Mathies, R. A. Anal. Chem. 1998, 70, 684-688. (11) Wang, J.; Tian, B.; Sahlin, E. Anal. Chem. 1999, 71, 3901-3904. (12) Martin, R. S.; Gawron, A. J.; Lunte, S. M.; Henry, C. S. Anal. Chem. 2000, 72, 3196-3202. (13) Gavin, P. F.; Ewing, A. G. Anal. Chem. 1997, 69, 3838-3845. (14) Gawron, A. J.; Martin, R. S.; Lunte, S. M. Electrophoresis 2001, 22, 242248. (15) Wang, J.; Pumera, M.; Chatrathi, M. P.; Escarpa, A.; Konrad, R.; Griebel, A.; Dorner, W.; Lowe, H. Electrophoresis 2002, 23, 596-601. (16) Vandaveer IV, W. R.; Pasas, S. A.; Martin, R. S.; Lunte, S. M. Electrophoresis 2002, 23, 3667-3677. (17) Chen, D. C.; Hsu, F. L.; Zhan, D. Z.; Chen, C. H. Anal. Chem. 2001, 73, 758-762. (18) Wu, C. C.; Wu, R. G.; Huang, J. G.; Lin, Y. C.; Chang, H. C. Anal. Chem. 2003, 75, 947-952. (19) Tokel, N. E.; Bard, A. J. J. Am. Chem. Soc. 1972, 94, 2862-2863. (20) Lee, W. Y.; Nieman, T. A. J. Chromatogr., A 1994, 659, 111-118. (21) Noffsinger, J. B.; Danielson, N. D. J. Chromatogr. 1987, 387, 520-524. (22) Lee, W. Y.; Nieman, T. A. Anal. Chem. 1996, 68, 1530-1535. (23) Uchikvra, K.; Kirisawa, M. Anal. Sci. 1991, 7, 971-973.
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analysis,24-26 and CE.27-34 A variety of substances,35,36 such as alkylamines, amino acids, oxalate, NADH, and various antihistamine drugs, can be detected without derivatization. ECL is the production of light by an oxidation or reduction reaction at the electrode surface. Chemiluminescence (CL), however, is the emission of light as a result of chemical reaction by mixing the analytes with some necessary reagents. Therefore, ECL offers the ability to generate luminescence in a defined location on the surface of the electrode and control the reaction by alterations of the applied potential. Recently, CL detection coupled with microchip CE has been reported by a few groups.9,37,38 Mangru and Harrison37 first demonstrated CL detection for microchip CE to monitor horseradish peroxidase (HRP) and fluorescein-conjugated HRP. Liu et al.9 demonstrated CL detection of dansylphenylalanine enantiomers and metal ions with a microchip CE system fabricated in poly(dimethylsiloxane) (PDMS). Comparatively, the application of Ru(bpy)32+ ECL detection in microchip CE is very limited.39,40 Manz and co-workers39 described a microfluidic system with a U-shaped floating platinum electrode ECL detector and detected amino acids with indirect mode. Although the original design was apparently effective and simple, this approach was not readily available for normal analysis. The fabrication of the chip and electrode was complex and costly. Furthermore, the adhesion of ECL reagents such as Ru(bpy)32+ in the flowing stream at the electrode or the walls of the separation channel was unavoidable, which could affect the EOF27 and possibly resulted in the slow response in indirect determination of amino acids using this system.39 Huang et al.40 reported a low-cost miniaturized CE system developed on a chip platform with flow injection sample introduction and ECL detection by a platinum wire serving as the working electrode fixed and aligned with the separation capillary. In their work, an optical fiber was introduced to conduct ECL signals, which made the operation process (optimization of relative positions of capillary outlet, working electrode, and optical fiber) complex and time-consuming. More recently, a novel microfluidics-based sensing system that relied on EC detection and ECL reporting was reported by Crooks and co-workers.41 In their work, the ECL reporting reaction was chemically decoupled from the electrochemical sensing reaction. Similarly, two-channel42 and (24) Noffsinger, J. B.; Danielson, N. D. Anal. Chem. 1987, 59, 865-868. (25) Greenway, G. M.; Knight, A. W.; Knight, P. J. Analyst 1995, 120, 25492552. (26) Brune, S. N.; Bobbit, D. R. Anal. Chem. 1992, 64, 166-170. (27) Wang, X.; Bobbit, D. R. Anal. Chim. Acta 1999, 383, 213-220. (28) Wang, X.; Bobbit, D. R. Talanta 2000, 53, 337-345. (29) Tsukagoshi, K.; Miyamoto, K.; Saiko, E.; Nakajima, R.; Hara, T.; Fujinaga, K. Anal. Sci. 1997, 13, 639-642. (30) Chiang, M.; Whang, C. J. Chromatogr., A 2001, 934, 59-66. (31) Liu, J.; Cao, W.; Qiu, H.; Sun, X.; Yang, X.; Wang, E. Clin. Chem. 2002, 48, 1049-1058. (32) Cao, W.; Liu, J.; Qiu, H.; Yang, X.; Wang, E. Electroanalysis 2002, 14, 15711576. (33) Liu, J.; Cao, W.; Yang, X.; Wang, E. Talanta 2003, 59, 453-459. (34) Cao, W.; Liu, J.; Yang, X.; Wang, E. Electrophoresis 2002, 21, 3683-3691. (35) Lee, W. Y. Mikrochim. Acta 1997, 127, 19-39. (36) Knight, A. W. Trends Anal. Chem. 1999, 18, 47-62. (37) Mangru, S. D.; Harrison, D. J. Electrophoresis 1998, 68, 2301-2307. (38) Hashimoto, M. H.; Tsukagoshi, K.; Nakajima, R.; Kondo, K.; Arai, A. J. Chromatogr., A 2000, 867, 271-279. (39) Arora, A.; Eijkel, J. C. T.; Morf, W. E.; Manz, A. Anal. Chem. 2001, 73, 3282-3288. (40) Huang, X.; Wang, S.; Fang, Z. Anal. Chim. Acta 2002, 456, 167-175. (41) Zhan, W.; Alvarez, J.; Crooks, R. M. J. Am. Chem. Soc. 2002, 124, 1326513270.
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multichannel43 microfluidic sensors based on this principle were also described in their later work. In the initial studies, most of microchip CE devices were fabricated from glass and quartz because of the mature micromachining technology of these materials, but their high cost and complicated fabrication procedures led to the investigation of alternate materials such as plastics and polymers. A wide range of polymer materials, such as polycarbonate44 and poly(methyl methacrylate),15 have been evaluated for the fabrication of microchips. In particular, PDMS, as one of these polymer materials, has been well studied and reviewed by Whitesides and co-workers.45,46 Effenhauser47 first reported the microchip CE device that combined a PDMS layer and a glass substrate. The advantages of PDMS are that they are less expensive, rapidly prototyping complex devices and are able to reversibly/irreversibly bind to smooth surfaces. In addition, the fabrication procedures based on rapid prototyping and replica molding are accessible to a general laboratory without ultraclean conditions.48 In this paper, we describe an integrated indium tin oxide (ITO) electrode-based Ru(bpy)32+ ECL detector for a PDMS microchip CE device. The microchip CE-ECL system utilizes an ITO-coated glass slide as the chip substrate with a photolithographically fabricated ITO electrode located at the end of the separation channel. Although the microchip components described in our experiments have some commonality with the work reported by Crooks and co-workers,41-43 the detection principle and the management of the flowing system are different. Proline and tripropylamine (TPA) are selected as model analytes to perform this microchip CE-ECL system in the experiments. EXPERIMENTAL SECTION Chemicals. Tris(2,2′-bipyridyl)ruthenium(II) chloride hexahydrate and TPA were purchased from Aldrich (Milwaukee, WI). Sylgard 184 silicone elastomer and curing agent were obtained from Dow Corning (Midland, MI). RZJ-390 photoresist (for TN/ STN ITO) was purchased from Suzhou Ruihong Electronic Chemicals Co., Ltd. (Suzhou, China). Proline was obtained from Shanghai Biochemical Co. Other reagents and chemicals were at least analytical reagent grade. ITO-coated (150 nm thick and resistance of
