Chemiluminescence Detection for a Microchip Capillary

Application of a fixed electrode offers a fully integrated18 and disposable16 .... a novel wireless electrochemiluminescence (ECL) detector for. MCE w...
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Anal. Chem. 2003, 75, 36-41

Chemiluminescence Detection for a Microchip Capillary Electrophoresis System Fabricated in Poly(dimethylsiloxane) Bi-Feng Liu,*,† Motoaki Ozaki,† Yuichi Utsumi,‡ Tadashi Hattori,‡ and Shigeru Terabe†

Graduate School of Science and Laboratory of Advanced Science and Technology for Industry, Himeji Institute of Technology, Kamigori, Hyogo, 678-1297, Japan

Chemiluminescence (CL) detection integrated with a microchip capillary electrophoresis (MCE) system that was fabricated in poly(dimethylsiloxane) was demonstrated for chemical and biochemical analyses. Two model CL systems were involved here: metal ion-catalyzed luminol-peroxide reaction and dansyl species conjugated peroxalate-peroxide reaction. Different strategies based on three chip patterns (cross, cross combining with Y, and cross combining with V) to perform on-line CL detection for MCE were evaluated and compared in terms of sensitivity, reproducibility, and peak symmetry. The chip pattern of cross combining with Y proved to be promising for the luminol-peroxide CL system, while the chip pattern of cross combining with V was preferred for the peroxalate-peroxide system where CL reagent could not be effectively transported by electroosmotic flow. A detection limit down to submicromolar concentrations (midattomole) was achieved with good reproducibility and symmetric peak shape. Successful separation of three metal cations such as Cr(III), Co(II), and Cu(II) and chiral recognition of dansyl phenylalanine enantiomers within 1 min revealed distinct advantages of combining MCE with CL detection for rapid and sensitive analyses. The past decade has witnessed rapid progress toward a miniaturized total analysis system (µTAS),1-4 referred to as labon-a-chip. It prompts a profound revolution in the conception of an instrument, based on which microchip capillary electrophoresis (MCE) is currently experiencing a great leap from academic success to commercialization since its introduction by Manz et al.1 in 1990. Due to its high performance, high speed, low sample, and reagent requirement as well as integration ability and compactness, MCE currently has been widely used to perform * Corresponding author. E-mail: [email protected]. Tel: +81-79158-0171. Fax: +81-791-58-0493. † Graduate School of Science. ‡ Laboratory of Advanced Science and Technology for Industry. (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.

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chemical and biochemical analyses5,6 and also has been recognized as a powerful tool for genomics,7 proteomics,8 and metabolomics.9 However, the extremely small sample size (usually at the picoliter level) makes it a challenge to achieve high detection sensitivity. Common detection schemes for MCE are laser-induced fluorescence (LIF)10 and mass spectroscopy (MS).11 Although submicromolar detectability can be readily obtained with these two detection methods, the high cost and large size of the instrument are quite incompatible with the concept of µTAS. Consequently, development of alternative detectors that can be directly microfabricated on a chip has attracted a great of interest. For instance, electrochemical detection (ED)12-19 has achieved a dramatic advance in recent years because of its high sensitivity, simplicity, and low cost. Many ED formats have been proposed on glass17,18 or polymer chips,15,16 with fixed16,18 or replaceable17 electrodes. Application of a fixed electrode offers a fully integrated18 and disposable16 potential while a replaceable one can be rapidly changed to meet the needs of different experiments.17 Like LIF, a limitation of ED is that a derivatization procedure is usually required for compounds without electroactivity. Chemiluminescence (CL) detection, emerging as a very sensitive mode of detection, has many advantages that have been (5) Dolnı´k, V.; Liu, S.; Jovanovich, S. Electrophoresis 2002, 21, 41-54. (6) Khandurina, J.; Guttman, A. J. Chromatogr., A 2002, 943, 159-183. (7) Medintz, I. L.; Paegel, B. M.; Blazej, R. G.; Emrich, C. A.; Berti, L.; Scherer, J. R.; Mathies, R. A. Electrophoresis 2001, 22, 3845-3856. (8) Figeys, D.; Pinto, D. Electrophoresis 2001, 22, 208-216. (9) Ramseier, A.; vonHeeren, F.; Thormann, W. Electrophoresis 1998, 19, 29672975. (10) Jacobson, S. C.; Hergenro ¨der, R.; Moore, A. W.; Ramsey, J. M. Anal. Chem. 1994, 66, 18-22. (11) Li, J.; Kelly, J. F.; Chernushevich, I.; Harrisom, D. J.; Thibault, P. Anal. Chem. 2000, 72, 599-609. (12) Gavin, P. F.; Ewing, A. J. Am. Chem. Soc. 1996, 118, 8932-8936. (13) Wolley, A. T.; Lao, K.; Glazer, A. N.; Mathies, R. A. Anal. Chem. 998, 70, 684-688. (14) Wang, J.; Tian, B.; Sahlin, E. Anal. Chem. 2000, 72, 3196-3202. (15) Martin, R. S.; Gawron, A. J.; Lunte, S. M.; Herry, C. S. Anal. Chem. 2002, 74, 3670-3697. (16) Wang, J.; Pumera, M.; Chatrashi, M. P.; Escarpa, A.; Konrad, R.; Griebel, A.; Do ¨rner, W.; Lo ¨ne, H. Electrophoresis 2002, 23, 596-601. (17) Zeng, Y.; Chen, H.; Pang, D. W.; Cheng, J. K. Anal. Chem. 2002, 74, 24412445. (18) Baldwin, R. P.; Roussel, T. J., Jr.; Crain, M. M.; Bathlagunda, V.; Jackson, D. J.; Gullapalli, J.; Conklin, J. A.; Pai, R.; Naber, J. F.; Walsh, K. M.; Keynton, R. S. Anal. Chem. 2002, 74, 3690-3697. (19) Backofen, U.; Matysik, F. M.; Lunte, C. E. Anal. Chem. 1999, 71, 39013904. 10.1021/ac026096s CCC: $25.00

© 2003 American Chemical Society Published on Web 11/28/2002

demonstrated in conventional capillary electrophoresis (CE):20-27 (i) High detection sensitivity with a detection limit down to the single-molecule26 level can be accomplished in CE, which is comparable with that achieved using LIF.28 (ii) Wide linear range of responding signal will be beneficial to analyte quantitation. (iii) No light source is required, which makes instrument configuration very simple. (iv) Many CL systems have been well characterized for a variety of species from the beginning of the last century. Although CL detection method is very promising, as mentioned above, little attention has been paid to its application in MCE. Mangru and Harrison29 first demonstrated excellent work on CL detection for MCE to monitor horseradish peroxidase (HRP) and fluorescein-conjugated HRP using a luminol CL system. A postseparation reactor for CL was designed in the shape of a “Y”. Hashimoto et al.30 recently presented a contribution on an endchannel reactor to achieve CL detection for dansyl amino acids using a peroxalate CL system. However, the sensitivity of ∼10 µM was relatively low. More recently, Arora et al.31 demonstrated a novel wireless electrochemiluminescence (ECL) detector for MCE with a U-shaped floating electrode. That MCE-ECL system was applied to an analysis of three amino acids with an indirect detection mode. All these reports used glass microchips. As a chip material, glass has many advantages, such as low electric conductivity and high thermal conductivity for the use of high voltage, transparency for optical detection, well-developed surface chemistry, and so forth. However, its disadvantages are also very apparent such as high cost, complicated and harmful fabrication procedures, and limitation on the geometry of the chip channel. There is an increasing interest in the use of polymers instead of glass. A wide variety of polymer materials32 have been evaluated for the fabrication of microchips, in which poly(dimethylsiloxane) (PDMS)33 has been widely accepted due to its low cost, easy fabrication method, and transparent character in a wide range of wave bands. In this work, a deep insight was given into CL detection for MCE that fabricated in PDMS. Different strategies based on three chip patterns (cross, cross combining with Y, cross combining with V) to perform on-chip CL detection for MCE were evaluated by comparing the sensitivity, reproducibility, and peak symmetry, involving two model CL systems: metal ion-catalyzed luminolperoxide reaction and dansyl species conjugated peroxalateperoxide reaction. A separation of cations and chiral recognition (20) Liu, Y. M.; Cheng, J. K. J. Chromatogr., A 2002, 959, 1-13. (21) Huang, X. J.; Fang, Z. L. Anal. Chim. Acta 2000, 414, 1-14. (22) Campan ˜a, A. M. G.; Baeyens, W. R. G.; Zhao, Y. Anal. Chem. 1997, 69, 83A-88A. (23) Tsukagoshi, K.; Nakamura, T.; Nakajima, R. Anal. Chem. 2002, 74, 41094116. (24) Ren, J.; Huang, X. Anal. Chem. 2001, 73, 2663-2668. (25) Huang, B.; Li, J.; Zhang, L.; Cheng, J. Anal. Chem. 1996, 68, 2366-2369. (26) Liu, Y. M.; Liu, E. B.; Cheng, J. J. Chromatogr., A 2001, 939, 91-97. (27) Dadoo, R.; Seto, A. G.; Colo´n, L. A.; Zare, R. N. Anal. Chem. 1994, 66, 303-306. (28) Shortreed, M. R.; Li, H.; Huang, W. H.; Yeung, E. S. Anal. Chem. 2000, 72, 2879-2885. (29) Mangru, S. D.; Harrison, D. J. Electrophoresis 1998, 68, 2301-2307. (30) Hashimoto, M. H.; Tsukagoshi, K.; Nakajima, R.; Kondo, K.; Arai, A. J. Chromatogr., A 2000, 867, 271-279. (31) Arora, A.; Eijkel, J. C. T.; Morf, W. E.; Manz, A. Anal. Chem. 2001, 73, 3282-3288. (32) Becker, H.; Ga¨rtner, C. Electrophoresis 2000, 21, 12-26. (33) McDonald, J. C.; Duffy, D. C.; Anderson, J. R.; Chiu, D. T.; Wu, H.; Schueller, O. J.; Whitesides, G. M. Electrophoresis 2000, 21, 27-40.

Figure 1. Schematic layouts of MCE system coupled with CL detection. (A) Chip pattern of a cross. (B) Chip pattern of a cross combining with Y. (C) Chip pattern of a cross combining with V. Channel dimension, 40 µm in width and 50 µm in depth, except that the channel width of the stem in the Y shape of chip B was 80 µm. The scale using for the channel length in the figure was in millimeters.

were also discussed to show the advantages and facilitation originating from the hybrid of MCE as a high-performance separation tool and CL as a high-sensitivity detection scheme. EXPERIMENTAL SECTION Fabrication of the PDMS Device. Three PDMS microchips with different patterns were fabricated in the laboratory as schematically described in Figure 1, following the method published in a previous paper.34 Briefly, chip layout made by autoCAD was printed onto a glass mask for UV photolithography. A glass wafer was used as a substrate of UV-cured polymer mold for the replication of the channel pattern to PDMS. First, the glass wafer was coated with silanocoupling reagent (KBM-403) using a spin-coater and baked at 65 °C for 15 min after pretreatment with acetone, ethanol, water, alkaline solution, water, and N2 gas (for drying) in that order. Second, the wafer was further spincoated with negative photoresist (SU8 100). After a preexposure bake at 75 °C for 3 h, the wafer was exposed to UV light through the photomask using an aligner (MA55C/C, Karl Suss). Following a postexposure bake at 95 °C for 15 min to bridge epoxy polymer, the wafer was developed for 3 min in γ-butyrolactone, subsequently rinsed with isopropyl alcohol, and hard baked for 2 h to remove organic solution. Thus, a patterned SU8 microstructure was formed and used as the channel mold for thermal cured Si elastomer-PDMS that was prepared by mixing Sylgard 184 silicone elastomer and curing agent at a ratio of 10:1 (w/w). Third, the PDMS was poured onto the patterned SU8 structure and baked at 65 °C for 2 h. After cooling to room temperature, the PDMS layer that transcribed the channel pattern was peeled from the SU8 mold and sequentially washed with acetone and methanol. Holes were directly drilled on the PDMS layer to access the channels as buffer reservoirs. Finally, another flat PDMS layer was bonded to the patterned PDMS layer to form closed microchannels. Before bonding, both two PDMS layers were washed (34) Ustumi, Y.; Ozaki, M.; Terabe, S.; Hattori, T. Proc. 19th Sens. Symp. 2002, 215-218.

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with ethanol to remove the contaminants and further treated with KOH and pure water in that order. The two layers were then exposed to air plasma for 10 min in an air plasma chamber (model PC-101A, Yamato). The aim of this procedure was to form a silanol group on the surface of the PDMS layer that would lead to a tight seal between the two layers by dehydration when bonding at 65 °C in the presence of vacuum. Moreover, the channels exhibited a more hydrophilic nature and had less chance to form bubbles compared to untreated PDMS. Materials. Sylgard 184 silicone elastomer and curing agent were obtained from Dow Corning Corp. (Midland, MI). SU8 100 negative photoresist was purchased from Microchem Crop. (Newton, MA). Luminol (Wako, Japan) was dissolved in 0.1 M NaOH solution at a concentration of 10 mM. A 5 mM solution of bis[2-(3,6,9-trioxadecanyloxycarbonyl)-4-nitrophenyl] oxalate (TDPO, Wako, Japan) was prepared in acetonitrile. A 30% H2O2 solution was used as the stock solution. Co(NO3)2, CuSO4, and CrCl3 were used for the preparation of the stock solution of metal ions in pure water, respectively. Dansyl amino acids (Sigma, St. Louis, MO) were dissolved in pH 7.0 phosphate-buffered saline (PBS) solution. All other reagents were of analytical grade. Ultrapure water purified by the Milli-Q Labo system was used for the preparation of all solutions. Experimental Procedures. For a new microchip, channels were washed sequentially using methanol, water, 1 M NaOH, 0.1 M HCl, 0.1 M NaOH, and water. Then they were rinsed and conditioned with buffer solution. Between runs, channels were rinsed in order using 0.1 M NaOH, water, and buffer to ensure the reproducibility. Gated injection proposed by Jacoboson et al.35 was employed for injections in all experiments. For chip A in Figure 1, the channels and reservoirs were first filled with buffer solution. Then the buffer solutions in the sample reservoir (SR) and buffer waste reservoir (BWR) were replaced by sample solution and CL reagent, respectively. For initialization, voltages for all reservoirs were +620 V at SR, +120 V at sample waste reservoir (SWR), +720 V at buffer reservior (BR), and grounding at BWR. When injecting, the voltages were changed to +620, +410, and +430 V and grounding for SR, SWR, BR, and BWR, respectively. After injection, the voltages at all reservoirs were restored to the values at the initialization phase for separation. For chip B, all channels and reservoirs were first filled with buffer. Buffer solution was then replaced by CL solution in the CL reagent reservoir (CRR). Afterward, a vacuum was given at BWR to drive CL solution into the channel connecting with CRR. Thus, the solution in the channel connecting with BWR would be a mixture of buffer and CL reagent. Then the buffer solution in SR was changed to sample. For initialization, voltages for all reservoirs were +940 V at SR, +590 V at SWR, + 1010 V at BR, grounding at BWR, and + 485 V at CRR. For injection, the voltages were changed to +940, +793, and +807 V, grounding, and +485 Vrespectively. After injection, the voltages at all reservoirs were back to the values at the initialization phase for separation. For chip C, the procedures were similar to the chip cross combining with Y channels except that CL reagent was also present in BWR. In the initialization phase, voltages for all reservoirs were +740 V at SR, +320 V at SWR,

+880 V at BR, and grounding at BWR. For injection, the voltages were changed to +740, +593, and +607 V and grounding, respectively. For separation, the voltages at all reservoirs were back to the values of the initialization phase. No voltage was provided at CRR. A pressure was used to deliver the CL reagent. Instrument. All analyses were performed on a home-built microchip system. In brief, A PMT (Hamamatsu) was mounted to an inverted microscope (Carl Zeiss) that was housed in a black box. CL light was collected by a 20× objective. No filter was used in the optic path. The PMT current was converted and amplified into a voltage by a picoamperometer (Takeda) and then acquired by a data acquisition board (National Instrument) at a sampling rate of 50 Hz. A moving average (35 points) was used for all data process. Programs locally written in Labview 6.0 (National Instrument) were used to control the high-voltage suppliers (Matsusada Precision device, Kusatsu, Japan) and data acquisition.

(35) Jacobson, S. C.; Koutny, L. B.; Hergenro ¨der, R.; Moore, A. W.; Ramsey, J. M. Anal. Chem. 1994, 66, 3472-3476.

(36) Campbell, A. K. Chemiluminescence: Principles and Applications in Biology and Medicine; Ellis Horwood and VCH: Weinheim, 1988.

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RESULTS AND DISCUSSION The aim of this work was to develop an effective on-line CL detector for MCE. Based on the practice of CL detection in CE,20-22 two crucial factors governed the achieved sensitivity. The first was the dynamics of the CL reaction. Only those with rapidly responding reaction dynamics could be successfully applied. The second was the configuration of the CL detector, that is, the way for mixing CL reagent and the analytes. Consequently, two model CL systems that have been well-characterized, namely, a metal ion-catalyzed luminol-peroxide reaction and dansyl species conjugated peroxalate-peroxide reaction, were first used to evaluate different on-chip configurations of a CL detector in terms of sensitivity, reproducibility, and peak symmetry. Another important reason to choose the above-mentioned CL systems was that they took place in different solution phases that would have a great impact on the design of the CL detector as discussed below. Evaluation of Strategies for On-Chip CL Detection. In alkaline solution, the CL reaction occurs between luminol and peroxide with the help of a catalyst.36 Because a linear quantitation relationship exists within a certain range between concentration of catalyst and CL intensity, this reaction could be conveniently used to analyze the catalyst. The most popular catalyst is metal ions, such as, Co(II). Thus, we first examined how to perform CL detection on a microchip using a Co(II)-catalyzed luminolperoxide CL system, which could be considered as representative of CL systems in aqueous phase. Initially, a chip with a cross channel pattern as described in Figure 1A was investigated. The strategy to achieve CL detection was similar to the end-column scheme27 that was used in CE. The CL reagent was directly added into the BWR, where it mixed and reacted with the analyte pumped out from the separation channel and then produced CL emission (strategy I). Here, 0.5% peroxide dissolved in 20 mM NaAc-NaOH (pH 11.7) solution was added into the BWR as the CL reagent. A 2 mM solution of luminol was present in the running buffer 20 mM HAc-NaAc (pH 6.0) containing 5 mM R-HIBA. The sample 0.2 mM Co(II) was prepared in the buffer solution. Usually, an acidic buffer ranged from pH 4.0 to 5.025,26 was selected for the separation of metal ions in CE to avoid hydrolysis of the

Table 1. Comparison of Detection Limits and Reproducibility in Various Strategies for MCE-CL System

DL

S/N ) 3

RSD (n ) 5)

/µM /amol time/% height/%

strategy I for Co2+

strategy II for Co2+

strategy I for Dns-Gly

strategy III for Dns-Gly

strategy IV for Dns-Gly

8.28 2643 8.7 5.4

0.493 157 6.8 5.2

8.56 1309 4.6 5.0

0.683 104 5.8 -

0.390 59.3 4.2 5.0

Figure 2. Electropherograms by different strategies for achieving on-chip CL detection. (A) Peak for 0.2 mM Co(II) by strategy I. (B) Peak for 0.2 mM Co(II) by strategy II. (C) Peak for 0.25 mM Dns-Gly by strategy I. (D) Peak for 0.25 mM Dns-Gly by strategy III. (E) Peak for 0.25 mM Dns-Gly by strategy IV. Buffer in (A) and (B), 20 mM NaAc-HAc (pH 6.0) containing 2 mM luminol and 5 mM R-HIBA. CL reagent, 0.5% H2O2 in 20 mM NaAc-NaOH (pH 11.7). Buffer in (C)(E), 25 mM PBS (pH 7.0). CL reagent in (C), (D) and (E), 1% H2O2 and 5 mM TDPO in pure acetonitrile. Gated injection, 0.2 s. Electric field strength for injection and separation, 200 V/cm.

analytes that would further influence the detection sensitivity. However, in such acidic buffer, the reproducibility of the separation and the detection was found to be very poor in this work. A main reason was that our microchip was fabricated in PDMS. The inner surface of the channels still had a strong affinity for hydrophobic species,37 despite treatment by the plasma. In an acidic buffer of pH 4.0-5.0, luminol was very hydrophobic, which caused a strong adsorption onto the channel wall, which in turn influenced the stability of electroosmotic flow (EOF) and luminescence efficiency. Consequently, a buffer with relatively higher pH value was selected.The results are shown in Figure 2A. An asymmetric peak (tailing) was obtained with low detection sensitivity as given in Table 1. This phenomenon also occurred in CE with end-column CL detection. A reasonable explanation was that the buffer continuously pumped out from the separation channel diluted the local concentration of CL reagent. Analytes should have diffused to a broader area to catalyze the CL reaction. This would lead to band broadening over time and also a lower CL intensity for the sensitivity. Based on such a consideration, another channel pattern, a cross combining with Y shape as shown in Figure 1B, was employed, which was very similar to the chip adopted by Mangru and Harrison.29 With such a design, the CL reagent was not present in BWR. It was added in the CRR from where EOF pushed the CL reagent through one arm of the Y to the joint where peroxide instantly was mixed with luminol and Co(II) from another (37) Duffy, D. C.; McDonald, J. C.; Schueller, O. J.; Whitesides, G. M. Anal. Chem. 1998, 70, 4974-4984.

arm of Y and then gave out luminescence at the stem of Y (strategy II). As shown in Figure 2B, a symmetric peak was achieved with ∼20-fold improvement in sensitivity as given in Table 1. Although the detection sensitivity was significantly enhanced, it was still much higher than that obtained by CE.26 The reason for this difference was apparent. First, the sample volume at about the picoliter level was much smaller than that in CE, where several nanoliters or more sample was injected without sample overloading danger. Second, the on-chip mixing efficiency of analyte and CL reagent was not efficient as a coaxis design in CE. An angle of 360-deg diffusion of analytes to the flowing CL reagent occurred with such a configuration of CL detector in CE, regardless of the possible occurrence of turbulence that would further improve the mixing efficiency. But for an on-chip scheme with the Y mixer, side-by-side laminar flows38 occurred in the stem channel of the Y shape. Diffusion only took place at the interface between the analyte stream and CL reagent stream. Third, no on-column concentration method like electrostacking and field amplification that was readily performed in CE was employed. The results obtained by strategy II for the luminol CL system made us further consider its use for the dansyl species conjugated peroxalate CL system, in which the dansyl species absorbs the energy of reaction between TDPO (a kind of oxalate ester) and peroxide and then emits a green light.36 However, the major problem encountered here was that the CL reagent could not be effectively delivered by EOF, because TDPO could only be dissolved in an organic solvent without any buffer. The presence of the buffer would completely hydrolyze TDPO and further lead to a failure of the CL reaction. Thus, strategy II could not be applied to such a CL system. So we reexamined experiments using strategy I. The CL reagent, 5 mM TDPO dissolved in acetonitrile containing 1% H2O2 was added in BWR. A 0.25 mM solution of dansyl-glycine (Dns-Gly) was prepared in running buffer (25 mM PBS, pH 7.0). Results similar to the cobalt ion-catalyzed luminolperoxide CL system were found (low sensitivity and peak tailing), as shown in Figure 2C. The buffer continuously pumped out from the separation channel diluted and, more seriously, hydrolyzed CL reagent TDPO in a local area. Since the low detection sensitivity and asymmetric peak in strategy I were caused by above-mentioned reasons, we tried an active method to transport CL reagent to the outlet of the separation channel instead of passive diffusion, to instantly supply fresh CL reagent. As a result, a capillary was used to deliver the CL reagent by pressure (strategy III). One end of the capillary was etched by HF to reduce the outer diameter so that it could be as close as possible to the outlet of the separation channel. The other end of the capillary was connected with a raised vial or (38) Johnson, T. J.; Ross, D.; Locascio, L. E. Anal. Chem. 2002, 74, 45-81.

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syringe micropump. Using such a configuration of CL detector for MCE, higher sensitivity and better peak shape as shown in Figure 2D was accomplished, as expected. Unfortunately, no reproducible peak height was found. Experiments revealed that the peak height was quite sensitive to the position of the capillary end relative to the outlet of the separation channel. To overcome the problem in strategy III, the end of the capillary transporting the CL reagent should be effectively fixed at the outlet of the separation channel, which would be very easily accomplished by fabricating the capillary onto the microchip. This was the right strength a microchip possessed. As shown in Figure 1C, a cross combining with a V pattern was fabricated. The CL reagent was added in both CCR and BWR. Using pressure, one arm of the V would act as a tube to transport the CL reagent to the outlet of the separation channel (strategy IV). In practices, the pressure could be provided by either micropump or gravity. Figure 2E showed the electropherogram. High sensitivity was achieved together with good reproducibility as given in Table 1. It is seriously stressed here that the introduction of CL reagent into the separation channel should be avoided as much as possible. Because no electrolyte was presented in this organic phase, the insertion of an organic segment would change the distribution of the electric field through the separation channel and then cause irreproducible separation. Moreover, the organic plug inserted into the separation channel had very low conductivity, which would lead to a high potential drop and formation of bubbles to block the separation. In this work, a flux lower than 500 nL/min in the channel to deliver the CL reagent could ensure the avoidance of such a condition. This design could be also viewed as a revision of strategy II. Based on a chip pattern of cross combining with Y, EOF could not be used to effectively transport CL reagent thoroughly dissolved in organic solution. If directly using pressure instead of EOF, the flux in the channel delivering CL reagent should be controlled with respect to EOF in the separation channel. Otherwise, the CL reagent would be continuously driven into the separation channel, which would lead to a failure of separation and detection. More importantly, even under wellcontrolled pressure, the introduction of pure organic solvent into the electric field easily caused bubble formation and then blockage of the channel. In a case where the stem of Y was cut off, that was, using V instead of Y, the pressure would be released in the reservoir. Thus, CL reagent would not easily enter into the separation channel. Because the reservoir was electrically grounded, the chance for bubble formation was also avoided. A calibration curve was further constructed to test the quantitative validity of this strategy. A linear dynamic range of 2 orders of magnitude between peak height and concentration of Dns-Gly was found from 1 µM to 0.1 mM, with a correlation coefficient of 0.9903. Data comparison was detailed in Table 1. The detection limits were calculated based on a peak height of three times the baseline noise. The sample volume of injection for the estimation of the absolute detection limit was counted from the mobility of analyte, injection time, and electric field strength. Table 1 verified that the performance of the on-chip CL detector was quite configuration-dependent. It was concluded that strategy II based on the chip pattern of cross combining with Y proved to be promising for a CL system in an aqueous phase, while strategy IV based on the chip pattern of cross combining with V was preferred for a 40 Analytical Chemistry, Vol. 75, No. 1, January 1, 2003

Figure 3. Electropherogram for separation and detection of cations by MCE-CL. Sample concentration, 10 µM Cr(III), 2 µM Co(II), and 20 µM Cu(II) prepared in buffer. Electric field strength for injection, 100 V/cm. Other conditions were the same as in Figure 2B.

CL system in an organic phase or other cases where the CL reagent could not be effectively transported by EOF. Cation Analysis. A separation of cations by MCE with CL detection was further investigated based on strategy II. Metal ions such as Cr(III), Co(II), and Cu(II) that had rapidly responded to CL dynamics were examined as shown in Figure 3. Three species were well resolved in 40 s. The condition was similar to that used in Figure 2B with a modification of electric field strength (100 V/cm) for injection. A concentration of analyte close to the detection limit was selected mainly to show the detection sensitivity. Chiral Recognition. Chiral recognition39 is attractive in the area of bioanalysis and pharmaceuticals. Using CE,40 the enantiomers could be effectively resolved by the employment of a chiral additive in buffer such as crown ethers,41 cyclodextrin (CD) and its derivatives,42 surfactants,43 and so on, or by coating the selector on the surface of the capillary inner wall44 and stationary phase45 in capillary. There were recently several papers46,47 dealing with separations of fluorescein isothiocyanate (FITC)-labeled chiral amino acids by MCE coupled with LIF detection. A selector of γ-CD was used because the cavity diameter of γ-CD was suitable for the FITC-tagged amino acid. In this work, chiral recognition of Dns-amino acid was first reported using MCE coupling with CL detection based on the well-characterized strategy IV above. Dns-phenylalanine (Dns-Phe) was chosen as a representative test analyte. Many papers on CE have discussed the separation of chiral Dns-amino acid using different CDs and found that the cavity size of β-CDs was suitable for the dansyl species. We chose hydroxypropyl-β-CD (HP-β-CD)48 as the chiral selector here, which (39) Ward, T. J. Anal. Chem. 2002, 74, 2863-2870. (40) Amini, A. Electrophoresis 2001, 22, 3107-3130. (41) Tanaka, Y.; Otsuka, K.; Terabe, S. J. Chromatogr., A 2000, 875, 323-330. (42) Fanali, S. J. Chromatogr., A 2000, 875, 89-122. (43) Otsuka, K.; Terabe, S. J. Chromatogr., A 2000, 875, 163-178. (44) Liu, Z.; Otsuka, K.; Terabe, S. J. Sep. Sci. 2001, 24, 17-26. (45) Laemmerhofer, M.; Svec, F.; Frechet, J. M. J.; Lindner, W. Anal. Chem. 2000, 72, 4623-4628. (46) Hutt, L. D.; Glavin, D. P.; Bada, J. L.; Mathies, R. A. Anal. Chem. 1999, 71, 4000-4006. (47) Rodrı´guez, I.; Jin, L. J.; Li, S. F. Y. Electrophoresis 2000, 21, 211-219. (48) Razzi, A. M.; Kremser, L. Electrophoresis 1999, 20, 2715-2722.

Figure 4. Influence of chiral selector HP-β-CD concentration on separation resolution. Sample, 5 µM D/L-Dns-Phe (D/L, 1/1) prepared in buffer. Electric field strength for injection, 100 V/cm. Other conditions were the same as in Figure 2E.

Figure 5. Electropherogram of chiral recognition for 5 µM D/L-DnsPhe (D/L, 1/1) by MCE-CL based on strategy IV. Buffer, 25 mM PBS (pH 7.0) containing 2 mM HP-β-CD. Electric field strength for injection and separation, 100 and 300 V/cm, respectively. Other conditions were the same as in Figure 2E.

was quite hydrophilic. For chiral separation, the most important factor was the concentration of the selector. The influence of concentration of HP-β-CD on resolution was first examined from 0 to 40 mM as shown in Figure 4. With the increase of selector concentration, the resolution was rapidly increased at the beginning and then gradually decreased. The optimized concentration of HP-β-CD was 2 mM. The effect of electric field strength on the separation quality was also investigated from 100 to 500 V/cm. An electric field strength of 300 V/cm was found to be the optimum. Although we attempted to further improve the resolution by addition of an organic additive such as acetonitrile, no obvious enhancement was achieved, with a longer separation time. An increase in the length of the separation channel46 might be a good choice but was not tested in this work. Under the optimized conditions, the two Dns-Phe enantiomers were recognized within 1 min as depicted in Figure 5.

simplest way to perform end-column CL detection, but with low sensitivity, whereas a chip with cross combining with a Y layout showed higher sensitivity. A chip with cross combining with a V pattern was specially designed for that CL reagent could not be effectively transported by EOF, where a pressure was employed. A detection limit down to the submicromolar (midattomole) level was realized. A separation of cations and a chiral recognition of dansyl amino acid exhibited advantages and facilitation stemming from the hybrid of MCE as a highly efficient separation tool and CL as a highly sensitive detection scheme in integration. The success of this work revealed good prospects for the MCE-CL system in the field of chemical and biochemical analyses.

CONCLUSION A MCE system fabricated in an elastomer PDMS with CL detection was demonstrated. By comparison of different strategies based on three chip patterns for achieving on-line CL detection, it was concluded that the mixing configuration had great influence on detection sensitivity. A chip with a cross layout provided the

ACKNOWLEDGMENT The authors gratefully acknowledge the postdoctoral fellowship (to B.-F.L.) and the Grant-in-Aid for Scientific Research (P01082) supported by Japan Society for the Promotion of Science (JSPS). The authors also thank Dr. Philip Britz-McKibbin for his valuable comments on this paper. Received for review September 1, 2002. Accepted October 29, 2002. AC026096S

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