High-Speed, Whole-Column Fluorescence Imaging Detection for

Jul 20, 2006 - Bo Yao,† Huihua Yang,† Qionglin Liang,† Guoan Luo,*,† Liduo Wang ... Department of Chemistry, Tsinghua University, Beijing 1000...
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Anal. Chem. 2006, 78, 5845-5850

High-Speed, Whole-Column Fluorescence Imaging Detection for Isoelectric Focusing on a Microchip Using an Organic Light Emitting Diode as Light Source Bo Yao,† Huihua Yang,† Qionglin Liang,† Guoan Luo,*,† Liduo Wang,‡,§ Kangning Ren,† Yudi Gao,§ Yiming Wang,† and Yong Qiu‡,§

Department of Chemistry, Tsinghua University, Beijing 100084, China, Key Lab of Organic-Optoelectronics & Molecular Engineering of Ministry of Education, Department of Chemistry, Tsinghua University, Beijing 100084, China, and Beijing Visionox Technology Co., Ltd., Beijing 100085, China

An integrated and simplified microfluidic device using a 250 µm × 1-4 cm of organic light emitting diode (OLED) array as a two-dimensional light source for single-channel and multichannel whole-column imaging detection was developed. This fluorescence detection system was used for isoelectric focusing (IEF) of R-phycoerythrin in a microchip. The IEF conditions were optimized, and the total analysis time was extremely reduced to 30 s for 2-cm-long microchannels at 700 V/cm of electric field strength without the presence of electroosmotic flow. The compression of pH gradient caused by electrolytes drawing into the microchannels was efficiently restrained when 1% hydroxylpropylmethyl cellulose in 2% ampholyte was used as the carrier for IEF. Under optimized IEF conditions, the detection limit of this system was ∼0.6 µg/mL or 45 pg at 75 nL/column injection of R-phycoerythrin. This OLED-induced fluorescence detection system for WCID provides a high-speed IEF technique with quantitative ability and the potential for high integration and throughput microchip systems. Capillary isoelectric focusing (cIEF) is recognized as a promising separation technique for proteins, peptides, and other zwitterionic biomolecules,1,2 which not only provides a powerful means to determine sample composition, but also can be used to measure the isoelectric point (pI). Different from other capillary electrophoresis (CE) modes, cIEF is an equilibrium technique where the analysis proceeds until equilibrium is reached with each component focusing and settling down at its pI, instead of moving out of the capillary like other CE modes. Therefore, how to detect the focused bands inside capillary becomes an important problem for cIEF systems. * To whom correspondence should be addressed. Fax: +86-10-62781688. Tel: +86-10-62781688. E-mail: [email protected]. † Department of Chemistry, Tsinghua University. ‡ Key Lab of Organic-Optoelectronics & Molecular Engineering of Ministry of Education, Department of Chemistry, Tsinghua University. § Beijing Visionox Technology Co., Ltd. (1) Liu, X.; Sosic, Z.; Krull, I. S. J. Chromatogr., A 1996, 735, 165-190. (2) Kilar, F. Electrophoresis 2003, 24, 3908-3916. 10.1021/ac060445r CCC: $33.50 Published on Web 07/20/2006

© 2006 American Chemical Society

Several detection schemes have been developed for cIEF. Since most of the commercial CE instruments are coupled with an online detection system at a fixed point along the capillary, cIEF may employ a method to transport focused zones passing through the detection point. Three approaches including chemical,3 hydraulic,4 and electroosmotic5 mobilization have been used to transport focused zones in two-step or one-step cIEF. This detection scheme is most used in commercial CE systems although the mobilization process extremely lengthens analysis time and causes distortion of the sample bands. So, whole-column detection without a mobilization step appears more efficient for cIEF, which offers substantial potential advantages both in time and in maintenance of separation integrity. Hartwick6 presented a capillary scanning detection method in which the entire column was transported passing through a single detection point after focusing. This method, in principle, obviates the need for mobilization of the focused zones, but inhomogeneity of the capillary wall may cause relatively high noise and band broadening may occur, as the voltage is not maintained during detection.7 Pawliszyn et al. developed a series of whole-column imaging detection (WCID) systems without the need of mobilization steps by using a bundle of optic fiber to direct laser beam8 or lamp light9,10 to a short separation column, and a charge-coupled device (CCD) was employed to image the focused sample zones within the column. They also proposed an axially illuminated laserinduced fluorescence (LIF) WCID for cIEF by employing Teflon capillary, which could facilitate total internal reflection.11-14 Compared with transversely illuminating WCID, the axial illumina(3) Zhu, M. D.; Wehr, T.; Levi, V.; Rodriguez, R.; Shiffer, K.; Cao, Z. A. J. Chromatogr., A 1993, 652, 119-129. (4) Huang, T. L.; Shieh, P. C.H.; Cooke, N. Chromatographia 1994, 39, 543548. (5) Tang, Q.; Lee, C. S. J. Chromatogr., A 1997, 78, 113-118. (6) Wang, T. S.; Hartwick, R. A. Anal. Chem. 1992, 64, 1745-1747. (7) Liu, Z.; Pawliszyn, J. Anal. Chem. 2003, 75, 4887-4894. (8) Wu, X. Z.; Wu, J.; Pawliszyn, J. Electrophoresis 1995, 16, 1474-1978. (9) Wu, J.; Pawliszyn, J. Anal. Chem. 1994, 66, 867-873. (10) Wu, J.; Pawliszyn, J. Analyst 1995, 120, 1567-1571. (11) Huang, T.; Pawliszyn, J. Analyst 2000, 125, 1231-1233. (12) Huang, T.; Pawliszyn, J. J. Sep. Sci. 2002, 25, 1119-1122. (13) Liu, Z.; Pawliszyn, J. Anal. Chem. 2003, 75, 4887-4894. (14) Liu, Z.; Pawliszyn, J. Anal. Chem. 2005, 77, 165-171.

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tion configuration exhibits great advantages, including ease in light alignment and reduction of background noise. In addition, Das and co-workers also reported a LIF imaging system for IEF by using beam expander and cylindrical lens.15 Over the past decade, miniaturization and integration of some traditional chemistry and biology methods into a microscaled chip has abstracted remarkable interest by using standard photolithography and microfabrication techniques. Microchip electrophoresis is a popular area among those interests for its potential to provide rapid analysis, straightforward integration, and small sample requirement. However, compared with the abundant research work on microchip CZE,16 MEKC,17 and CGE,18 the IEF mode is relatively seldom carried out in microchip systems. Hofmann et al.19 first adapted cIEF to microchip where three common methods of chemical, hydrodynamic, and electroosmotic mobilization were investigated in a 7-cm-long column with singlepoint fluorescence detection. Cy5-labeled peptides were focused within 30 s with the presence of EOF, although baseline fluorescence plateau and sample loss were found related to N,N,N,Ntetramethylethylenediamine (TEMED), a commonly used additive that could prevent the peptides/proteins from focusing in sections of the capillary beyond the detection point. Later, Tan et al.20 performed chemical mobilization in a plastic microchip with crossnetwork for IEF of three proteins within 150 s. Image detection is also frequently employed in microchip systems by using an epifluorescence microscope with a 100-W mercury lamp and CCD camera for cIEF21-23 and multidimensional separations.24-26 However, the mercury lamp could not provide uniform excitation for imaging detection without a fiber bundle and sufficient view for whole-column imaging.21 Besides the above work, Mao and Pawliszyn27 demonstrated whole-column imaging detection for IEF in a quartz microchip by using an optical fiber bundle to guide the Xe lamp light beam to the microchannel. Total analysis of pI markers and proteins was within 10 min. To simplify the imaging detector and miniaturize the total system, they also developed a whole-column absorbance imaging detection system, which employed a short capillary (1.2 cm) as separation column and a lightemitting diode (LED) as light source, which also had the problem of nonuniform excitation.28 (15) Das, C.; Xia, Z.; Stoyanov, A. Instrum. Sci. Technol. 2005, 33, 379-389. (16) Jacobson, S. C.; Hergenro¨der, R.; Koutny, L. B.; Ramsey, J. M. Anal. Chem. 1994, 66, 1114-1118. (17) von Heeren, F.; Verpoorte, E.; Manz, A.; Thormann, W. Anal. Chem. 1996, 68, 2044-2053. (18) He, Y.; Pang, H. M.; Yeung, E. S. J. Chromatogr., A 2000, 894, 179-190. (19) Hofmann, O.; Che, D. P.; Cruickshank, K. A.; Mu ¨ ller, U. R. Anal. Chem. 1999, 71, 678-686. (20) Tan, W.; Fan, Z. H.; Qiu, C. X.; Ricco, A. J.; Gibbons, I. Electrophoresis 2002, 23, 3638-3645. (21) Cui, H. C.; Horiuchi, K.; Dutta, P.; Ivory, C. F. Anal. Chem. 2005, 77, 13031309. (22) Cui, H. C.; Horiuchi, K.; Dutta, P.; Ivory, C. F. Anal. Chem. 2005, 77, 78787886. (23) Macounova´, K.; Cabrera, C. R.; Yager, P. Anal. Chem. 2001, 73, 16271633. (24) Wang, Y. C.; Choi, M. H.; Han, J. Y. Anal. Chem. 2004, 76, 4426-4431. (25) Herr, A. E.; Molho, J. I.; Drouvalakis, K. A.; Mikkelsen, J. C.; Utz, P. J.; Santiago, J. G.; Kenny, T. W. Anal. Chem. 2003, 75, 1180-1187. (26) Chen, X. X.; Wu, H. W.; Mao, C. D.; Whitesides, G. M. Anal. Chem. 2002, 74, 1772-1778. (27) Mao Q. L.; Pawliszyn, J. Analyst 1999, 124, 637-641. (28) Wu, X. Z.; Sze, N. S. K.; Pawliszyn, J. Electrophoresis 2001, 22, 39683971.

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As to miniaturization of the optical detector for adaptation to a microchip system, we have recently reported an integrated fluorescence detection system that used a green organic light emitting diode (OLED) as light source.29 OLED has a flat surface, which makes it easy to integrate with microfluidic devices and flexible to fabricate into the required size and shape by photolithography techniques. Therefore, it is feasible to fabricate a twodimensional light source for whole-column imaging chip IEF detection without an optic fiber bundle that also is uniform. In this paper, we established a miniaturized and simplified wholecolumn fluorescence imaging detection system using twodimensional OLED consisting of 250 × 250 µm2 unit array as light source, which has a peak wavelength at 520 nm. R-Phycoerythrin as model fluorescent protein in 2% ampholyte and 1% HPMC carrier was focused and imaged, and the total analysis time was reduced to 15 s in a 1-cm-long column. Simultaneously, imaging of triple-channel IEF was also achieved by turning on three OLED arrays for excitation at the same time. EXPERIMENTAL SECTION Reagents and Protein Preparation. R-Phycoerythrin (MW ∼240 000) at 1 mg/mL (in 1 M NH4SO4) was purchased from Auwei Bioengineering (Hangzhou, China), and Bio-Lyte pH 3-10 carrier ampholytes were obtained from Bio-Rad Laboratories (Hercules, CA). Hydroxylpropylmethyl cellulose (HPMC) was bought from Shin-Etsu (viscosity of 2% solution at 20 °C is ∼4000 cP). Acrylamide monomer and N,N,N′,N′-tetramethylethylendiamine (TEMED) were bought from Promega (Madison, WI), and ammonium persulfate was from Amresco (Solon, OH), and [γ-(methacryloyloxy)propyl]trimethoxysilane (MAPS) was the product of Fluka (Buchs, Switzerland). All other chemicals were of analytical reagent grade, and Milli-Q water (18.2 MΩ, Millipore, MA) was used throughout. Before use, the original R-phycoerythrin solution was desalted with Microcon centrifugal filter devices (YM-30, Millipore, MA) at 6000 and 1000 rpm, respectively, and then diluted in aqueous solution containing 2% carrier ampholyte and 1% HPMC to a final concentration of 1-50 µg/mL. Desalted R-phycoerythrin should be stored at 4 °C and used in 1 week. Microchip Fabrication and Coating. The glass/PDMS microchip used in following experiments was designed and homemade as described before.29 In brief, glass plate with single or triple channels for IEF was fabricated by photolithography and wet chemical etching technique.30 While the opposite plate was a piece of 100-µm-thick PDMS replica from a flat glass wafer, which was silanized in 3% (v/v) octadecyltrichlorosilane (Sigma, St. Louis, MO) in dry toluene for 2 h beforehand. A 10:1 ratio of the silicone elastomer and curing agent (Sylgard 184, Dow Corning, Midland, MI) was mixed and poured onto the wafer after being stirred and degassed. The solution was baked in a vacuum oven at 65 °C for 4 h. Immediately, PDMS was sealed to the glass plate after peeling off the wafer, and then the microchip was exposed to ultraviolet light (UV, 254 nm) for 9 h. The final chip is shown in Figure 1. Four groups of microchannels with different lengths (1, 2, 3, 4 cm) were designed and (29) Yao, B.; Luo, G. A.; Wang, L. D.; Gao, Y. D.; Lei, G. T.; Ren, K. N.; Chen, L. X.; Wang, Y. M.; Hu Y.; Qiu, Y. Lab Chip 2005, 5, 1041-1047. (30) Yao, B.; Luo, G. A.; Feng, X.; Wang, W.; Chen, L. X.; Wang, Y. M. Lab Chip 2004, 4, 603-607.

Figure 2. Schematic diagram of the instrumental setup for wholecolumn imaging detection using OLED as light source. Figure 1. Scheme of microchip designed for single- or triple-channel IEF. (a) From the top view consisting of two groups of channels with different length (3 and 4 cm); (b) from the side view with 50-depth channel, 2-mm-diameter reservoirs, and 100-µm-thick PDMS plate.

fabricated. Each group had three columns standing side by side as shown in Figure 1a; the other two groups of chips were not presented here. Several 2-mm-i.d. reservoirs were fabricated at each terminal of channels by an desk drill at 5000 rpm (Z4006, Tianjin No.4 Machine Tools Works, Tianjin, China) with an emery drill bit. The distance between two columns was 3 mm. All channels were etched to 50 µm deep, 100 µm at bottom, and 200 µm at top (see Figure 1b); glass plate is on the top and PDMS is at the bottom in the experiment as shown. Before use, each channel was filled with 5 µL of 5% MAPS (in acetic acid, pH 3.5) with a syringe for pretreatment.31 After that, they were left at room temperature for reaction overnight. Then they were rinsed with methanol followed by water for 2 and 10 min, respectively, and filled with freshly prepared reaction buffer (3% acrylamide, 0.6% ammonium persulfate and 0.2% TEMED in water) for 3 h as reported by Han et al.32 Finally, the channels were rinsed with water for 10 min, dried with nitrogen for 10 min, and were ready for use. Instrument Setup for WCID. The OLEDs used in the experiments were fabricated by lithographical patterning and organic molecular beam deposition on indium tin oxide-coated glass plate described previously,33,34 which had an array of 250 × 250 µm2 illuminant units connected with deposited electrodes emitting an intensity of 20 000 cd/m2 and irradiance of 7.5 mW/ cm2 (at 12 V of driving voltage) green fluorescence with a peak emission at 520 nm and ∼60 nm bandwidth (fwhm). Different from the single-point fluorescence detection system recently reported by us,29 in this work, anode electrodes of the illuminant units were jointed by deposition of a gold layer to the desired length (1∼4 cm). Cathode electrodes were selected connecting with the homemade power source (0-25 V dc; see Figure 2 power source 2) in order to carry out single- or triple-channel imaging. As (31) Hjerte´n, S. J. Chromatogr. 1985, 347, 191-198. (32) Han, F. T.; Wang, Y.; Sims, C. E.; Bachman, M.; Chang, R. S.; Li, G. P.; Allbritton, N. L. Anal. Chem. 2003, 75, 3688-3696. (33) Qiu, Y.; Gao, Y. D.; Wang, L. D.; Wei, P.; Duan, L.; Zhang D. Q.; Dong, G. F. Appl. Phys. Lett. 2002, 81, 3540-3542. (34) Qiu, Y.; Gao, Y. D.; Wei, P.; Wang, L. D. Appl. Phys. Lett. 2002, 80, 26282630.

optimized before, 12 V was used to drive OLEDs throughout the experiments unless stated otherwise. The instrument setup for whole-column imaging is presented in Figure 2, where on top of the OLEDs was a piece of 0.3-mmthick short-pass interference filter (550 nm) designed and fabricated by Optical Coating Center of the Film Machinery Research Institute (Beijing, China), which consisted of 30 alternating layers of SiO2 and TiO2 and was ∼4.5 µm thick. When 12 V dc was applied to selected anode and cathode electrodes, the OLED array was illumined in single or triple line. Green emission transited the interference filter by which the unwanted excitation light was removed and covered whole microchannels. A color CCD camera (DH-HV1302UC, Daheng image, Beijing, China) coupled with a TV zoom lens (MLM3X-MP, Computar) was employed to capture fluorescence signals excited by OLEDs and image the process of fluorescent proteins transporting and focusing inside columns at real time. Between the microchip and CCD camera, a long-pass emission filter (XF3089 575ALP, Omega Optical) was placed for elimination of the excitation light. The exposure time of the CCD camera was set at 5-100 ms, depending on the fluorescence intensity of the sample, the frame rate was locked at 2 fps, and the resolution of each image was 1280 × 1024 to provide enough information for data processing with MATLAB off-line. IEF Conditioning. The microchip was rinsed with water and IEF buffer (2% ampholyte in 1% HPMC) for several minutes before each performance. Sample solution containing R-phycoerythrin in IEF buffer was introducted into the microchannels with a syringe, and extra sample solution in reservoirs was eliminated by pipets. Then the reservoirs were filled with 4 µL of 100 mM phosphoric acid and 200 mM sodium hydroxide, respectively, as anolyte and catholyte. High voltage was provided with a regulated high-voltage power supply (DW-P203-1AC, 0-20 kV, Dongwen, Tianjin, China) (see Figure 2 power source 1). RESULTS AND DISCUSSION Miniaturization of WCID for Microchip. WCID has been already proved an efficient method for capillary and chip IEF systems because of obviating the need for mobilization steps, reducing the total analysis time and maintaining the resolution of focused zones. However, the existing strategies for WCID including optical fiber bundle guiding and axial illumination are both complicated and highly precise, which has restricted them from wide applications. As a matter of fact, the most crucial Analytical Chemistry, Vol. 78, No. 16, August 15, 2006

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Figure 3. Images (a) and electropherograms (b) of the dynamic IEF process in a 2-cm-long microchannel. CCD captured a frame every 4.5 s from top to bottom. IEF conditions: 700 V/cm electric field strength; 2% ampholyte (pH 3-10) and 1% HPMC; 25 µg/mL R-phycoerythrin; 100 mM H3PO4 as anolyte; 200 mM NaOH as catholyte.

problem is the light source. Laser and lamp as commonly used light source in a single-point detection system are not inherently designed for WCID and cannot provide the appropriate radiation covering the whole column uniformly. Therefore, this problem can be easily resolved by providing a two-dimensional light source. OLED is such an alternative that can be fabricated into the required size and shape by photolithography technology. Furthermore, it is low cost and miniaturized, which can greatly simplify the whole system. That is valuable, especially for microchip systems. WCID for microchip using OLED as a light source without any optical arrangements is shown in Figure 2, and real-time images of IEF in the microchannel are presented in Figure 3a, which described the whole process more clearly and vividly than only electropherograms.11 From top to bottom, each frame demonstrated the exact state of R-phycoerythrin in the focusing process when CCD captured one frame every 4.5 s. As voltage applied to the anode and cathode reservoirs, proteins began to migrate to their pI and two protein zones met at ∼20 s. Then they focused and separated into three zones, and at ∼30 s, those sample bands immobilized while current decreased ∼87% from 35.4 to 4.9 µA. In this experiment, R-phycoerythrin was divided into three adjacent zones, which differed by a single peak as reported by other groups,21,22 however, consistent with the results of Kang and Yeung.35 That might be because the product was not purified enough before coming on the market. Figure 3b shows electrophoregrams of this focusing process by extracting the gray value of the image and calculating with MATLAB. From the position they focused in, we can calculate that the pIs of three R-phycoerythrin bands are ∼4.9, 5.0, and 5.1 in this experiment and the reference value provided by manufacturer is ∼4.6. (35) Kang, S. H.; Yeung, E. S. Anal. Chem. 2002, 74, 6334-6339.

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Compared with those previous light sources including laser, lamp, and LEDs for WCID, OLED is a promising alternative, which is naturally two-dimensional and readily integrated with a microchip without external optical elements. IEF Conditions. There is a problem of compression of the pH gradient reported in some capillary or chip IEF systems,21,27 which is assumed to be as a result of anolyte or catholyte unexpectedly flowing into the IEF column from the reservoirs. Since compression of the pH gradient appears to require a flow of anolyte or catholyte into the separation columns, Cui et al. replaced the high viscosity of polymer solutions (2.5% methylcellulose) in the two reservoirs, like two plugs, to prevent electrolyte from being drawn into the channels and to extend the pH gradient to ∼3-fold compared with the absence of MC.21 In the experiments, it is found that high-viscosity electrolytes will influence the IEF process by extending the period of current attenuation and resulting in a longer analysis time. Actually, to increase the viscosity of the dynamic coating solution is another simple way to mitigate the pH compression, which has the similar function of restraining pH gradient shrinking. Furthermore, high-viscosity ampholyte carrier will efficiently maintain the resolution of IEF by eliminating convection at the interface of the highly concentrated sample and the low-viscosity ampholyte solution and by reducing diffusion.36 The influence of HPMC concentration from 0.25 to 1.5% on the IEF focusing process of R-phycoerythrin was studied in this experiment, and the results are shown in Figure 4. When the viscosity of the sample solution was low, compression of the pH gradient was obvious and R-phycoerythrin focused at about the middle of the 2-cm column (pI ∼6.5). While the concentration increased to 1.5%, the pI measured by isoelectric (36) Wehr, T.; Rodrı´guez-Dı´az, R.; Zhu M. D. Capillary Electrophoresis of Proteins; Marcel Dekker: Basel, 1998.

Figure 4. Influence of electric field strength from 400 to 1200 V/cm (left) and concentration of HPMC from 0.25 to 1.5% (right) on IEF focusing process. Other conditions are the same as in Figure 3 (n ) 3).

focusing was ∼4.8 (first band of three). Focusing time is found shorter at low-viscosity HPMC but, however, is accompanied with drift toward the cathode after focusing. That we think might be contributed by remnant electroosmostic flow (EOF), although the internal surface of the microchanels was coated beforehand and dynamic coating was also employed. When HPMC concentration was reduced, cathode drift was observed due to the increasing of EOF. Therefore, the concentration of HPMC was optimized to 1% because higher viscosity would make it troublesome to fill the IEF columns. The electric field was also studied from 400 to 1200 V/cm, and no Joule heat effect was found inside the microchannels. Also, more serious cathode drift was found when electric field strength increased above 1000 V/cm across a 2-cm-long column (see Figure 4) due to the higher electric field caused by higher EOF. So a relatively weak electric field of 700 V/cm was finally selected. Under the optimized conditions, the total analysis of IEF was reduced to 30 s for a 2-cm-long microchannel and 15, 45, and 60 s for 1-, 3-, and 4-cm columns, respectively. Column Length. Since IEF is a concentrating technique, increased column length may lead to more quantity of sample focused, thus enhanced fluorescence responses. That was verified by the following experiment in which the column length of the IEF column was increased from 1 to 4 cm for 25 µg/mL R-phycoerythrin under a 700 V/cm electric field. Figure 5a is the comparison of electropherograms at different column lengths, and Figure 5b shows the results displayed respectively in peak height and area. It appears that the peak height stopped climbing when column length extended to 2 cm. However, this is not the result of CCD’s response reaching its saturation because the peak area was found increased with column length proportionately. That means the quantity of protein sample reached its upper limit for IEF and resulted in extension of the focused band in peak width. That phenomenon was mitigated when low a concentration of proteins was filled in the microchannels. Performance of the Detection System. To obtain a high fluorescence response for R-phycoerythrin, a 4-cm column microchip was used in the following experiment where a different concentration of R-phycoerythrin was focused in microchannels and imaged by the detection system. The electropherograms are shown in Figure 6 from 3.1 to 50 µg/mL. When peak area was

Figure 5. Comparison of electropherograms (a) and fluorescence signal in peak height and area (b) under different column lengths (1, 2, 3, and 4 cm). Other conditions are the same as in Figure 3 (n ) 3).

Figure 6. Electropherograms of increased concentration from 3.1 to 50 µg/mL R-phycoerythrin focused in a 4-cm-long microchannel. Other conditions are the same as in Figure 3.

used for quantitative analysis, the coefficient of correlation (r) achieved in the standard curve was 0.99627 for three repetitions (n ) 3). As the ratio of fluorescence signal-to-noise (S/N) at 3.1 µg/mL is ∼15, the detection limit of this system can be calculated to ∼0.6 µg/mL (S/N ) 3). For a 4-cm-long column, the total volume of sample solution filled in the channel is ∼75 nL. So the Analytical Chemistry, Vol. 78, No. 16, August 15, 2006

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Figure 7. Images (a) and electropherograms (b) of triple-channel IEF mode with three 2-cm-long columns, and other IEF conditions are the same as in Figure 3.

lowest amount of R-phycoerythrin that can be detected by this system is ∼2.5 × 10-9 M and ∼45 pg. This performance of detection is still 2 orders of magnitude higher than average laserinduced fluorescence WCID systems37 and ∼3 orders higher than the axial illuminant WCID13 because of the poor excitation and spectral characteristics of OLEDs. However, it has been extremely improved over that of the single-point OLED system developed by our group recently,29 in which the limit of detection achieved was roughly 6 orders poorer than good laser-induced fluorescence detection in capillary electrophoresis. This progress might be due to the energy loss of the laser WCID system with fiber bundle and other optical guiding arrangements. Furthermore, the OLED induced fluorescence WCID system is advantageous for quantitative analysis of IEF because of its uniform background, which is problematic in axial illuminant13 and lamp systems.21 Multiple-Channel Imaging. Compared with other WCID light sources, OLEDs are also advantageous to easily develop a multiple channel imaging system by photolithography without additional labor and arrangements. In this experiment, a triple-channel WCID device was set up by connection of three cathode electrodes 3 mm away from each other, and IEF electrophoresis was performed and imaged synchronously in three microchannels. Panels a and b in Figure 7 are respectively the images of focused 25 µg/mL R-phycoerythrin zones in microchannels and the electropherograms. High producibility of fluorescence intensity and focused position are both obtained. Microchips with radiative 6- and 12channel design were also studied for higher throughput IEF. However, the sensitivity achieved was found to be fairly poor due to the scattering light coming from the wall of the channels, which is illuminated by not only the OLED source but also by the fluorescence of R-phycoerythrin protein.

CONCLUSIONS In this study, a noval whole-column imaging detection system using a green organic light emitting diode as two-dimensional light source for on-chip isoelectric focusing of fluorescent proteins was developed, and the whole detector was extremely integrated and simplified by fabricating OLED into a two-dimensional array. Light sources for single- or triple-channel IEF were obtained by selected connection of the electrodes of OLED array, and imaging of singleand triple-channel IEF was achived in this experiment. IEF conditions were optimized, and the total analysis time was reduced to less than 30 s for a 2-cm column at high electric field strength. The results proved that OLEDs were a promising light source for WCID, especially for microchip systems, although the intensity and stability of its illuminant is expected to be more powerful.38 To develop the potential ability of OLEDs for high-throughput multichannel detection, the problem of scattering light from the inside wall should be resolved as soon as possible.

(37) Wu, J. Q.; Tragas, C.; Watsona, A.; Pawliszyn, J. Anal. Chim. Acta 1999, 383, 67-78. (38) Wei. B.; Ichikawa, M.; Furukawa, K.; Koyama, T.; Taniguchi, Y. J. Appl. Phys. 98, 044506.

Received for review March 10, 2006. Accepted June 7, 2006.

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ACKNOWLEDGMENT This research was supported by projects of the National Science Foundations of China (Grant 20299036, 20475031), National Basic Research Program (973 Program) of China (2005CB523503, 2001CB510305), and Key Technology Program of China (2004BA721A13). The authors thank Dr. Yi Gao at Department of Biomedical Engineering, Georgia Institute of Technology, for the help on data processing. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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