Laser-Induced Fluorescence Detection System for Microfluidic Chips

In this work, a LIF detection system for microfluidic chips with a simple ... A 473-nm diode pumped laser (Changchun New Industries Optoelectronics Te...
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Anal. Chem. 2006, 78, 3827-3834

Laser-Induced Fluorescence Detection System for Microfluidic Chips Based on an Orthogonal Optical Arrangement Jing-Lin Fu,† Qun Fang,*,† Ting Zhang,† Xin-Hua Jin,‡ and Zhao-Lun Fang†

Institute of Microanalytical Systems, Zhejiang University, Hangzhou, 310028 China, and Department of Optical Engineering, Zhejiang University, Hangzhou, 310027 China

In this work, a simple LIF detection system based on an orthogonal optical arrangement for microfluidic chips was developed. Highly sensitive detection was achieved by detecting the fluorescence light emitted in the microchannel through the sidewall of the chip to reduce scattered light interference from the laser source. A special crossedchannel configuration, with a 1.5-mm distance from the separation channel to the sidewall of the glass chip, was designed in order to facilitate collection of emitted fluorescence light through the sidewall. The significant difference in intensity distribution of scattered laser light on the chip plane observed in this study was fully exploited to optimize S/N ratio of detected signals by rejection of scattered light, both through systematic measurements and employing ray-tracing simulation. A fluorescence collection angle of 45° in the chip plane gave the best result, with a scattered light intensity 1/38 of that obtained at an angle of 90°. Sodium fluorescein and fluorescein isothiocyanate-labeled amino acids were used as model samples to demonstrate the performance of the LIF system. A detection limit (S/N ) 3) of 1.1 pM fluorescein was obtained, which is comparable to that of optimized confocal LIF systems for chip-based capillary electrophoresis. Apart from the high detection power, the system also has the advantages of simple optical structure, compactness, and ease in building. In recent years, microfluidic chip-based analysis has made great developments in realizing the lab-on-a-chip or µTAS concept of having all major analytical functional components integrated on a single chip.1-6 However, highly sensitive detection methods, * To whom correspondence should be addressed. E-mail: fangqun@ zju.edu.cn. Tel.: +86-571-88273496. Fax: +86-571-88273496. † Institute of Microanalytical Systems. ‡ Department of Optical Engineering. (1) Manz, A.; Harrison, D. J.; Verpoorte, E. M. J.; Fettinger, J. C. J. Chromatogr. 1992, 593, 253. (2) Harrison, D. J.; Fluri, K.; Seiler, K.; Fan, Z.; Effenhauser, C. S.; Manz, A. Science 1993, 261, 895. (3) Jacobson, S. C.; Hergenroeder, R.; Koutny, L. B.; Warmack, R. J.; Ramsey, J. M. Anal. Chem. 1994, 66, 1107. (4) Woolley, A. T.; Mathies, R. A. Anal. Chem. 1995, 67, 3676. (5) Reyes, D. R.; Lossifidis, D.; Auroux, P.-A.; Manz, A. Anal. Chem. 2002, 74, 2623. (6) Auroux, P.-A.; Lossifidis, D.; Reyes, D. R.; Manz, A. Anal. Chem. 2002, 74, 2637. 10.1021/ac060153q CCC: $33.50 Published on Web 05/03/2006

© 2006 American Chemical Society

such as laser-induced fluorescence (LIF), have to be employed in such systems in order to detect the minute amounts of analyte in the micrometer scale channels in the chips. Various LIF detection systems6 based on different optical arrangements were developed and have been successfully and broadly applied in the microfluidic chip-based analysis systems, particularly in chip-based capillary electrophoresis (CE) systems. The optical arrangements of hitherto three major types of LIF detection system employed in chip devices are shown schematically in Figure 1a-d. The confocal LIF system4,7-11 (as shown in Figure 1a) has proved to be an effective arrangement to perform highly sensitive detection on microchips. Ramsey’s group10 reported an on-chip detection of limit (LOD) of 1.7 pM rhodamine 6G and 8.5 pM rhodamine B in a chip-based CE system with a confocal LIF detector. Harrison’s group developed a series of confocal epifluorescence microscope systems for microchip LIF detection by which LODs of 300 fM fluorescein8 and 9 pM Cy-5 9 were obtained. However, the complicated and critical structure as well as difficulties associated with construction and miniaturization of the optical system for confocal LIF systems appears to have limited their wider application in microfluidic chips. Besides the confocal arrangement, many nonconfocal LIF detection systems based on bevel incident laser or orthogonal arrangement were also developed for coupling to chip-based systems, which have advantages of relatively simple structure and ease in system building. In the former LIF system, the laser beam was usually focused on the microchannel with a bevel incident angle, such as 45°, and the emitted fluorescence was detected in the direction perpendicular to the microchip (as shown in Figure 1b and c).12-17 However, this approach suffered from rather high background levels generated from reflections (7) Haab, B. B.; Mathies, R. A. Anal. Chem. 1999, 71, 5137. (8) Ocvirk, G.; Tang, T.; Harrison, D. J. Analyst 1998, 123, 1429. (9) Jiang, G. F.; Attiya, S.; Ocvirk, G.; Lee, W. E.; Harrison, D. J. Biosens. Bioelecton. 2000, 14, 861. (10) Fister, J. C.; Jacobson, S. C.; Davis, L. M.; Ramsey, J. M. Anal. Chem. 1998, 70, 431. (11) Dang, F. Q.; Zhang, L. H.; Hagiwara, H.; Mishina, Y.; Baba, Y.; Electrophoresis 2003, 24, 714. (12) Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1996, 68, 720. (13) Fister, J. C.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1999, 71, 4460. (14) Schrum, D. P.; Culbertson, C. T.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1999, 71, 4173. (15) Roulet, J. C.; Volkel, R.; Herzig, H. P.; Verpoorte, E.; de Rooij, N. F.; Dandliker, R. Anal. Chem. 2002, 74, 3400. (16) Sanders, J. C.; Huang, Z. L.; Landers, J. P. Lab Chip 2001, 1, 167. (17) Fang, Q.; Xu, G. M.; Fang, Z. L. Anal. Chem. 2002, 74, 1223.

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EXPERIMENTAL SECTION

Figure 1. Schematic diagrams of typical optical arrangements of microfluidic chip LIF detection systems: E, excitation source; F, fluorescence; L, lens; O, optical fiber.

and refraction from the cover plate and channel walls. Even if some data processing approaches, such as employing a crosscorrelation method,13 were adopted to improve the detection performance, its application in performing highly sensitive fluorescence detection appears to be rather limited. In the LIF detection systems based on an orthogonal optical arrangement, an optical fiber fixed in a guiding hole fabricated on the chip is usually adopted to guide the laser beam to the microchannel from a horizontal direction, and the fluorescence signal was detected in the direction perpendicular to the chip (as shown in Figure 1d).18-21 The structure of the LIF system is significantly simplified by using such an arrangement; however, the reported LODs in most of these systems were usually in the nanomolar range, which are 2-4 orders of magnitude higher than those obtained in the confocal LIF systems, presumably owing to the relatively weak focusing properties of optical fibers compared with optical lenses, leading to deterioration of the S/N ratio of the detection system. In this work, a LIF detection system for microfluidic chips with a simple orthogonal optical arrangement, characterized by detection of the fluorescence transmitted through the polished sidewall of a chip, was developed. The interference from scattered laser light was significantly reduced by optimizing the fluorescence collection angle in the plane of chip plate, perpendicular to the incident laser beam. (18) Liang, Z. H.; Chiem, N.; Ocvirk, G.; Tang, T.; Fluri, K.; Harrison, D. J. Anal. Chem. 1996, 68, 1040. (19) Chabinyc, M. L.; Chiu, D. T.; McDonald, J. C.; Stroock, A. D.; Christian, J. F.; Karger, A. M.; Whitesides, G. M. Anal. Chem. 2001, 73, 4491. (20) Camou, S.; Fujita H.; Fuji, T. Lab Chip 2003, 3, 40. (21) Li, H. F.; Lin, J. M.; Su, R. G.; Uchiyama, K.; Hobo, T. Electrophoresis 2004, 25, 1907.

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Reagents. All reagents used were of analytical reagent grade, and demineralized water was used throughout. A 5 mM sodium tetraborate buffer (pH 9.2) was used as working electrolyte for CE separation. The stock solution of 10 mM sodium fluorescein was prepared by dissolving 37 mg of the dye in 10 mL of 5 mM sodium tetraborate buffer (pH 9.2). Stock solutions of 40 mM amino acids were prepared from L-arginine and D,L-β-phenylalanine purchased from Kangda Amino Acid Works (Shanghai, China). The labeling reagent of 40 mM fluorescein isothiocyanate (FITC, Sigma Chemicals, St. Louis, MO) was prepared by dissolving 9.5 mg of FITC in 0.6 mL of acetone. The 4 mM FITC-labeled amino acid solutions were prepared by mixing 0.3 mL of stock solutions of each of the amino acids with 0.6 mL of 50 mM sodium tetraborate buffer (pH 9.2) and 0.1 mL of FITC reagent, and the resultant mixtures were allowed to stand overnight in the dark. The standard series of sodium fluorescein or FITC-labeled amino acids were prepared by sequentially diluting the stock solutions with 5 mM sodium tetraborate buffer (pH 9.2). The stock solutions were filtered through a membrane filter (0.45-µm pore size, Xinya Co., Shanghai, China) before use. The diluted working standards in the concentration range lower than 1 µM were freshly prepared and used within 2 h following preparation. Apparatus and Equipment. A schematic diagram of the LIF detection system is shown in Figure 2. A 473-nm diode pumped laser (Changchun New Industries Optoelectronics Tech. Co., Changchun, China) was used as light source. The laser beam was passed through a 471-nm band-pass filter (8-nm band-pass, Shenyang HB Optical Technology Co., Shenyang, China), reflected by a mirror and focused by a 40× microscope objective (0.65 NA, 3.0-mm-long working distance, Chongqing MIC Optical & Electrical Instrument Co., Chongqing, China) at a right angle to the chip plane in the center of the separation channel with a focusing point of ∼20 µm. Alignment of the microchip position was achieved by adjusting an X-Y-Z translation stage (model TSM-4A-XYZ, Zolix Instruments Co., Beijing, China) on which the chip was horizontally mounted. The fluorescence light transmitted through the sidewall of the chip was collected by a 25× microscope ocular (Xi’an Sicong Laser Co., Xian, China) with an angle of 45° between the separation channel and detection light path in the plane of the chip plate (as shown in Figure 2) and imaged upon a 0.9-mm pinhole. The light passing through the hole was transmitted through a 510-nm long-pass filter and a 525nm band-pass filter (15-nm band-pass, Shenyang HB Optical Technology Co.) before being detected by a photomultiplier tube (PMT R928, with integrated amplifier CC171, Hamamatsu, Japan). During optimization of the optical system, a sheet of translucent paper was used to facilitate alignment of optical components by imaging the fluorescence beam upon the paper placed at the position of the component. The output signals from the PMT were recorded by a PCI6013 16 A/D board (National Instruments, Austin, TX) and processed using a program written with LabVIEW (National Instruments). All collected data were filtered by a Butterworth 5-Hz low-pass filter and smoothed using a 50-point adjacent averaging smoothing program, included respectively in the LabVIEW and Origin softwares. A homemade high-voltage

Figure 2. Schematic diagram of the optical setup of the LIF detection system.

Figure 3. Schematic diagram of the microchannel layout on the etched plate.

power supply,17 variable in the range of 0-2000 V, was used for on-chip sample injection and CE separation. Fabrication of the Microchip Device. Standard photolithographic and wet chemical etching techniques17 were used for fabricating channels onto a 1.7-mm-thick 12 × 60 mm glass plate with chromium and photoresist coating (Shaoguang Microelectronics Co., Changsha, China). A special crossed-channel configuration (as shown in Figure 3) for the CE chip was designed for the present LIF detection system with a short distance of 1.5 mm from the separation channel to the sidewall of the chip. The channels were etched into the plate in a well-stirred, dilute HF/ NH4F bath to a depth of 30 µm and a width of 90 µm. Four access holes of 1.8-mm diameter for the reservoirs were drilled at the

terminals of the crossed channels in the etched plate. The etched plate was bonded to the base plate with low-temperature prebonding and subjected to high-temperature treatment (550 °C) to achieve permanent bonding.22 The four reservoirs were produced from 5-mm-i.d., 10-mm-section plastic tubes, cut from commercial pipet tips, affixed with epoxy on the etched plate, surrounding the access holes. The sidewall proximate to the separation channel on the chip was polished to ensure unimpeded light transmission using cerium oxide polishing powder (R111 Repowder, 0.8-1.2 µm, Shanghai Gona Powder Technology Co., Shanghai, China). Ray-Tracing Simulation. TracePro software (Lambda Research Co.) was used for ray-tracing simulation of the reflection and refraction of the excitation laser beam within the chip (as shown in Figure 4). The studied zone on the chip measured L 20 × W 10 × D 3.4 mm. The parameters employed for simulation are as follows: refractive index of chip material (glass), 1.52; medium filled in the microchannel, water; excitation beam assumed as grid ray trace of a Gaussian beam with 473-nm wavelength and 1.1-mm radius of the grid boundary; laser beam orientation assumed to converge to a point in air with a focal length of 3 mm. Other simulating parameters and detailed simulation procedure are available as Supporting Information. Procedures. Chip-Based CE Separation. Before use, the microchannels in the CE chip were sequentially flushed with concentrated sulfuric acid for 30 min and 1 M NaOH, water, and 5 mM borate buffer, each for 3 min. Reservoir 1 was filled with 200 µL of sample solution, and reservoirs 3, 4, and 2 with 200 µL of borate buffer, respectively. Four platinum electrodes were inserted into the individual reservoirs. The position of the microchip fixed on the X-Y-Z stage was adjusted relative to the focus point of the laser beam to illuminate the detection point on the separation channel with an effective separation length of 30 (22) Jia, Z. J.; Fang, Q.; Fang, Z. L. Anal. Chem. 2004, 76, 5597.

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Figure 4. Schematic diagrams of (I) ray-tracing simulation of intensity distribution of the scattered light in the LIF system and (II) sectional view of (a) the actual channel and (b) simulated channel.

mm. Gated sample injection23 was carried out by applying 1200 V between reservoirs 1 and 4, with reservoirs 2 and 3 maintained at floating state, and then separation by applying 1200 V to reservoir 1 and 1500 V to reservoir 2, while reservoirs 3 and 4 were maintained at zero potential. The sample injection and separation times were 2 and 90 s, respectively. For optimizing the fluorescence collection angle, 10 µM sodium fluorescein standard was used as a sample for the measurement of fluorescence intensity using a 510-nm long-pass filter, and 5 mM borate buffer (pH 9.2) was used for the measurement of scattered light intensity. The S/N ratio for each electropherogram was calculated by dividing the average peak height by the standard deviation of the background noise, determined from the section of the electropherogram immediately before and after the peak. Safety Considerations. Wet etching of the glass chips should be performed in a well-ventilated hood, while wearing protective gloves and goggles. In setting up of the LIF detection system, dark glasses should be worn to avoid hurting the eyes with the laser beam. (23) Jacobson, S. C.; Koutny, L. B.; Hergenroder, R.; Moore, A. W.; Ramsey, J. M. Anal. Chem. 1994, 66, 3472.

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RESULTS AND DISCUSSION Design of the LIF Detection System. Orthogonal optical arrangements24-28 for LIF systems, with which fluorescence emissions are detected from the direction orthogonal to the incident laser beam, have proved to be an effective approach for reducing scattered light interference from the laser source. Owing to the simpler arrangement and structure compared with the confocal LIF systems, the orthogonal LIF systems have been widely applied in conventional CE systems.25-28 An extremely low LOD of ∼10 molecules for a CE system was reported by Dovichi’s group,28 using an orthogonal LIF system with a sheath flow design for reducing light scattering. However, to build a LIF system for microfluidic chips, the orthogonal arrangement may pose difficulties owing to the poor transparency of the sidewalls of most microchips, including glass and polymer chips, which may result in strong scattering of the incident laser beam or excited fluorescence while transmitting through the chip sidewall. A further limitation could be the larger distance of the channel to the sidewall of most chips, as compared with that to the upper or lower plate surfaces of 1-2 mm. Thus, in most of hitherto reported orthogonal LIF systems designed for microchips, optical fibers were often adopted for transmitting the laser light to the vicinity of the microchannel on the chip. However, the LODs obtained using such systems were often 2-4 orders of magnitude worse than those obtained for most of the confocal LIF systems for both chip-based and conventional CE systems. Presumably, this is due to the relatively weak focusing capabilities of optical fibers, which may result in excessive energy dispersion of the laser beam and increase of light scattering at the detection point in the microchannel, both leading to a deterioration of S/N level. Therefore, the important advantages pursued by orthogonal LIF systems, i.e., low scattering background and high S/N ratio, were not realized in such systems. Another drawback of LIF systems using optical fibers for transmission of laser light is that the position of the detection point on the channel is decided by that of the fixed optical fiber, which is difficult to adjust once the chip is fabricated and leaves no chance for further optimization.20,21 In this work, instead of using optical fibers for light transmission, a simple and sensitive orthogonal LIF detection system for microchips was developed by directly utilizing the optical path through the chip sidewall.29 Microfluidic chips produced from glass were adopted to demonstrate the performance of the present LIF system. For most of the microchips produced using common microfabrication techniques, the sidewalls of the chip are often rough and uneven, which may result in strong light scattering. Therefore, prior to use, the chip sidewall through which the laser/ fluorescence light was transmitted was subjected to intensive polishing, to form a smooth and highly transparent surface. At a preliminary stage of this work, two orthogonal arrangements were tested. In one of the arrangements, the incident laser beam was focused on the channel by an objective lens through the sidewall (24) Dovichi, N. J.; Martin, J. C.; Jett, J. H.; Trkula, M.; Keller, R. A. Anal. Chem. 1984, 56, 348. (25) Cheng, Y. F.; Dovichi, N. J. Science 1988, 242, 562. (26) Swerdlow, H.; Zhang, J. Z.; Chen, D. Y.; Harke, H. R.; Grey, R.; Wu, S. L.; Dovichi, N. J. Anal. Chem. 1991, 63, 2835. (27) Ueno, K.; Yeung, E. S. Anal. Chem. 1994, 66, 1424. (28) Chen, D. Y.; Dovichi, N. J. Anal. Chem. 1996, 68, 690. (29) Fang, Q.; Fu, J. L.; Huang, Y. Z. Chinese Patent, Appl. 03141572.5, 2003.

Figure 5. CCD images of the chip sidewall with the fluorescence spot produced by excitation of 10 µM fluorescein.

of the chip along the horizontal axis, and the emitted fluorescence was detected on the axis perpendicular to the chip plane (as shown in Figure 1e). In the other arrangement, the incident laser beam was focused on the channel along the axis perpendicular to the chip plane, and the fluorescence transmitted along horizontal axis through the sidewall of chip was collected by an objective lens placed close to the chip sidewall and detected by a PMT (as shown in Figure 1f). Owing to strong scattering of the incident laser beam at the sidewall of the etched microchannel, the LIF system with the former arrangement showed S/N ratios that were 1/5 of those obtained with the latter arrangement under identical conditions. Thus, orthogonal LIF systems using the latter design were employed in all later studies, unless mentioned otherwise. Optimization of Fluorescence Collection Angle. It is welldocumented that, for orthogonal LIF detection systems, suppression of scattered laser light could be achieved by observing the fluorescence on the plane perpendicular to the incident laser beam.24-28 However, to our best knowledge, little, if any, studies have been made on the degree of scattered light rejection at different observation angles on the plane perpendicular to incident laser beam. In this work, we found a significant difference in distribution of the scattered laser intensity on the chip plane and conducted a systematic study on the phenomenon to exploit its potentials. Figure 5 shows three CCD images of the chip sidewall with the laser-excited fluorescence image of a 10 µM fluorescein solution filled in the chip channel, observed at a collection angle (defined as the angle between the separation channel and detection path) of 45°, 90°, and 135°, respectively. A significant difference in the color of the images could be observed. The image observed at a collection angle of 90° appeared predominantly blue instead of the green of fluorescein fluorescence, owing to the domination of strong blue scattered light under such conditions. The fluorescence image observed at collection angles of 45° and 135°, although not as bright, showed relatively green in the images, qualitatively demonstrating that the intensity of the scattered light were much lower at these observation angles. A more quantitative evaluation on the effect of collection angle on the scattered laser intensity was undertaken within a range of 30°150° observation angle through intensity measurements as well as via ray-tracing simulation. The results are as shown in Figure 6a1, a2, and c. In Figure 6a1, the scattering intensity of the laser

beam significantly increased with the increase of angle in the range of 30°-90°, reached maximum at 90° with an intensity 38fold of that at 45°, and decreased with the increase of angle in the range of 90°-150°. Similar results were obtained from the ray-tracing simulation as shown in Figure 6a2. In the present LIF system, the laser beam was focused in the microchannel by the microscope objective with a focused beam diameter of about 20 µm and half-angle of convergent laser beam of 13°, which were similar to those in most previous reported high-sensitivity LIF detection systems.8,12,24-26 The reasons for the difference in scattered light intensity at different collection angle in the plane of the chip were further studied using the ray-tracing simulation procedure under various conditions, including diameter of the focused laser beam, half convergent angle, and shape of the channel cross section. The simulation results showed that the difference in the intensity distribution of scattered light in the chip plane was mainly related to the half-angle of the convergent laser beam. In a half-angle range of 0°-34°, which included all of the convergent laser beams focused by common objectives including those with 10×, 20×, and 40× magnifications, the scattered light exhibited significantly different intensity distributions on the chip plane. This implies that different intensity distributions of scattered light on the chip plane would always be observed in LIF detection systems with the laser beam perpendicularly focused at the channel of the chip. When half-angles are higher than 34°, the scattered light distribution tended to be uniform. In addition to the studies performed above with the convergent laser beam striking through the smooth cover plate of the chip on the microchannel, further studies were conducted with the laser beam striking through the etched chip substrate on the microchannel by placing the chip upside down on the X-Y-Z stage. Owing to the roughness of the etched surface of the microchannel bottom, the results showed ∼2-3-fold increases in the intensity of scattered light compared with those under right side conditions. Similar results were also obtained with ray-tracing simulation under these conditions. The effects of the collection angle in the range of 30°-150° on the distribution of fluorescence excited from a 10 µM fluorescein standard filled in the channel were also studied with the same chip and LIF detection system, except that a 510-nm long-pass filter was employed to eliminate the scattered light. The Analytical Chemistry, Vol. 78, No. 11, June 1, 2006

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Figure 6. Measured intensity distribution of scattered light (a1) and fluorescence (b1), and fluorescence-to-scattered light ratio (c) at different collection angles; ray-tracing simulation intensity distribution of scattered light (a2) and fluorescence (b2) at different collection angles.

profile for the distribution of fluorescence (as shown in Figure 6b1) also showed a peak shape with maximum at 90° as for the scattered light. However, owing to the uniform spatial emitting property of the fluorescence light, the variations on fluorescence intensity with collection angle were much smaller than that for the scattered laser light. Using ray-tracing simulation, a similar fluorescence distribution profile (as shown in Figure 6b2) was obtained, assuming that the fluorescence was emitted from a cylindrical source with a diameter of 30 µm and a height of 30 µm, estimated according to the geometry of the laser beam crossing the channel. Figure 6c shows the measured intensities of fluorescence and scattered laser light as well as their ratios at different collection angles. The latter is directly correlated to the S/N ratio and the LOD of the LIF detection system. The 45° and 135° collection angles demonstrated the highest fluorescence/scattered light ratio with an improvement of 25-fold over that obtained at 90°. 3832

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In the present LIF detection system, a collection angle of 45° was employed instead of 135°, purely for the sake of convenience in building the optical system. Optimization of Filters. There are three main sources contributing to the background in high-sensitivity LIF detection systems using microfluidic chips,30 i.e., scattered laser light, Rayleigh and Raman scattering from the sample, and background fluorescence emission from the optical lens and chip. Filter selection plays a crucial role in reducing these interferences in these systems. In the present work, the effects of the filter combination on the intensities of background and fluorescence light as well as S/N ratio were studied by using a 471-nm bandpass filter for the laser beam and 100 pM sodium fluorescein solution as sample. A series of emission filter combinations were examined with the results shown in Table 1. Compared with the various single emission filter arrangements, the emission filter (30) Lyons, J. W.; Faulkner, L. R. Anal. Chem. 1982, 54, 1960.

Table 1. Evaluation of Filter Sets long-pass filter

band-pass filter

background signala (mV)

fluorescence signal b(mV)

S/N

510 LP 510 LP none 510 LP none 510 LP

none 525 BP35 546 BP8 546 BP8 525 BP15 525 BP15

856.0 ( 8.6 105.1 ( 0.9 20.2 ( 0.2 17.4 ( 0.2 61.5 ( 0.5 32.2 ( 0.3

147.0 87.6 15.8 14.8 61.1 50.6

17 97 79 74 122 169

a Background signal was measured while filling the separation channel with buffer solution. b Fluorescence signal with 100 pM sodium fluorescein solution. Conditions: fluorescence collection angle, 45°; PMT operating high-voltage, -880 V; pinhole diameter, 0.9 mm.

Figure 7. Typical electropherograms of 2.5 pM sodium fluorescein solutions with 2-s gated injections. Working electrolyte, 5 mM sodium tetraborate buffer; injection voltage, 1200 V; separation voltage, 1500 V; effective separation length, 31 mm; PMT voltage, -1000V.

Table 2. Evaluation of the Channel-to-Sidewall Distance distancea (mm)

calcd fluorescence collection effic (%)b

background signalc (mV)

fluorescence signald (mV)

S/N

1.5 5 10

6.2 2.4 0.9

32.2 ( 0.3 24.1 ( 0.3 18.2 ( 0.2

50.6 16.4 6.1

169 55 31

a Channel-to-sidewall distance. b Fluorescence collection efficiency was calculated according to eq 1. c Background signal measured while filling the separation channel with buffer solution. d Fluorescence response with 100 pM sodium fluorescein solution. Conditions: fluorescence collection angle, 45°; PMT operating high voltage, -880 V; pinhole diameter, 0.9 mm.

combination composing of a long-pass filter and a band-pass filter always demonstrated higher S/N ratios under the same conditions, since the background fluorescence produced from the optical lens and glass chip could be effectively reduced by using the second band-pass filter. Among the series of filter combinations using 546 BP8, 525 BP35, and 525 BP15 band-pass filters as second emission filter, respectively, the filter combination composing of 510 LP and 525 BP15 filters produced the best S/N ratio. Although the lowest background signal was obtained by using a 546 BP8 band-pass filter as second filter, the fluorescence signal transmitted through this filter was also reduced to the lowest level, resulting in a decrease of S/N ratio. Effects of Fluorescence Collection Lens Distance. In the present LIF detection system, a 25× microscope ocular was placed close to the sidewall of the chip to collect the fluorescence light transmitted through the sidewall with a collection angle of 45°. In this section, for convenience, the term “detection distance” is defined as the distance between the fluorescence emitting point and fluorescence collection ocular lens, which is mainly determined by the distance between the separation channel and the chip sidewall at a fixed collection angle. The effects of detection distance on the fluorescence and scattered light signal as well as the S/N ratio were studied by employing three types of chips with channel-to-sidewall distances of 1.5, 5, and 10 mm, respectively, using 100 pM sodium fluorescein solution as sample. The results are shown in Table 2. The channel configuration for the first type of chip (as shown in Figure 3) was specially designed with an asymmetrical layout to obtain a short channel-to-sidewall distance of 1.5 mm; the other two types of chips had a regular symmetrical crossed-channel configuration for CE separations. The results in Table 2 show that both the fluorescence and scattered light signal

Figure 8. Typical electropherograms of a mixture of 100 pM FITC-Arg and 100 pM FITC-Phe solutions. Conditions as shown in Figure 7.

as well as the S/N ratio decreased with the increase of detection distance, i.e., channel-to-sidewall distance. The fluorescence collection efficiency could be approximately estimated by the following equation:

η)

R12 4(R2 + x2d)2

(1)

where η is the fluorescence collection efficiency, R1 the inner radius of the collection ocular lens, R2 the outer radius of the lens in the ocular, and d the channel-to-sidewall distance. Thus, employing chips with specially designed channel configurations with short channel-to-sidewall distances is beneficial for obtaining high detection power. However, even for the chips with regular crossed-channel designs, with 10-mm distance from channel to the chip sidewall, S/N ratios of 1/5 that obtained using the chip with 1.5-mm channel-to-sidewall distance could still be obtained. Performance of LIF Detection System. The limit of detection for the present LIF detection system was demonstrated in the CE separation of fluorescein using a gated injection mode. The linear regression equation for fluorescein within 1 pM to 50 pM range was, F ) 3.2C, r2 ) 0.9992, where F is the fluorescence intensity (in mV) and C the concentration of fluorescein (in pM), with PMT voltage of -1000 V and gated injection time of 2 s. Figure 7 shows a typical electropherogram of repetitively introduced sample of 2.5 pM fluorescein solution. The average S/N ratio was 6.8, corresponding to a LOD of 1.1 pM (S/N ) 3), which Analytical Chemistry, Vol. 78, No. 11, June 1, 2006

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is comparable to most of the optimized confocal LIF systems for chip-based capillary electrophoresis. The performance of the present LIF detection system was further demonstrated in the CE separation of FITC-labeled amino acids. Figure 8 shows a typical electropherogram of repetitively introduced samples of a mixture of 100 pM FITC-arginine and 100 pM FITC-phenylalanine solutions with a separation voltage of 1500 V and gated injection time of 2 s. RSD values of the fluorescence intensity were 3.0 and 3.6% (n ) 5) for FITC-Arg and FITC-Phe, respectively. CONCLUSIONS In this work, highly sensitive LIF detection was achieved using an orthogonal optical arrangement, by collecting the emitted fluorescence at optimized angles through the sidewall of a microchip with an asymmetrical channel layout. The pM level LODs are comparable to those achieved by employing more sophisticated confocal optical arrangements. The new setup has the advantages of simple structure, compactness, and being easily constructed in routine chemical laboratories. A further potential advantage of such LIF systems is that the optical arrangement allows the setting of multiple detection paths through one or both sidewalls of the chip, making possible for simultaneous detection at multiple wavelengths using a series of detectors installed around the sidewalls, such as for chip-based DNA sequencing or flow cytometry. Although glass chips were used in this work to demonstrate the performance of the system, chips produced from other materials, such as quartz, polymer (poly(methyl methacry-

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late) or polycarbonate) could be easily coupled with the present LIF system. Apart from the chip-based system studied, we also observed the phenomenon of varied distribution of light scattering on the plane perpendicular to the laser beam using fused-silica capillaries for CE separations. This implies that the present LIF orthogonal optical arrangement could also be applicable to conventional CE systems. Preliminary results showed that LODs in picomolar fluorescein range could also be obtained in conventional capillary zone electrophoresis. ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China under Projects 20299030 and 20575059, and Ministry of Science and Technology of China under Projects 2004AA404240 and 2005AA2Z2042. The authors are grateful to Mr. Chuanliang Fan and Lei Chen of the Optical Engineering Department of Zhejiang University for inspiring discussions. The authors also thank Mr. Bo Kong and Wenbin Du for their help in the writing of the data processing program. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review January 21, 2006. Accepted March 29, 2006. AC060153Q