Acousto-Optical Deflection-Based Laser Beam Scanning for

Nov 2, 1999 - Acousto-Optical Deflection-Based Laser Beam. Scanning for Fluorescence Detection on. Multichannel Electrophoretic Microchips. Zhili Huan...
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Anal. Chem. 1999, 71, 5309-5314

Acousto-Optical Deflection-Based Laser Beam Scanning for Fluorescence Detection on Multichannel Electrophoretic Microchips Zhili Huang,† Nicole Munro,‡ Andreas F. R. Hu 1 hmer,‡,§ and James P. Landers*,†,‡

Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, and Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260

Laser beam scanning driven by an acousto-optical deflector (AOD) is presented for multimicrochannel laserinduced fluorescence (LIF) detection during microchipbased electrophoresis. While fast laser beam scanning for LIF detection on capillary or microchannel arrays can been achieved with galvanometric scanning or a translating stage, it can also be accomplished by using acoustic waves to deflect the laser beam in a manner that is dependent on the acoustic frequency. AOD scanning differs from other approaches in that no moving parts are required, and the scan frequency is faster than conventional approaches. Using a digital/analog (D/A) converter to provide addressing voltages to a voltage/frequency converter, rapidly changing the frequency input to the AOD allows the laser beam to be addressed accurately on a microchip. With the ability to change the frequency on the nanosecond time scale, scanning rates as high as 30 Hz for Windows-based LabView programming are possible, with much faster scan rates achievable if a microprocessor-embedded system is utilized. In addition to spatial control, temporal control is easily attainable via raster scanning or random addressing, allowing for the scanning process to be self-aligning. Since the D/A output voltages drive the scanning of the laser beam over all channels, the software can define addressing voltages corresponding to the microchannel centers and, subsequently, fluorescence data can be collected from only those locations. This method allows for flexible, highspeed, self-align scanning for fluorescence detection in capillary or microchip electrophoresis and has the potential to be applied to a number of applications. The ability to carry out electrophoretic separations in microcolumns1 or microstructures etched into planar substrates2,3 has * To whom correspondence should be addressed. Department of Chemistry, University of Virginia. Tel.: 804-243-8658. Fax: 804-243-8852. E-mail: jpl5e@ virginia.edu. † University of Virginia. ‡ University of Pittsburgh. § Current address: Microcomponents Technology Center, Mixed-Signal Product Development, Semiconductor Group, Texas Instruments, Dallas, TX. (1) Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1981, 53, 1298-1302. (2) Manz, A.; Graber, N.; Widmer, H. M. J. Chromatogr. 1990, 1, 244-252. (3) Harrison, D. J.; Fluri, K.; Seiler, K.; Fan, Z.; Effenhauser, C. S.; Manz., A. Science (Washington, D.C.) 1993, 261, 895-897. 10.1021/ac990740u CCC: $18.00 Published on Web 11/02/1999

© 1999 American Chemical Society

begun to revolutionize analytical separations. Capillary electrophoresis (CE) has demonstrated that high-efficiency separation of DNA, proteins, and small molecules can be achieved with low volumes of reagents and minute sample volumes in a rapid, costeffective manner.4,5 Capillary electrophoresis has been extrapolated to microfabricated chips where capillary-like microchannels etched in a planar glass substrate allow for similar separations to be carried out with roughly an order of magnitude decrease in analysis time compared with that of the capillary format. The collation of capillaries or microchannels into arrays has provided a platform where microcolumn technology cannot only compete effectively with slab gels for the parallel processing of multiple samples, but also reduce total analysis times by as much as 2 orders of magnitude.6,7 Sensitivity limitations associated with the small dimensions of capillaries and channels used for separation can be circumvented by the use of laser-induced fluorescence (LIF), which can routinely provide attomole sensitivity with a welldesigned optical system.8-11 The multiplexing of electrophoresis in multiple capillaries or multiple channels on a microchip is not only complex from a sample injection and separation perspective, but also complicated by the fact that rapid and effective fluorescence detection must be achieved. Several reports in the literature have described low intensity level LIF detection in CE with the use of charge-coupled device (CCD) cameras7,12 as well as various laser beam scanning approaches.13-15 Mathies’ group13,14 has described effective fluorescence detection in multiple microchannel chip devices by (4) Gordon, M. J.; Huang, X.; Pentoney, S. L., Jr.; Zare, R. N. Science (Washington, D.C.) 1988, 242, 224-228. (5) Karger, B. L.; Cohen, A. S.; Guttman, A. J. Chromatogr. 1989, 492, 585614. (6) Huang, X. C.; Quesada, M. A.; Mathies, R. A. Anal.Chem. 1992, 64, 21492154. (7) Tan, H.; Yeung, E. S. Anal. Chem. 1998, 70, 4044-4053. (8) Hadd, A. G.; Raymond, D. E.; Halliwell, J. W.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1997, 69, 3407-3412. (9) Ocvirk, G.; Tang, T.; Harrison, D. J. Analyst (Cambridge, U.K.) 1998, 123, 1429-1434. (10) Munro N. J.; Snow, K.; Kant, J. A.; Landers, J. P. Clin. Chem. 1999, in press. (11) Hofgaertner, W.; Hu ¨ hmer, A. F. R.; Landers J. P.; Kant, J. Clin. Chem. 1999, in press. (12) Wu, J. Q.; Tragas, C.; Watson, A. Anal. Chim Acta 1999, 383, 67-78. (13) Huang, X. C.; Quesada, M. A.; Mathies, R. A. Anal. Chem. 1992, 64, 967972. (14) Woolley, A. T.; Sensabaugh, G. F.; Mathies, R. A. Anal. Chem. 1997, 69, 2181-2186.

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translating the microchip over a stationary laser beam. A number of other methods for laser-beam scanning exist including resonant scanning, polygon scanning, double-pass scan lens scanning, holographic scanning systems, internal drum system scanning, electro-optical scanning, and acousto-optic scanning.16,17 With the exception of electro-optical and acousto-optical devices, all of these methods use a mirror-reflecting principle in which the mirror movement is motor-driven. Most commercially available scanning microscopes are based on this principle.17 While reflection-based scanning approaches are advantageous because they are wavelength-independent, they too are mechanically controlled and, therefore, it is difficult to achieve ultrafast scanning rates. Furthermore, it is difficult to cancel out the mechanical noise, e.g., jitter and wobble, which can result in distortion of a scanned image or signal and, with capillaries/microchannels, can be associated with defocusing and displacement of the laser beam during detection. Mathies and co-workers18 have developed an elegant solution to this problem via a “spinning objective” system which allows for LIF detection on 96 microchannels in a silica microchip. However, this optical approach is limited to circular designs with microchannels in a circular array. The power of electro-optical and acousto-optical scanning approaches have not yet been exploited for capillary or microchip electrophoresis. While electro-optical (EO)-based scanning would be ideal for microchip applications, few materials exhibit an E-O effect of substantial magnitude, and the physical dimensions of commercially available devices are too large to be applied to microscopy. This contrasts with the case of acousto-optic deflectors (AODs) which are compact in design and available commercially. AODs utilize the diffraction effect induced by optical gratings to achieve a deflection of the laser beam.19-22 The acoustooptic (A-O) effect occurs when a light beam passes through a material in which acoustic waves are also present. Acoustic waves are generated by a piezoelectric transducer that is driven by a radio frequency signal. The spatially periodic density variations in the material corresponding to compressions and reduction of the travelling acoustic wave are accompanied by corresponding changes in the index of reduction for propagation of light in the medium. For acoustic waves of sufficiently high power, most of the light incident on the acousto-optic device will be diffracted and, therefore, deflected from its incident direction. The efficiency of deflection is dependent on the wavelength of light, while the magnitude of the deflection is mainly dependent on the frequency (∆f) centered at f0. This is defined by

∆Θ ) λ ∆f/V

where V is the acoustic velocity within the optical medium, λ is (15) Simpson, P. C.; Roach, D.; Woolley, A. T.; Thorsen, T.; Johnston, R.; Sensabaugh, G. F.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 2256-2261. (16) Marshall, G. Optical Scanning; Marcel Dekker, Inc.: New York, 1991. (17) Bass, M. Handbook of Optics, Volume II; McGraw-Hill: New York, 1995; Chapter 19. (18) Trepte, O.; Lijeborg, A. Opt. Eng. 1994, 33, 3774-3780. (19) Xu, J. P.; Stroud, R. Acousto-optic Devices: Principles, Design, and Applications; John Wiley & Sons: New York, 1992. (20) Bullen, A.; Patel, S. S.; Saggau, P. Biophys. J. 1997, 73, 477-491. (21) Gass, P. A.; Schalk, S.; Sambles, J. R. Appl. Opt. 1994, 33, 7501-7509. (22) Isomet Corp. Manual of Acousto-optical Deflectors, Springfield, VA, 1993.

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the laser wavelength within the medium, and ∆f is the bandwidth of the acoustic frequency. Since the deflection angle is proportional to the acoustic frequency and inversely proportional to the acoustic velocity, generation of relatively large scan angles requires that the deflectors operate with a large acoustic-frequency bandwidth and/or a low acoustic velocity. Moreover, since the acoustic frequency can be altered rapidly, these devices should be effective as ultrafast deflectors for rapid, one-dimensional, random-access laser-beam scanning. In this paper, we describe the use of an acousto-optic deflector and the associated optical configuration for high-speed laser scanning applied to fluorescence detection of multiple channels on a microfabricated chip. We demonstrate the effectiveness of acousto-optic deflection for scanning as fast as 200 Hz and illustrate a rudimentary application of this technology for simultaneous fluorescence detection from multiple channels on a microchip. EXPERIMENTAL SECTION Acousto-Optic Device and Scanning System Configuration. The scanning system consisted of a laser source, a scanning unit, and the appropriate optics. A continuous-wave argon ion laser (532R-BS-A04, Omnichrome) is used for excitation. The output wavelength is 488 nm and the power 25 mW. A 4× beam expander (Spindler & Hoyer) is used to expand the laser beam diameter from 0.6 to 2.4 mm to more effectively focus the laser on the microchip. The scanning unit is based on a precision-optimized achromats lens (Melles Griot) with a 250-mm focal length, an acousto-optic deflector (1205C, ISOMET Co., Springfield, VA), a driver (D320, ISOMET), and a spatial filter. The detection unit includes a 2.5× microscope objective with a 42-mm working distance and φ20-mm diameter (Melles Griot), a long-pass filter with a 515-nm cutoff wavelength (Melles Griot), a photomultiplier tube (PMT) (R3896, Hamamatsu) and a socket amplifier (C1053, Hamamatsu), and a low-pass filter (AMLP8B-15 Hz, Avens Signal Co., New York, NY). The objective focuses on the microchannel plane and gathers the fluorescence emission; the long-pass filter is placed after the objective. The PMT converts the fluorescence emission to an electrical signal, which is amplified by the socket amplifier and filtered with a low-pass filter. For video capture, a CCD camera (OS-40D, World Precision Instruments) and a video capture device (LAV-1000, Dazzle Multimedia) were used in place of the PMT. A computer (Pentium-II-233, Windows 95) is used to control the scanning unit, with a 12-bit D/A converter board (DT302, Data Translation) and an in-house-manufactured highvoltage power supply with a 12-bit 6-channel D/A converter board (AT-AO-6, National Instruments); to sample data from the detection unit with a 12-bit A/D converter board (DT302, Data Translation); and complete data processing, analysis, and display. The current software is written in LabView for Windows. Microchip Electrophoresis. Multichannel electrophoretic microchips were fabricated as described previously.10,11 Electrophoretic chips consisted of an eight channel arrangement with each channel containing an injection cross; the sample to sample waste distance was 1.45 cm, the inlet to outlet distance was 6.65 cm, and the junction was 0.5 cm from the inlet. Detection occurred 4.2 cm from the injection cross. The sample channel was 50 µm wide and 20 µm deep, and the separation channel 50 µm wide and 20 µm deep. Reservoirs were crafted from 1 mm holes drilled in the top glass plate to allow access to the channels.

All channels were coated using a modified Hjerte´n method,23 with 1% HEC in 1X TBE used as a sieving buffer and YO-PRO-1 (Molecular Probes, Eugene, OR) added as a fluorescent intercalated at a concentration of 1 µM. The DNA standard, HaeIII digest of pBR322 (Boehringer Mannheim Biochemicals, Indianapolis, IN), was diluted 1:10 with 10 mM Tris/1 mM EDTA to a final concentration of 42.0 µg/mL. The separation voltages were supplied by an in-house-manufactured high-voltage power supply controlled by a program written in LabView. Chip sample injection was performed by applying a 400 V (275 V/cm) potential across the sample and sample waste reservoirs, with the sample at ground. For separation, the sample and sample wastes were grounded and -200 V was applied to the inlet and 900 V to the outlet (165 V/cm). RESULTS AND DISCUSSION When comparing and contrasting the various scanning approaches for possible implementation in multichannel microchip electrophoresis, several factors need to be considered. These include spatial resolution, sensitivity, temporal resolution, scatter, and cross-talk; ease of use and cost are additional factors that need to be addressed. AODs possess very high temporal bandwidth and are relatively easy to use, but are potentially limited to a small scanning range; this is perfectly tailored to microchannel or capillary arrays. One of the advantages unique to AOD scanning is that it is capable of any of three different scanning modes: raster (uni- or bidirectional), step, and random addressing, a mode difficult to achieve with translating stage- and galvanometer-based scanning. Consequently, acousto-optic-based scanning possesses most of the characteristics desired for detection during electrophoresis with capillary arrays or multichannel microchips. In order for AOD based scanning to be effective for an application such as microchip electrophoresis, the AOD must first be integrated into the appropriate optical system. Figure 1A presents a schematic of the AOD driven laser scanning system configuration. In this system, a continuous-wave argon-ion laser was employed. To optimize focusing, the laser beam was expanded before presentation to the optimized achromatic lens. This step allowed the beam to be focused within the microchip channel with a final diameter of less than 50 µm. The acousto-optic deflector was placed after the lens to deflect the laser beam; however, a spatial filter was required to select the first-order diffraction beam and obstruct the zero-, second-, and higher-order diffractions to reduce extraneous scattered light on the microchip. The accuracy of the laser spot position on the microchip was governed by a voltage that was output from a computer. This output voltage was converted by a D/A converter, and then sent to the AOD driver where the voltage-frequency converter output the desired RF signal to the AOD. Fluorescence emitted from the excited sample was collected by an objective, passed through a dichroic beam splitter, and spectrally filtered before being received by the PMT. The PMT information was amplified and electronically filtered to reduce environmental noise for signal optimization. Several parameters specific to application of the AOD microchip scanning technology were evaluated. Although distances were small for microchip applications, scanning range and speed were (23) Hjerte´n S. J. Chromatogr. 1985, 347, 191-198.

Figure 1. Acousto-optical deflection-based laser-beam scanning. (A) Optical setup for AOD scanning. Components are the following: 1-beam expander, 2-lens, 4-AOD, 5-spatial filter, 6-dichroic beam splitter, 7-objective, 8-filter, 9-PMT, 10-amplifier and filter, 11computer, 12-voltage-frequency converter. (B) Microchannel configuration on an 8-channel microchip. (C) Detection window for laserbeam scanning.

important criteria, as was compensation for laser intensity variations due to the scanning method used. Prototype software must also be developed for AOD control, channel identification, and data collection in an interfaceable environment in a manner that does not limit the number of microchannels interrogated during electrophoretic separation. Scanning Range. The scanning range is governed by the deflection angle (θ) and the distance between the AOD and the microchip. The maximum one-dimensional scanning distance possible with the AOD can be approximated by the relationship

Scanning distance ≈ D tan θ

where D represents the distance between the AOD and the microchip and θ is the deflection angle of the first-order laser beam from the AOD. To evaluate the optical setup for AOD scanning, the scanning process was visualized by substituting a CCD camera with a video capture device for the PMT, amplifier, and filter shown in Figure 1A. Using an AOD with θ ) 0.28° and having the AOD and the microchip separated by 230 mm (D), a calculated scanning range of 1.12 mm results. The microchip possessed microchannels that were 50 µm (w) × 20 µm (d) with center-tocenter spacing of 150 µm (Figure 1B). Consequently, the 1.12mm scan distance was adequate to cover the 1.1-mm distance between channel 1 and channel 8 in the detector window (Figure 1C). The channels were filled with an aqueous solution of 10-5 M Analytical Chemistry, Vol. 71, No. 23, December 1, 1999

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Figure 3. PMT voltages as the laser beam scans across 6 channels filled with 10-5 M 6-carboxyfluorescein. Rawswithout processing shows fluorescence intensity attenuation. Compensatedsintensity software compensation as described in text. The dotted line is the D/A output voltage to drive laser-beam raster scanning.

Figure 2. Laser-beam scanning of 6 channels filled with 10-5 M 6-carboxyfluorescein. Frames of the laser-beam step scanning across the microchannels, captured from the video record.

6-carboxyfluorescein (6-CF) by filling the inlet and sample reservoirs and applying a vacuum at the outlet reservoir. Frame 1 of Figure 2 shows a white light micrograph of the detection window with the channels filled with 6-CF. The translucent appearance of channels 1 through 6 indicates successful filling with 6-CF while the opaque appearance of channels 7 and 8 likely represents particulates in the microchannel. These channels were found to be irreversibly occluded, and hence, only channels 1-6 were used for scanning experiments. AOD scanning was initiated by having the D/A converter output a voltage from 0 to 10 V linearly to a voltage-to-frequency (V-F) converter driver which controlled the laser beam scanning across the six functional channels on the microchip in a linear fashion. The video signal from a CCD camera (320 × 240 pixel resolution) was captured at 30 frames per second, displayed on a screen, and recorded by the computer. The remaining frames in Figure 2 show sequential video-captured images as the laser beam is acousto-optically scanned from channel 1 through channel 6. Note how the beam is focused on the center of each channel (channels 1-6) as it scans across the detector window at 1 mm/s. Intensity Compensation. It is known that the power of the first-order deflected laser beam from the AOD is proportional to the acoustic power in the AO material, the elasto-optic figure of merit of the interaction medium, and a geometric factor and inversely proportional to the square of the wavelength.22 This can be visualized in Figure 2 where the fluorescence intensity of the focused laser beam decreases markedly as the laser beam progresses from channel 1 to channel 6. This angle-dependent 5312 Analytical Chemistry, Vol. 71, No. 23, December 1, 1999

Figure 4. PMT voltage shows the lack of cross-talk between the channels. AOD laser-beam scan across the channels with 10-5 M 6-carboxyfluorescein in channels 1, 2, 5, and 6, and 10-6 M 6-carboxyfluorescein in channels 3 and 4.

attenuation of the laser beam intensity can be seen graphically in Figure 3 where the microchannels have been filled with 10-5 M 6-CF. The laser intensity attenuation showed an approximately linear reduction with the deflection angle and can be corrected by a linear compensation, which multiplies the raw input data by a coefficient which transforms the data to nullify the deflectionangle-dependent attenuation of the fluorescence intensity (Figure 3). The slight peak-to-peak dissimilarities probably result from subtle differences in the physical characteristics of the channels, e.g., width, depth, and channel-wall conditions. With respect to channel-to-channel cross-talk, the following indicate that this is not problematic. First, the sequential video-captured images given in Figure 2 show no signs of cross-talk problems using this scanning approach. This was confirmed by nonwhite light illuminated scanning where the only observed effect was a slight reflection of light on the wall when the laser beam was focused on a neighboring channel (data not shown). Second, channels 1, 2, 5, and 6 were filled with the 10-5 M 6-CF while channels 3 and 4 were filled with 10-6 M 6-CF and AOD scanning was initiated (Figure 4). Fluorescence from each of the channels appears distinct, and the apparent lack of cross-talk indicates that this is not likely to be a problem. AOD Scanning Speed. As electrophoretic analyses are reduced from minutes to seconds and even to a millisecond time scale,24 laser-beam scanning speed for fluorescence detection in multichannel/multifunctional platforms becomes critically impor(24) Jacobson, S. C.; Culbertson, C. T.; Daler, J. E.; Ramsey, J. M. Anal. Chem. 1998, 70, 3476-3480.

tant. This not only applies to fluorescence detection in multiple microchannels on a single microchip, but also to fluorescence detection in multiple domains on a microchip as a means of monitoring the execution of other on-chip processes. For multichannel electrophoretic separations with total analysis times of at least several hundred seconds, fluorescence detection can be successfully accomplished using a CCD camera,7,12 a translating stage,13,14 or a galvanometer.15 While the CCD approach is effective for these analyses, it is limited to exposure times on the order of hundreds of milliseconds and, therefore, is associated with a frequency of data collection of ∼3 Hz.12 The translating-stage approach has been demonstrated with a bidirectional scan rate of 3.3 Hz13 and has not been shown to be functional at higher scan rates, presumably due to mechanical-movement limitations. Thus far, galvanometers have provided the most effective solution, with laser or optical beam scanning rates that are much higher.15 While some galvanometric scanners are capable of scanning speeds of more than 1 kHz with large scanning ranges, this speed has not yet been demonstrated in the literature for microchip applications, probably due to the fact that a ball-bearing-type galvanometric scanner exhibits a noticeable jitter and wobble. For high-cycle applications, these scanners will eventually wear and show a dramatic decrease in the smoothness of scanning. A flexure galvanometer, without wear, shows a much smoother motion throughout its life, but still suffers jitter and wobble for microscopic-range applications. Finally, it is worthy of note that the fast step scanning with micrometer-range accuracy required for microchannel detection on microchips is difficult to realize with galvanometers. The attractiveness of acousto-optic scanning is rooted in its simplicity, as well as in its ability to circumvent many of the limitations associated with conventional scanning technologies. For example, the access time for an AOD represents the time required for the acoustic wave to fill the aperturesthis is in the microsecond or nanosecond range for most AODs. The inexpensive AOD employed here has an access time of 180 nanosecondss this has two important ramifications. First, the time spent traversing the “dead space” between channels (i.e., re-addressing the laser beam to the next channel) is negligible at only a fraction of a microsecond. This allows for the vast majority of the scanning time to be utilized for capturing fluorescence from the channels, not traversing the channels. Second, with access times on the nanosecond time scale, higher scanning frequencies should be attainable. Figure 5 shows a single frame of a video signal captured from a CCD camera when AOD scanning was carried out with a frequency of 200 Hz over 6 channels filled with 6-CF small (10-5 M). Any attenuation of the laser beam intensity due to deflection of the beam can be compensated for effectively as discussed earlier. With scan rates in this frequency range clearly attainable, the possibility of doing ultrafast multichannel separations (faster than 10 s and into the millisecond time regime) with peak widths as small as 100-500 milliseconds10,11 becomes feasible. Consequently, the speed of AOD based scanning will allow it to address multichannel fluorescence detection for fast separations in a large number of channels or ultrafast separations (second to millisecond time scale) in a smaller number of channels.

Figure 5. Ultrafast AOD scanning across the microchannels, captured from the video record with the laser-beam scanning rate of 200 Hz.

Accurate Laser Beam Addressing Allows for Self-Aligned Fluorescence Detection. Of the raster (uni- or bidirectional), step, or random scanning modes possible with acousto-optical deflection, the most efficient of these modes is the randomaddressing mode, where the laser beam moves from one position to another rapidly with the dwell time at each position controlled independently. With the AOD used in these experiments, the addressing time is dependent on and calculated from the AOD access time (180 ns at 488 nm) and the frequency tuning slew rate of the AOD driver (>10 MHz/µs). Therefore, addressing the laser beam from channel 1 to channel 3 requires an applied voltage difference of ∼4 V, corresponding to a driver output frequency difference of ∼10 MHz and a resultant addressing time of less than 1 µs. Furthermore, an ON/OFF function on the AOD can block the laser beam completely when changing the address from one channel to another, minimizing scatter and cross-talk during multichannel scanning. This capability does not exist with galvanometric- or translation-stage-based scanning. One of the inherent advantages of the random addressability of AOD based scanning is that automated channel alignment is possible for optimizing detection. The simplest approach for accomplishing this is to utilize a multichannel microchip where the two outside channels, filled with a fluorescent solution, function as “alignment channels”. Following a slow AOD-based scan of all the channels, the alignment channel centers are identified and the centers of all channels in between calculated on the basis of the physical configuration of the microchannels. This was demonstrated with the eight-channel microchip shown in Figure 1 by using channels 1 and 6 (filled with 10-5 M fluorescein) for alignment and using channels 2 and 5 (acrylamide-coated using a modified Hjerte´n method23) for electrophoretic separation of a Hae III digest of pBR322. Prior to initiation of the separation, the computer, through the D/A converter, output voltages from 0 to 10 V to drive the laser-beam raster scanning over all channels, while simultaneously collecting fluorescence data from the PMT via the A/D converter. Through the software program, fluorescence peaks were detected as the laser beam scanned across the alignment channels, and the D/A output voltages at the peak were stored as “addressing voltages” for the alignment channel centers. On the basis of the physical configuration of the microchannels on the microchip (width, center-to-center spacing), the addressing Analytical Chemistry, Vol. 71, No. 23, December 1, 1999

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Limitations of AOD Scanning with LabView Driven Applications. Attempts to execute the dsDNA fragment separation in four channels (using the two outside channels for alignment) were found to be problematic due to software limitations associated with the use of LabView on the Microsoft Windows platform. The reasons underlying this is that LabView, an effective development environment for Windows-based control and measurement, has “user-friendly” sub-vi icons that cloak a number of unused codes which must be executedsthis seriously limits rapid control, sampling, and data analysis. For this reason, the multichannel electrophoretic separations carried out utilizing real-time control of high-voltage application to the microchip, laser scanning, data sampling, display and processing, had a maximum rate of 30 Hz for each cycle. This limitation can be overcome with C Programming or by adopting a microprocessor-embedded system. Figure 6. Separation of pBR322 Hae III digest in two microchip channels employing the AOD scanning system. Channels were polyacrylamide-coated and filled with 1% HEC in 1× TBE as the sieving matrix. A 275 V/cm potential was applied for 120 s for the injection and a 165 V/cm potential for 230 s for the separation. All other channels were empty except the alignment channels filled with 10-6 M 6-carboxyfluorescein. The data sampling rate was 10 Hz.

voltages for the separation-channel centers (microchannels 2-5) were automatically calculated and stored. Once sample was injected and separation initiated, these addressing voltages were sent sequentially to the AOD, driving the laser beam directly to the center of separation channel 2 and 5 with a 1-ms software delay (due to system dependence) at each channel, where laserinduced fluorescence was exacted through an intercalating dye added to the buffer. The A/D converter sampled the PMT signals as the laser beam stopped at each channel for the 1-ms dwell time with a data collection rate of 10 Hz. The data were deconvoluted so that electropherograms for each channel were obtained, and compensated to nullify the attenuation of the fluorescence intensity. As can be seen in Figure 6, the simultaneous separation and detection of the dsDNA fragments of pBR322 was accomplished. Similar migration times were observed with both channels with a sampling rate of 10 Hz sufficient for complete peak representation. This provides evidence that, via a single scan of the laser over all channels, the AOD based detection system can accurately locate the channel centers, assign addressing voltages, and execute LIF detection from multiple channels simultaneously. One can easily begin to envisage how alignment channels can be built-in to microchip designs as well as how rapid and frequent recalibration may be accomplished during the separation. Rescanning the alignment channels would ensure that the stored addressing voltages were still correctsany alteration would initiate a recalculation of the new addresses. (25) Fodor, S. A. Science (Washington, D.C.) 1997, 277, 393-395.

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CONCLUSIONS This study demonstrates the development of a novel approach to laser-beam scanning fluorescence detection on multichannel microchips based on acousto-optic deflection. The speed, accuracy, and flexibility of AOD scanning provide unique possibilities for simple fluorescence detection with large capillary arrays or on microchips with either a large number of channels or multifunctional domains. While conventional scanning approaches are based on mechanical motion and, hence, limited by mechanical speed, acousto-optic scanning produces very fast, high-resolution positioning of the laser beam without any moving parts. The temporal resolution achieved by the system at this stage of development is ∼ 200 ns, easily allowing for multichannel detection with relatively fast electrophoretic separations (100 seconds). An additional distinguishing feature of this system is the customized scanning rate, which not only allows considerable flexibility in separation speed and the number of channels interrogated, but also allows autoalignment for optimal detection. Applications for this scanning technology are numerous, ranging from applying it to existing capillary-based systems or to the evolving microchip technology. The latter holds the most promise, and one could envisage applying this to any microchip technology application that requires accurately controlled detection at multiple points on the device. It is also important to note that AOD scanning is not limited to electrophoresis. Extrapolation of this technology to other fluorescence-imaging applications such as detection on DNA hybridization chips25 or even retinal scanning holds great potential. Future efforts toward utilizing this system for high-speed, highthroughput electrophoretic analysis will be focused on adopting a microprocessor-embedded system to allow for ultrafast laser scanning of a large number of channels, as well as being focused on issues that will allow for sensitive fluorescence detection. Received for review July 7, 1999. Accepted September 30, 1999. AC990740U