Anal. Chem. 2006, 78, 5590-5596
Multicolor Fluorescence Detection on an Electrophoretic Microdevice Using an Acoustooptic Tunable Filter James M. Karlinsey† and James P. Landers*,†,‡
Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, and Department of Pathology, University of Virginia Health Science Center, Charlottesville, Virginia 22903
An acoustooptic tunable filter (AOTF) is used to detect multiple fluorescent signals on a fluidic microdevice. A confocal laser-induced fluorescence detection setup is used to excite fluorescent dyes in glass microchannels, presenting a streamlined and robust detection system consisting of the narrow-bandwidth AO filter and a single photodetector. The flexibility of the filter is demonstrated by alternating between wavelengths for precise microchannel alignment and sweeping through a range of wavelengths for preliminary spectral characterization of subnanoliter probe volumes of target analytes. The AOTF is also coupled with an electrophoretic separation for the multicolor detection of PCR-amplified DNA against a labeled sizing standard, the discrimination of multiple amplicons overlapped in time, and the identification of amplified biowarfare agents in a fluorescent spiking experiment. Finally, to demonstrate the multicolor capability of the system, 19-wavelength detection is performed during the separation of a three-dye sample mixture. Much of the analytical technology that has been developed for capillary electrophoresis separations is being transferred to a microchip format in an effort to realize a micro total analysis system.1-3 The most common form of detection on these microdevices is fluorescence, especially when high sensitivity is required.4 However, as samples become more complex, it is of great interest to perform multicolor detection for simultaneous monitoring of various analytes of interest. While few analytes exhibit native fluorescence, several methods are available to derivatize nucleic acids, proteins, and small molecules with a variety of fluorescent molecules.5 What remains to be developed, however, is a simple, flexible, and robust detection platform that can be paired with microchip separation technology in a manner that will enable user* To whom correspondence should be addressed. Phone: 434-243-8658. Fax: 434-243-8852. E-mail:
[email protected]. † University of Virginia. ‡ University of Virginia Health Science Center. (1) Vilkner, T.; Janasek, D.; Manz, A. Anal. Chem. 2004, 76, 3373-3386. (2) Auroux, P.-A.; Iossifidis, D.; Reyes, D. R.; Manz, A. Anal. Chem. 2002, 74, 2637-2652. (3) Reyes, D. R.; Iossifidis, D.; Auroux, P.-A.; Manz, A. Anal. Chem. 2002, 74, 2623-2636. (4) Johnson, M. E.; Landers, J. P. Electrophoresis 2004, 25, 3513-3527. (5) Haugland, R. P. The Handbook-A Guide to Fluorescent Probes and Labeling Technologies, 10th ed.; Invitrogen, Molecular Probes, 2006.
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selectable multiwavelength filtering. Using an acoustooptic tunable filter (AOTF) provides one simple approach that, in addition to other advantages, allows for the detection of multiple emission signals from laser-induced fluorescence on analytical microchips. Simultaneous monitoring of multiple wavelengths provides qualitative information about a species, but requires that a wavelength selector be introduced into the simple single-color systems routinely utilized in microchip applications. The selector may either be a series of filters with defined wavelength ranges or a grating or prism to detect over a range of wavelengths. For the most part, these techniques are first demonstrated on a capillary format, and several groups have implemented techniques for dispersing wavelengths onto array detectors such as charge-coupled devices (CCDs) and photodiode arrays.6-9 However, this technique has been seldom used for multicolor detection on microchip.10 Instead, several research groups have turned to filter sets, likely due to the sensitivity of the available photodetectors.4 In an attempt to avoid the moving parts associated with using a filter wheel, current methods for filtering fluorescence emission from microchip samples primarily include placing the appropriate filters before multiple photomultiplier tubes. Mathies and co-workers11,12 developed a functional and sensitive four-color confocal fluorescence scanner for DNA sequencing on capillary and then adapted it for the microchip format.13-15 This scanner is characterized by four separate photodetectors, along with several additional optics for splitting and filtering the emission beam. This same idea was used by other groups for both two-color16 and four-color17-20 de(6) Cheng, Y. F.; Piccard, R. D.; Vodinh, T. Appl. Spectrosc. 1990, 44, 755765. (7) Sweedler, J. V.; Shear, J. B.; Fishman, H. A.; Zare, R. N.; Scheller, R. H. Anal. Chem. 1991, 63, 496-502. (8) Karger, A. E.; Harris, J. M.; Gesteland, R. F. Nucleic Acids Res. 1991, 19, 4955-4962. (9) Zhang, X.; Stuart, J. N.; Sweedler, J. V. Anal. Bioanal. Chem. 2002, 373, 332-343. (10) Backhouse, C.; Caamano, M.; Oaks, F.; Nordman, E.; Carrillo, A.; Johnson, B.; Bay, S. Electrophoresis 2000, 21, 150-156. (11) Ju, J. Y.; Kheterpal, I.; Scherer, J. R.; Ruan, C. C.; Fuller, C. W.; Glazer, A. N.; Mathies, R. A. Anal. Biochem. 1995, 231, 131-140. (12) Kheterpal, I.; Scherer, J. R.; Clark, S. M.; Radhakrishnan, A.; Ju, J. Y.; Ginther, C. L.; Sensabaugh, G. F.; Mathies, R. A. Electrophoresis 1996, 17, 1852-1859. (13) Liu, S.; Shi, Y.; Ja, W. W.; Mathies, R. A. Anal. Chem. 1999, 71, 566-573. (14) Shi, Y. N.; Simpson, P. C.; Scherer, J. R.; Wexler, D.; Skibola, C.; Smith, M. T.; Mathies, R. A. Anal. Chem. 1999, 71, 5354-5361. (15) Paegel, B. M.; Emrich, C. A.; Wedemayer, G. J.; Scherer, J. R.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 574-579. 10.1021/ac0607358 CCC: $33.50
© 2006 American Chemical Society Published on Web 07/04/2006
tection. With this type of system, each photomultiplier tube (PMT) is continuously sampled, eliminating the possibility of temporal aliasing. However, it requires multiple optical components, including three beam splitters, four band-pass filters, and four detectors (for a four-color system). While these systems have been shown to be extremely effective, a single tunable filter and photodetector would offer a more rugged and adaptable system for multicolor applications. To this end, we propose the AOTF for multicolor fluorescence detection. With the AOTF, there are no moving parts, the filtering wavelengths are user-selectable, and there is no practical limit to the number of wavelengths that can be monitored. The theory and practice of acoustooptic filtering have been described thoroughly in the literature.21-25 Several AOTF-based detection systems have been developed by Tran and co-workers, with applications in spectroscopy and chemical separations.26-29 These include a quartz AOTF used in a collinear configuration as a rapid scanning detection system for HPLC in the UV-visible region27 and a noncollinear TeO2 AOTF used in a fluorescence detector for flow injection analysis.28 A polychromator was also developed where two AOTFs were used simultaneously for multiple excitation wavelengths and detection at multiple emission wavelengths, prompting the authors to theorize the possibility of analyzing a sample containing up to 100 different components.29 This system resembled earlier work by Kurtz et al., who used two AOTFs to perform fluorescence spectroscopy of a pH-sensitive dye.30 More recently, an AOTF has been evaluated as a spectrometer to detect the radiation emitted by thermally excited samples in the near-IR, creating an opportunity to investigate chemical systems at high temperature.31 Benefits of using an AOTF are that it is solid state, compact, and has no moving parts. This has been exploited by several groups in the development of portable Raman spectrometers,32-35 demonstrating the utility of the AOTF for remote sensing. While AOTF-mediated multiwavelength detection has previously been coupled with HPLC27 and FIA,28 it is perfectly suited to microchip electrophoresis, since it enables precise microchannel alignment, spectral characterization of subnanoliter probe volumes, and multicolor discrimination of electrophoretic species. Consequently, this work describes the first implementation of (16) Schmalzing, D.; Koutny, L.; Chisholm, D.; Adourian, A.; Matsudaira, P.; Ehrlich, D. Anal. Biochem. 1999, 270, 148-152. (17) Schmalzing, D.; Tsao, N.; Koutny, L.; Chisholm, D.; Srivastava, A.; Adourian, A.; Linton, L.; McEwan, P.; Matsudaira, P.; Ehrlich, D. Genome Res. 1999, 9, 853-858. (18) Liu, S. R.; Ren, H. J.; Gao, Q. F.; Roach, D. J.; Loder, R. T.; Armstrong, T. M.; Mao, Q. L.; Blaga, I.; Barker, D. L.; Jovanovich, S. B. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 5369-5374. (19) Koutny, L.; Schmalzing, D.; Salas-Solano, O.; El-Difrawy, S.; Adourian, A.; Buonocore, S.; Abbey, K.; McEwan, P.; Matsudaira, P.; Ehrlich, D. Anal. Chem. 2000, 72, 3388-3391. (20) Goedecke, N.; McKenna, B.; El-Difrawy, S.; Carey, L.; Matsudaira, P.; Ehrlich, D. Electrophoresis 2004, 25, 1678-1686. (21) Bei, L.; Dennis, G. I.; Miller, H. M.; Spaine, T. W.; Carnahan, J. W. Prog. Quant. Electron. 2004, 28, 67-87. (22) Chang, I. C. Opt. Eng. (Bellingham, Wash.) 1977, 16, 455-460. (23) Chang, I. C. Opt. Eng. (Bellingham, Wash.) 1981, 20, 824-829. (24) Tran, C. D. Anal. Chem. 1992, 64, 971A-978A, 980A-981A. (25) Tran, C. D. Anal. Lett. 2000, 33, 1711-1732. (26) Tran, C. D. Talanta 1997, 45, 237-248. (27) Tran, C. D.; Lu, J. Anal. Chim. Acta 1995, 314, 57-66. (28) Tran, C. D.; Lu, J. Appl. Spectrosc. 1996, 50, 1578-1584. (29) Tran, C. D.; Furlan, R. J. Anal. Chem. 1993, 65, 1675-1681. (30) Kurtz, I.; Dwelle, R.; Katzka, P. Rev. Sci. Instrum. 1987, 58, 1996-2003. (31) Gonzaga, F. B.; Pasquini, C. Anal. Chem. 2005, 77, 1046-1054.
Figure 1. Experimental setup for multicolor LIF detection. Excitation light shown in black, emission in gray. Components: (1) dichroic beam splitter, (2) microscope objective, (3) long-pass filter, (4) mirror, (5) pinhole, (6) achromat lens, (7) AOTF, (8) beam block, and (9) PMT. Inset: cross-tee chip design, with sample (S), waste (W), buffer inlet (I), buffer outlet (O), and alignment channels (A) on either side of the separation channel. S, W, and I are 6 mm long, and O is 75 mm.
AOTF technology in a fluorescent detection system for electrophoresis on the microscale. The AOTF is alternated between wavelengths for rapid alignment, scanned to collect spectral information from target analytes, and employed for multicolor detection during the separation of fluorescently labeled DNA. This highlights the multifunctional nature of the AOTF as a simple and effective tool for microdevice-based fluorescence detection. EXPERIMENTAL SECTION AOTF. The AOTF (model TEAF5-0.45-0.70-S, Brimrose Corp., Baltimore, MD) consists of a tellurium dioxide (TeO2) crystal bonded to a lithium niobate (LiNbO3) crystal, which acts as a piezoelectric transducer. Parameters for the TeO2 crystal are an acoustic velocity νa ) 6.17 × 104 cm/s and indices of refraction ni ) 2.45 and nd ) 2.26. The AOTF was calibrated using three selectable laser lines (457, 488, and 514 nm) from an argon ion laser and two HeNe lasers (594 and 632.8 nm). Experimental Setup. A conventional confocal detection setup was used,36 and a schematic is provided in Figure 1. A multiline argon ion laser (Laser Physics, West Jordan, UT) was used as the excitation source. Excitation light was reflected off of a dichroic filter (cut on 525 nm) (Omega Optical) and focused onto the microchannel with a 40× objective (LD Achroplan 44 08 64, Zeiss). Emission light was filtered with a 525-nm long-pass filter (Omega Optical) after passing through the dichroic and focused onto a 1-mm pinhole (National Aperture). The emission light was then collimated with an Achromat lens (Rolyn Optics) and directed into the AOTF window. After the AOTF, a beam block was placed to eliminate undiffracted light (0th order). The two first-order diffracted beams were collected and focused onto an H5784 PMT (Hamamatsu, Bridgewater, NJ) using two Achomat lenses (Rolyn Optics). The diffraction angle requires that the beam block be sufficiently distanced from the AOTF to avoid clipping the wavelengths of interest; thus, large (35 mm) lenses were used to collect the diffracted beams. Since each wavelength is diffracted to the same spot (within 1°), the AOTF, collection lenses, and PMT were connected to reduce mechanical instability. (32) Lewis, E. N.; Treado, P. J.; Levin, I. W. Appl. Spectrosc. 1993, 47, 539543. (33) Gupta, N.; Fell, N. F. Talanta 1997, 45, 279-284. (34) Cullum, B. M.; Mobley, J.; Chi, Z. H.; Stokes, D. L.; Miller, G. H.; Vo-Dinh, T. Rev. Sci. Instrum. 2000, 71, 1602-1607. (35) Mobley, J.; Cullum, B. M.; Wintenberg, A. L.; Frank, S. S.; Maples, R. A.; Stokes, D. L.; Vo-Dinh, T. Rev. Sci. Instrum. 2004, 75, 2016-2023. (36) Ocvirk, G.; Tang, T.; Harrison, D. J. Analyst 1998, 123, 1429-1434.
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Computer Control. LabVIEW code was written in-house to control the voltage application, AOTF switching, signal acquisition, and data analysis. Four 5-kV power supplies (Spellman, Hauppauge, NY) were connected to the analog output pins on a PCI6713 card (National Instruments), and relays were used to switch between voltage, ground, and float. An SPS controller system (Brimrose) was connected through an RS-232 port and delivered the appropriate frequency to the AOTF. Signal collected by the PMT was sent to a PCI-6014 analog input card (National Instruments, Austin, TX). Microchip Fabrication. The microchannels were fabricated in Borofloat glass coated with chrome and photoresist (Telic, Valencia, CA) using standard photolithography and wet chemical etching.37 The chip is patterned with a cross-tee architecture, with 6-mm-long sample, sample waste, and buffer inlet arms, and a 75mm-long separation channel. Initial line widths were patterned at 50 µm and then etched 50 µm deep. The chip design incorporates additional channels of the same width and depth as the separation channel, which can be used for alignment or spectral characterization of analytes of interest. Access holes were drilled at the end of each arm of the cross-tee and at the ends of the alignment channels with 1.1-mm diamond-tipped bits (Abrasive Technology, Lewis Center, OH), and the etched plate was then thermally bonded to a cover glass in a furnace. Nanoport reservoirs (Upchurch Scientific, Oak Harbor, WA) were placed at the fluidic access holes. Reagents. Ultrapure water (Barnstead, Dubuque IA) was used to prepare all aqueous solutions. Methanol, hydrochloric acid, sulfuric acid, and sodium hydroxide used to condition the microchips were obtained from Fisher Scientific (Pittsburgh, PA). The microchip for single-stranded DNA separations was coated using the silanizing agent TMSPM (Sigma-Aldrich, St. Louis, MO) followed by a solution of poly(vinyl alcohol) (Fluka, Milwaukee, WI), ammonium persulfate and TEMED (Fisher). Fluorescently labeled PCR primers were obtained through MWG Biotech (High Point, NC). POP-4 sieving matrix, TBE buffer, and GeneScan 500 Size Standard were purchased from ABI (Foster City, CA). Hidiformamide was obtained through Fisher. Mixed borate buffer was prepared from boric acid and Borax (Sigma-Aldrich). Stock solutions of fluorescein (Sigma-Aldrich), rhodamine-6G (R6G, Acros Organics, Morris Plains, NJ), and carboxy-X-rhodamine (ROX, Fluka) were dissolved in buffer and stored at -20 °C when not in use. Methods. For alignment and spectral scanning, 1 µL of dye solution was placed in the alignment channel and a drop of mineral oil was placed above the access holes to prevent evaporation. To focus the detection system within the microfluidic channel, the AOTF was operated in a hopping mode between an emission wavelength and a “dark” wavelength where no emission was expected (i.e., 450 nm). The z-position was adjusted manually using an actuator with coarse 10-µm-scale resolution to obtain the optimum signal. To collect emission spectra on analytes in the alignment channel, the AOTF was swept through a wavelength region where fluorescence was expected (530-620 nm). Prior to coating the microchannels, the surface was conditioned with 1:1 MeOH/HCl, rinsed with ddH2O, and then rinsed with concentrated sulfuric acid. This method has been shown to clean (37) Manz, A.; Fettinger, J. C.; Verpoorte, E.; Ludi, H.; Widmer, H. M.; Harrison, D. J. TrAC-Trend. Anal. Chem. 1991, 10, 144-149.
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the surface and increase the number of exposed silanol groups on glass slides.38 The microchannels were then coated using the Hjerten method.39 POP-4 was pulled into the channels using vacuum, and 1× TBE was added to the reservoirs as the run buffer. DNA amplicons for Bacillus anthracis pXO1 (211 bases) and Salmonella typhirium Invasiveness A (278 bases) were labeled through a PCR reaction using TET- or ROX-labeled forward primers. Product was filtered on a Microcon spin-column (Millipore, Billerica, MA) to remove salts and subsequently diluted in hidi-formamide, heat denatured, and snap cooled. Injections were performed by applying a forward bias of 420 V/cm across the sample and waste arms, while a field of 200 V/cm from the buffer inlet to the outlet was used to perform the DNA separations. A second uncoated cross-tee chip was used for the separation of the three dyes using electroosmotic flow. The channel surface was conditioned with 1:1 MeOH/HCl and concentrated sulfuric acid, as described above, followed by 1 M NaOH and buffer. Injection was performed at 500 V/cm for 10 s, followed by a 400 V/cm separation with a pullback of 67 V/cm. Data analysis was performed postcollection using a LabVIEW program designed to correlate wavelength, time, and signal. RESULTS AND DISCUSSION For utilization of the AOTF as an emission filter in the confocal detection setup (Figure 1), it was placed after the pinhole and a collimating lens that forms a spot, roughly 3 mm in diameter, on the AOTF window. The diffracted beam exiting the AOTF is spatially separated from the transmitted beam with a separation angle of 7.2-6.2° and a peak diffraction efficiency of 85%. The crystal diffracts not a single wavelength, but a wavelength band, the width of which is determined by the crystal properties. For the crystal and wavelength regime (530-620 nm) used in this work, the resolution range of the system is 2.5-4.5 nm with the spectral resolution degrading with increasing wavelength.40 It is possible to achieve higher resolution with an AOTF, but even the present device offers an improvement over conventional bandpass filters (∼10-nm bandwidth). The data acquisition rate utilized in these experiments was 18 Hz, but this does not represent the data acquisition rates capable with the AOTF. The switching speed of the AOTF is limited by the transit time of the acoustic wave as it crosses the optical beam (i.e., at 6.17 × 104 cm/s, it takes 3.2 µs), which suggests a theoretical frequency limitation of ∼3 MHz. However, the speed is also dependent upon the rf generation and the computer processing speed; thus, this lower rate was chosen to prevent temporal aliasing of the fluorescence signal. While sensitivity is a function of the overall detection system, the detector itself plays a very important role. For this reason, a comparison between a PMT and a CCD array is rather arbitrary. A comparison between PMT systems is also difficult to achieve. In systems with multiple photodetectors, the large bandwidths that result from using the dichroic beam splitters inevitably allow more light to pass. In the present system, sensitivity is sacrificed somewhat for selectivity, as the bandwidth associated with the chosen wavelength is 2.5-4.5 nm. The benefit of using a system (38) Cras, J. J.; Rowe-Taitt, C. A.; Nivens, D. A.; Ligler, F. S. Biosens. Bioelectron. 1999, 14, 683-688. (39) Hjerten, S.; Elenbring, K.; Kilar, F.; Liao, J. L.; Chen, A. J. C.; Siebert, C. J.; Zhu, M. D. J. Chromatogr. 1987, 403, 47-61. (40) Application Note: Acousto-Optic Tunable Filters; Brimrose Corp., Baltimore, MD. 1999.
Figure 2. (A) Signal collected as the channel is translated through the focal point of the objective. ROX dye is in the alignment channel, and the AOTF is alternating between 600 nm (red) where emission is expected and 500 nm (blue) where signal is due to background. The difference in emission and background is shown (dotted line), and this maximum represents the optimal z-axis position. Inset: z-axis translation of the microchip for focusing. (B) Normalized emission spectra for TET and ROX labels, with 540- and 605-nm emission bands shaded.
with multiple photodetectors, of course, is that the individual detectors can be selected with enhanced sensitivity toward a specific wavelength region. The choice of sample is also important, and there is no standard analyte found in the literature. In this work, detection of the labeled PCR product is dependent upon the efficiency of the amplification, which makes it difficult to quantify a detection limit. However, fluorescein was detected at 10-10 M using this system, with a signal-to-noise ratio of >20. Alignment and Spectral Characterization. The fluorescence detection setup provides the ability to adjust the z-axis position of the chip to obtain maximum sensitivity. Solution containing fluorophore was added to one of the alignment channels and excited with the same wavelength and intensity to be used in the experiment. The AOTF was then alternated between an emission wavelength for the fluorophore and a second wavelength where no fluorescence signal was expected. This allowed the z-position to be optimized for the fluorescence signal by subtracting out background signal due to light scatter and reflection collected at the second wavelength. An example of this is provided in Figure 2A using ROX dye with 514-nm excitation. While there is little variation between the signal and difference maximums in this example, this is expected to be affected by channel geometry changes (in width or depth), different microchip substrates, or when different excitation wavelengths are selected. Because the AOTF provides multicolor detection, alignment could also be performed directly in the analysis channel using a fluorophore with different spectral character than the analytes of interest. This could be used when alignment adjustments need to be made in real time. The alignment channels were also used for spectral characterization of the TET and ROX fluorescent labels used in this work by sweeping the AOTF through the visible spectrum to collect emission spectra (Figure 2B). In this way, the AOTF was utilized as a microfluorometer with a subnanoliter probe volume. The fluorescence signal was collected as the AOTF was scanned multiple times, and the signal was then averaged and normalized taking into account an average bandwidth of ∼3 nm. The emission profile is only useful above 530 nm because of the 525-nm dichroic and emission filters required for excitation with the 514-nm line of the laser. However, this did not prevent comparisons between
the dye spectra and allowed the contributions from each dye at each of the wavelengths selected for monitoring to be determined. Due to the broadband emission of organic fluorophores, the problem of spectral overlap becomes more complex with an increasing number of fluorescent species. With this system, sufficient information can be obtained by including enough channels to accommodate each species of interest, and the appropriate monitoring wavelengths can then be selected before the separation and analysis. Separation of Fluorescently Labeled DNA. A common application for multicolor fluorescence detection is DNA separations in which DNA has been amplified using multiple labeled primers in a PCR reaction. Standards, labeled with a unique fluorescent tag, are often mixed with the product before separation to verify the size of fragments present in the amplified sample. In Figure 3, a ROX-labeled size standard (15-500 bases) is coinjected with a PCR-amplified fragment from the anthrax genome labeled
Figure 3. Amplified TET-anthrax fragment coinjected electrokinetically with a ROX sizing standard and separated under denaturing conditions. Data were obtained by hopping the AOTF between emission wavelengths at 18 Hz, and the electropherograms are offset for clarity. Inset: sizing plot with ladder (red diamond) and anthrax (green square) peaks.
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Figure 4. (A) Mixture of TET-labeled anthrax and ROX-labeled salmonella amplicons showing that while they do overlap in time, they can still be separated based upon their spectral character. Inset: the unfiltered signal is presented for comparison. (B) TET-labeled anthrax, and (C) ROX-labeled salmonella amplicons injected by themselves.
with TET dye. The raw signal was filtered using the spectral information collected for each label (Figure 2B, with shading over the selected wavelengths: 540 and 605 nm). Once the signal is filtered, the peaks specifically labeled with TET include primer and product, but a small amount of nonspecific product, which can be difficult to predict, also appears to be present. Using the sizing plot of the migration time versus fragment length generated from the ROX-labeled standards (Figure 3, inset), the large TETlabeled peak was determined to be 210 base pairs (which is close to the 211 bp expected for the anthrax fragment). The unknown smaller peak electrophoresed between the 150- and 160-bp standard peaks and sized at 157 bp. Single-wavelength detection would have made it difficult to identify the peaks with an intercalating dye, since the standard peaks would exhibit the same spectral character as the unknown. This type of application would prove even more useful when the amplicon of interest overlaps with one of the standard peaks or when multiple amplifications performed on the same template DNA increases the number of specific or nonspecific product peaks. Another example of multicolor DNA detection utilizes different labels on one of the primers for each target in a multiplex reaction. This makes time-based separation requirements less stringent because the DNA fragments can also be identified by their label. An example of two DNA fragments overlapped in time is shown in Figure 4A where there is no observable separation of a TETlabeled anthrax fragment and a ROX-labeled salmonella fragment in the time-based electropherogram. In Figure 4B and C, each sample is injected and electrophoresed by itself, with detection at two emission wavelengths (540 and 605 nm) filtered to eliminate spectral overlap. When the products are coinjected but not given the separation length required to resolve, the AOTF can detect both emission wavelengths and identify the presence of both anthrax and salmonella in the sample. Note that the profile of each is unique and shifted in time, and it is thus unlikely that one of the peaks is due to background signal. The reduction in effective separation length, with the appropriate spectral deconvolution, lends itself well to the microchip format for increased throughput. 5594
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An interesting application investigated in this work is the possibility of fluorescent spiking. With DNA, this was performed by including the amplicon of interest as part of the standard by labeling it with a specific fluorescent tag. The presence of the same amplicon, through PCR amplification using a primer labeled with a different tag, could then be easily determined. In this work, known anthrax and salmonella fragments were both amplified with a ROX label primer and sized for verification (Figure 5A). The amplicons sized at 219 and 279 bases, respectively. The ROXlabeled commercial size standard, with 11 fragments, was then replaced by the ROX-labeled mixture of only the anthrax and salmonella fragments. A TET-labeled anthrax product was coinjected with this new “standard”, and the separation is shown in Figure 5B. The TET-labeled product electrophoresed identical to the ROX-labeled anthrax amplicon, while there is no TET signal observed at the salmonella fragment peak. The zoomed in view in the inset shows the unknown sample to be both positive (for anthrax) and negative (for salmonella). The result is a streamlined identification system where multiple samples of interest can be probed directly with innocuous preamplified product. In addition, the separation requirements are less stringent because of the decrease in the number of fragments in the standard. Future applications of multicolor DNA detection using the AOTF will include microsatellite analysis (i.e., STRs) and DNA sequencing. In addition, efforts will be taken to increase the wavelength switching speed, which is currently limited by the response of the driver. This will enable more wavelengths to be addressed and increase the overall dwell time at any particular wavelength. 19-Wavelength Fluorescence Detection. To demonstrate the ability to detect several different wavelengths during an experiment, a mixture of fluorescein, R6G, and ROX was injected and separated on chip while the AOTF was swept continually through the emission range. The range was 530-620 nm, with 5-nm increments, for a 19-color experiment. The resulting data are given as a contour plot in Figure 6A, with a time-based separation along the x-axis and a wavelength-based separation along the y-axis (averaged over three wavelengths). The signal
Figure 5. (A) Amplified ROX-anthrax and ROX-salmonella fragments coinjected electrokinetically with a ROX sizing standard and separated to verify the products. Data were also collected for TET emission to demonstrate the absence of TET-labeled product. Inset: sizing plot with ladder (red diamond) and anthrax and salmonella (green square) fragment peaks. (B) Electropherograms for TET-anthrax coinjected with the ROX-labeled amplicons in a two-color spiking experiment. Inset: enlarged view to show the presence of TET-anthrax in the sample.
Figure 6. (A) Contour plot of intensity with wavelength along the horizontal axis and time along the vertical. The intensity scale is displayed in gray scale, with white representing the most intense signal. Peaks for fluorescein (FL), R6G, and ROX are labeled for clarity. The AOTF was scanned from 530 to 620 nm at 5-nm intervals during the separation, and the signal is averaged over five consecutive runs. (B) Spectral scan at three different start times: 1 (green), 4 (red), and 12 s (blue) outlined in the contour plot with dashed lines. (C) Temporal scan at three different wavelengths: 540 (blue), 555 (green), and 605 nm (red) outlined in the contour plot with solid lines.
intensity is represented by the contours and indicates the presence of a peak. The resulting data yields overlap in time for the ROX and R6G dyes and overlap in wavelength for the fluorescein and R6G dyes; however, the contour plot of the mixture clearly identifies the presence of three distinct peaks. The resolution of the plot could be increased in both axes with an increase in the AOTF switching speed, as it would enable smaller wavelength increments and more wavelength scans in the same amount of
wavelength. Individual plots of intensity versus time (Figure 6B) and time (Figure 6C) are provided of the regions of interest for comparison, with no baseline correction or spectral filtering applied. Two-dimensional separations are often used to increase the peak capacity of a separation system, and the AOTF enables this type of analysis with a temporal separation due to electrophoresis combined with a spectral separation due to emission character. Analytical Chemistry, Vol. 78, No. 15, August 1, 2006
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CONCLUSIONS We have demonstrated a versatile detection platform for microscale separations that can be used for precise alignment, spectral characterization of subnanoliter probe volumes, and simultaneous collection of electropherograms at multiple wavelengths. The system has been demonstrated for two-color detection of real-world samples of clinical and forensic interest and has also been extrapolated to 19-wavelength detection. Future work will include faster AOTF switching and increased fluorescence sensitivity of the detection system. Potential applications of this technology include four- and five-color detection for DNA sequencing and forensic identification on chip. This work represents a move toward rapid, point-of-care applications. ACKNOWLEDGMENT Initial work with the AOTF was performed by Dr. Zhili Huang, and the authors appreciate his initial efforts in the development. The authors also thank Dr. Sanford Feldman for the anthrax and salmonella samples, Joan Bienvenue for the purification of the
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DNA, and Lindsay Legendre for her assistance with the PCR amplifications. J.M.K. gratefully acknowledges the SELIM program for a fellowship from the NSF-sponsored IGERT program at UVA, which supported part of this work. We also thank Christopher Easley for helpful discussions regarding data analysis and LabVIEW programming, and Drs. Mitch Johnson and Jerome Ferrance for discussions and advice regarding the optics and aspects of the manuscript. Research funding from the U.S. Department of Justice, Federal Bureau of Investigation under contract J-FBI-03-084 is gratefully acknowledged. The views and conclusions contained in this document are those of the authors and should not be interpreted as necessarily representing the official policies, either expressed, or implied, of the U.S. Government. Received for review April 18, 2006. Accepted May 31, 2006. AC0607358
