Immobilization of DNA Hydrogel Plugs in Microfluidic Channels

Loyola College in Maryland, 4501 North Charles Street, Baltimore, Maryland .... Harry R. Allcock, Mwita V. B. Phelps, and Eric W. Barrett , Michael V...
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Anal. Chem. 2002, 74, 1436-1441

Immobilization of DNA Hydrogel Plugs in Microfluidic Channels Kimberly G. Olsen,*,† David J. Ross,*,‡ and Michael J. Tarlov‡

Loyola College in Maryland, 4501 North Charles Street, Baltimore, Maryland 21210, and National Institutes of Standards and Technology, 100 Bureau Drive, Gaithersburg, Maryland 20899

Acrylamide-modified DNA probes are immobilized in polycarbonate microfluidic channels via photopolymerization in a polyacrylamide matrix. The resulting polymeric, hydrogel plugs are porous under electrophoretic conditions and hybridize with fluorescently tagged complementary DNA. The double-stranded DNA can be chemically denatured, and the chip may be reused with a new analytical sample. Conditions for photopolymerization, hybridization, and denaturation are discussed. We also demonstrate the photopolymerization of plugs containing different DNA probe sequences in one microfluidic channel, thereby enabling the selective detection of multiple DNA targets in one electrophoretic pathway.

Advances in microchip technology are revolutionizing the field of bioanalytical chemistry. DNA microarrays have taken advantage of many of the benefits of miniaturization, including speed of analysis, smaller sample size, and decreased cost.1-8 Many microarray chips have DNA immobilized in a variety of polymeric surface pads to facilitate spatially localized detection of DNA hybridization.9-17 * Corresponding authors. E-mail: [email protected]; [email protected]. † Loyola College in Maryland. ‡ National Institutes of Standards and Technology. (1) Jakeway, S. C.; de Mello, A. J.; Russell, E. L. Fresenius J. Anal. Chem. 2000, 366, 525-39. (2) Wang, S. L.; Fang, Z. L. Spectrosc. Spectral Anal. 2000, 20, 143-8. (3) Sanders, G. H. W.; Manz, A. TrAC, Trends Anal. Chem. 2000, 19, 364-78. (4) Mastrangelo, C. H.; Burns, M. A.; Burke, D. T. Proc. IEEE 1998, 86, 176987. (5) Bruin, G. J. M. Electrophoresis 2000, 21, 3931-51. (6) Cunningham, D. D. Anal. Chim. Acta 2001, 429, 1-18. (7) Quake, S. R.; Scherer, A. Science 2000, 290, 1536-40. (8) McGlennen, R. C. Clin. Chem. 2001, 47, 393-402. (9) Edman, C. F.; Raymond, D. E.; Wu, D. J.; Tu, E. G.; Sosnowski, R. G.; Butler, W. F.; Nerenberg, M.; Heller, M. J. Nucleic Acids Res. 1997, 25, 4907-14. (10) Belosludtsev, Y.; Iverson, B.; Lemeshko, S.; Eggers, R.; Wiese, R.; Lee, S.; Powdrill, T.; Hogan, M. Anal. Biochem. 2001, 292, 250-56. (11) Mitra, Robi D.; Church, George M. Nucleic Acids Res. 1999, 27, e34. (12) Dubiley, S.; Kirillov, E.; Lysov, Y.; Mirzabekov, A. Nucleic Acids Res. 1997, 25, 2259-65. (13) Yershov, G.; Barsky, V.; Belgovskiy, A.; Kirillov, E.; Kreindlin, E.; Ivanov, I.; Parinov, S.; Guschin, D.; Drobishev, A.; Dubiley, S.; Mirzabekov, A. Proc. Nl. Acad. Sci. U.S.A. 1996, 93, 4913-8. (14) Livshits, M. A.; Mirzabekov, A. D. Biophys. J. 1996, 71, 2795-801. (15) Vasiliskov, A. V.; Timofeev, E. N.; Surzhikov, S. A.; Drobyshev, A. L.; Shick, V. V.; Mirzabekov, A. D. Biotechniques 1999, 27, 592. (16) Timofeev, E.; Mirzabekov, A. Nucleic Acids Res. 2001, 29, 2626-34. (17) Tillib, S. V.; Mirzabekov, A. Curr. Opin. Biotechnol. 2001, 12, 53-8.

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Microfluidic channels offer potential analytical advantages over planar arrays, including enhanced mass transfer, lower sample volumes, and ease of integration with miniaturized sample preparation modules. The transfer of DNA detection techniques to microfluidic channels has been limited, however. Delamarche and co-workers have modified microfluidic channel walls to facilitate the detection of proteins.18,19 DNA has also been detected both in solution in microfluidic channels and immobilized onto electrode surfaces by its interaction with tagged liposomes and by surface plasmon resonance spectroscopy. 20-22 Polymeric structures appear to be ideal candidates for immobilizing DNA in microfluidic structures.23-25 We describe here a method for selectively immobilizing plugs of DNA probe/ polyacrylamide copolymers in microfluidic channels. The hydrogel plugs, which are photopolymerized in the channels in 5 min, are permeable to DNA under electrophoretic conditions and hybridize with fluorescently tagged complementary DNA. The hybridized DNA can be denatured, and the DNA hydrogel plug may be reused with a new analytical sample. Using the DNA copolymer plugs, the efficient, directed mass-transfer characteristics of microchannels are fully exploited for rapid hybridization of targets present at low concentration and in small sample volumes. EXPERIMENTAL SECTION Chemicals and Materials. Tetraethylmethylenediamine (TEMED, Aldrich, Milwaukee, WI), acrylamide solutions (Sigma, St. Louis, MO), riboflavin (Sigma), NaOH (J. T. Baker, Phillipsburg, NJ), NaCl (Sigma), 10× TE (100 mM Tris-HCl, 10 mM EDTA, pH 7.4) buffer (Research Genetics, Huntsville, AL), and Fluoresbrite Polychromatic Red 1.0-µm beads, 2.67% solids (Polysciences, Warrington, PA) were used as received.26 Acrylamidemodified single-stranded DNA (ssDNA) probes and fluoresceinlabeled complements were generously provided by Mosaic Technologies (Waltham, MA).26 TAMRA-labeled complements (18) Papra, A.; Bernard, A.; Juncker, D.; Larsen, N. B.; Michel, B.; Delamarche, E. Langmuir 2001, 17, 4090-5. (19) Bernard, A.; Michel, B.; Delamarche, E. Anal. Chem. 2001, 73, 8-12. (20) Patolsky, F.; Lichtenstein, A.; Willner, I. J. Am. Chem. Soc. 2001, 123, 5194205. (21) Esch, M. B.; Locascio, L. E.; Tarlov, M. J.; Durst, R. A. Anal. Chem. 2001, 73, 2952-8. (22) Lee, H. J.; Goodrich, T. T.; Corn, R. M. Anal. Chem. 2001, 73, 5525-31. (23) Brahmasandra, S. N.; Ugaz, V. M.; Burke, D. T.; Mastrangelo, C. H.; Burns, M. A. Electrophoresis 2001, 22, 300-11. (24) Kim, Y. D.; Park, C. B.; Clark, D. S. Biotechnol. Bio. 2001, 73, 331-7. (25) Zhao, B.; Moore, J. S. Langmuir 2001, 17, 4758-63. 10.1021/ac0156969 CCC: $22.00

© 2002 American Chemical Society Published on Web 02/12/2002

were purchased from Operon Technologies (Huntsville, AL).26 All solutions were prepared using water from a Millipore Milli-Q system (Bedford, MA).26 Microchannels were formed using polycarbonate (McMaster-Carr, Altanta, GA) and UV-transparent acrylic (poly(methyl methacrylate), Acrylite OP-4, Cyro Industries, Mt. Arlington, NJ) sheets.26 Microchannel Fabrication. A 248-nm excimer laser system (LMT-4000, Potomac Photonics, Inc., Lanham, MD) was used to form microchannels in polycarbonate.26-28 The excimer laser system28 contains a laser light source, a circular aperture (1.5mm diameter) for reducing the size and shaping of the beam, a focusing lens (10× compound), a visible light source, a CCD camera to image the ablation process, and a computer-controlled X-Y stage with a vacuum chuck to hold the substrate in place. Also, a gas nozzle was present to sweep nitrogen over the substrate during processing, and a vacuum nozzle was located on the opposite side of the stage to remove debris. The average power level per pulse was set to 70 µJ as determined using an Energy Max 400 laser energy meter from Molectron Detector, Inc. (Portland, OR).26 The frequency of pulses was set to 200 Hz, with a constant pulse width of 7 ns. To form the channels, the polycarbonate sheet was placed on the X-Y stage and moved linearly at a rate of 0.5 mm/s. The laser was passed over the surface 8 times to form channels that were approximately 50 µm wide and 95 µm deep with a slightly rounded bottom. The polycarbonate microchannel chip was covered with an acrylic lid containing 2-mm-diameter holes to provide fluid access to the channels. The two pieces were clamped together between glass slides and bonded by placing in a circulating air oven at 103 °C for 30 min. DNA Copolymer Polymerization. A procedure similar to that described by Rehman et al.29 for fabricating DNA copolymers on optical fibers was used for forming hydrogel plugs in the polycarbonate channels. The microchannels were filled with a solution containing 0.0006% (w/v) riboflavin, 10% (w/v) 19:1 acrylamide/bis-acrylamide, 10-15 µM acrylamide-modified oligomer, 0.125% (v/v) TEMED, and 0.000 07% Fluoresbrite beads in 1× TE (10 mM Tris-HCl, 1 mM EDTA, pH 7.4) buffer, with an equivalent amount of the same solution placed into each of the fluid reservoirs. Fluoresbrite beads were used as visualization markers to minimize fluid flow prior to photopolymerization. The channels were illuminated with 515-560-nm light, and emission from the beads was detected at 590 nm. The movement of the Fluoresbrite beads was then monitored while the volumes of each of the fluid reservoirs were adjusted and the volumes of each of the fluid reservoirs were adjusted to balance the fluid reservoirs and minimize fluid flow in the channels. The channel was then illuminated with 340-380-nm light from a 100-W Hg arc lamp at 10× magnification focused on a portion of the channel for 5 min (26) Disclaimer: Certain commercial equipment, instruments, or materials are identified in this report to specify adequately the experimental procedure. Such identification does not imply recommendation or endorsement by the National Institutes of Standard and Technology, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose. (27) Roberts, M. A.; Rossier, J. S.; Bercier, P.; Girault, H. Anal. Chem. 1997, 69, 2035-42. (28) Johnson, T. J.; Waddell, E.; Kramer, G. W.; Locascio, L. E. App. Surf. Sci. 2001, 149-59. (29) Rehman, F. N.; Audeh, M.; Abrams, E. S.; Hammond, P. W.; Kenney, M.; Boles, T. C. Nucleic Acids Res. 1999, 27, 649-55.

Figure 1. (a) Schematic diagram of the side-by-side two-channel microfluidic chip configuration. Fluorescent images of a side-by-side two-channel microfluidic chip during (b) and after (c) photopolymerization with UV light for 5 min. The fluorescence observed in the top panel originates from riboflavin found in the photopolymerization solution and background fluorescence from the polycarbonate substrate. The resulting hydrogel plugs are 500 and 600 µm long.

to effect polymerization. An adjustable aperture in the microscope illumination path was used to define the size of the illuminated spot (typically between 500- and 600-µm diameter) and, therefore, the size of the resulting hydrogel plug. After polymerization, the photopolymerization solution was rinsed from the open channels on either side of the hydrogel plus and replaced with either a buffer solution containing 0.5 M NaCl and 1× TE buffer or the same buffer containing complementary DNA. All microfluidic chips were refrigerated and filled with 0.5 M NaCl and 1× TE buffer when not in use. Electrophoresis. Platinum electrodes were placed in contact with the solution in reservoirs A and D (and simultaneously A′ and D′) as shown in Figure 1a and connected to a high-voltage power supply (Keithley 247 HVS).26 The current through the microchannel was determined by measuring the voltage drop across a 100-kΩ resistor connected to the power supply in series with the microchannel. Image Acquisition. Fluorescence imaging of the microchannels was performed using a research fluorescence microscope equipped with a 10× objective, mercury arc lamp, appropriate filters, and two cameras. For color images, an MTI (Dage-MTI, Michigan City, IN) 3CCD camera was utilized, and a Cohu (San Diego, CA) 4900 gray-scale camera was used for correlating fluorescent intensities with fluorophore concentrations.26 RESULTS AND DISCUSSION Formation of the DNA Hydrogel Plugs. The formation of the DNA copolymer in microfluidic channels was investigated under a variety of conditions. Figure 1a displays a schematic of a microchannel geometry that was found to be useful for these Analytical Chemistry, Vol. 74, No. 6, March 15, 2002

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studies. This geometry permitted easy flushing and replacement of solutions in channels on both sides of the hydrogel plug after polymerization. In addition, the side-by-side microchannels (Figure 1a) facilitated the concurrent photopolymerization of two adjacent channels, allowing comparison of two channels photopolymerized under the same conditions, but containing different DNA copolymers. Also displayed in Figure 1 are fluorescent images of the copolymer formed in the side-by-side microchannels during (b) and after (c) polymerization of a 19:1 mixture of acrylamide to bisacrylamide. The illuminated circle in Figure 1b indicates the aperture size for our UV source, resulting in a 500-600-µm-long polymeric plug. The fluorescence is caused by riboflavin in the photopolymerization solution and background fluorescence of the polycarbonate substrate. Figure 1c was acquired after the photopolymerization process was complete under white light, and the edges of the hydrogel plugs, which appear to be denser than the interior of the hydrogel, are visible in the image. Photopolymerization of a 29:1 mixture of acrylamide to bis-acrylamide was also attempted to enhance the permeability of the polymer, but these plugs were not stable under mild electrophoretic conditions. Experiments with photopolymerization times longer than 5 min were also performed, but these polymers were substantially less permeable to DNA under electrophoresis. When a fluorescent solution was introduced into channels with less-permeable plugs and electrophoresis attempted, flow was not observed through the plug but between the plug and the channel walls, and a significant population of the fluorescent compound seemed to be located at the plug/channel wall interface. It should be noted that chemical modification of the channel walls is not required before photopolymerization to obtain stable plugs. We believe that the polymers are not chemically bound to the channel surface but nonetheless are able to withstand pressures up to 3 psi and voltages as high as 100 V for short periods of time. The polymers are routinely exposed to multiple 10-min applications of 10-25 V and are stable under these conditions for extended periods of time. One microchip was utilized for a total of several hours over the course of 2 days. However, the polymers are prone to fail if continually exposed to voltages of 10-25 V for periods greater than 25 min. The mode of polymer failure is not well understood but is indicated by the onset of electroosmotic flow and a drop in the microchannel resistance during electrophoresis. The polymer appears to be macroscopically intact after failure, and after a brief open-circuit relaxation period, the recovery of electrophoretic behavior has been observed. We speculate that over time electroosmotic flow initiates through microscopic pathways along the microchannel wall or a pore network that develops through the polymer plug. One possible mode of hydrogel plug failure is Joule heating of the polymer plug due to the increased resistance of the viscous polymeric material. Resistance measurements recorded before and after the polymerization process indicate an increase in resistance in the area of the hydrogel plug from approximately 40 Ω/µm before polymerization to 65-140 Ω/µm after polymerization. An average electrophoretic current of 16 µA for the hydrogelcontaining channels results in a dissipated power value of (2-4) × 10-4 W/cm across the hydrogel plug. Temperature measurements using a temperature-dependent fluorescent dye30 demon(30) Ross, D.; Gaitan, M.; Locascio, L. E. Anal. Chem. 2001, 73, 4117-23.

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strate that this magnitude of power dissipation typically results in a negligible temperature increase in the plug, indicating that Joule heating is probably not a factor in hydrogel plug failure. DNA Hybridization in Microfluidic Channels. Because the copolymer contains ssDNA, it is possible to use the polymer-filled microfluidic channels to detect complementary DNA via hybridization, as demonstrated in Figure 2. In all of the panels of Figure 2, the hydrogel plug in the top channel contains an immobilized acrylamide-modified 20-base oligomer GCA CCT TGT CAT GTA CCA TC identified as sequence 1 (S1), and the bottom channel holds a hydrogel plug containing a second, different immobilized acrylamide-modified 20-base oligomer AGG CCC GGG AAC GTA TTC AC, sequence 2 (S2). Initially the A-B and A′-B′ channels in the chip were filled with 12 µM S1 complement tagged with fluorescein in a solution containing 0.5 M NaCl and 1× TE buffer. The C-D and C′-D′ channels were filled with the 0.5 M NaCl/ 1× TE buffer alone. In panel a, the complementary DNA solution was electrophoresed into the polymer plug for 5 min at an applied potential of 25 V. Once the hydrogel plugs appeared to be full of the complementary DNA, the A-B, A′-B′, C-D, and C′-D′ channels were all rinsed with 0.5 M NaCl in 1× TE buffer and the clean buffer solution was then electrophoresed through both hydrogel plugs at 25 V for 5 min. The result is shown in Figure 2b. The fluorescence images indicate that S1 complement remains in the top plug containing the immobilized S1 probe, while it is largely flushed from the bottom plug containing the noncomplementary S2 probe. The remaining fluorescence at the edges of the bottom plug is a color slightly different from the fluorescein fluorescence of the top plug; we suspect that this results from riboflavin that is left in the plug from the polymerization process. The hybridization of complementary DNA targets with the probe DNA-containing hydrogel plug was also found to be reversible. Duplexes formed in the copolymer could be denatured either electrophorectically or chemically and the hybridization process repeated. If the channels are rinsed with 1× TE buffer containing no NaCl and electrophoresis is reinitiated, a gradual, but incomplete, loss of complement from the hydrogel plugs occurs over a span of 10-15 min. A faster and more efficient method for removing the hybridized target DNA is to electrophorese a denaturation solution of 0.4 M NaOH/0.5 M NaCl into the polymer plug at 10 V for 10 min, which removes not only the complement but also the riboflavin remaining from the photopolymerization process. The results of this protocol are represented in Figure 2c. Figure 2 panels d-f represent the repetition of the entire process, with one change; the rinsing of the A-B, A′-B′, C-D, and C′-D′ channels is eliminated, and the electrophoresis voltage is simply inverted, just as the end of the polymer fills with DNA. Thus, voltage inversion reverses the unbound DNA out of the polymer plug, as is evident in panel e. Treatment with aqueous NaOH is a commonly used DNA denaturation procedure in Southern blot chemistry, and acrylamide hydrogels are known to be chemically stable under these conditions. The DNA hydrogel plug also acts to scavenge complementary DNA and low concentrations of complementary DNA in solution can be accumulated and concentrated in the hydrogel plug. The ability of the plugs to concentrate DNA is demonstrated by the data presented in Figures 3 and 4. In Figure 3, a solution containing 150 nM TAMRA-tagged S2 complement in 0.5 M NaCl

Figure 2. Reproduceability and regeneration of DNA detection at polymer plugs. In all of the panels, the hydrogel plug in the top channel contains an immobilized acrylamide-modified 20-base oligomer GCA CCT TGT CAT GTA CCA TC identified as sequence 1 (S1), and the bottom channel holds a hydrogel plug containing a second, different immobilized acrylamide-modified 20-base oligomer AGG CCC GGG AAC GTA TTC AC, sequence 2 (S2). In panels a and b, the excess fluorescence observed on the edges of hydrogel plugs results from riboflavin that is left in the plug from the polymerization process and is removed in the denaturation process in panel c. A 12 µM solution of fluorescein-tagged S1 complement and 0.5 M NaCl in 1× TE buffer was electrophoresed into both plugs (a) and rinsed with 0.5 M NaCl in 1× TE buffer to remove unhybridized DNA (b). In panel c, the bound S1 complement was denatured by the electrophoresis of 0.4 M NaOH/0.5 M NaCl in 1× TE buffer through both plugs. The process is repeated in panels d and f, but in panel e, the unbound DNA is removed from the noncomplementary plug by inverting the polarity of the electric field for both channels and then reversing the movement of the unbound DNA out of the hydrogel plugs. The lower fluorescence intensity in panel a relative to the other panels is due to a 4-fold decrease in fluorescence excitation used for that panel only. All other panels have been imaged at the same fluorescence excitation level.

in 1× TE buffer was electrophoresed into an S2-containing hydrogel plug. This concentration is sufficiently low such that fluorescence was not observed from the solution in the open channel. However, with continued electrophoresis, the TAMRAtagged S2 complement accumulated in the hydrogel plug over time. Concentration profiles of accumulating TAMRA-tagged S2 complement with increasing time of electrophoresis are shown in Figure 3. Concentrations were determined using the following method. The fluorescent intensities of solutions of TAMRA-tagged S2 complement of varying known concentrations were measured in microchannels not containing hydrogel and a calibration curve of fluorescence intensity versus DNA concentration was generated. Then the fluorescence intensity of the hydrogel plug during the accumulation experiment was compared against the TAMRAtagged S2 complement calibration curve, resulting in concentration values for the TAMRA-tagged S2 complement hybridizing in the hydrogel plug. The fluorescence was averaged across the entire (north/south) width of the channel for each data point along the length of the hydrogel plug. The potential variation in TAMRA fluorescence intensity between solution and the hydrogel plug environments was not considered in calculating concentrations. The sharp peak of TAMRA-tagged S2 complementary DNA seen on the far left is the accumulation of the complement in the open channel at the solution/plug interface due to the interface acting as an electrophoretic dam. After 25 min of electrophoresis, the fluorescence intensity reaches a plateau at a distance of approximately 40-100 µm into the plug. The intensity of the plateau roughly corresponds to a concentration of 20 µM in the

plug, some 2 orders of magnitude higher than the initial 150 nM concentration of the solution in the microchannel. Although we do not currently know the exact concentration of the acrylamidemodified ssDNA in the hydrogel plug, the value of 20 µM for the captured S2 complement seems reasonable considering that the hydrogel plug photopolymerization solution contained 15 µM acrylamide-modified ssDNA and the hydrogel plug has not been rinsed with 0.5 M NaCl in 1× TE buffer. Work is in progress to determine the concentration of covalently bound DNA probes in the plugs and the associated hybridization efficiency of the DNA copolymers. It is possible to average the fluorescence intensity over a rectangle encompassing the entire hydrogel plug, thereby calculating the average concentration of complementary DNA throughout the entire hydrogel. It is also instructive to calculate the position of the leading edge of fluorescence along the hydrogel plug, which was defined from concentration profiles like those shown in Figure 3 as the distance from the left edge of the hydrogel at which the concentration first reaches 3 µM. The average concentration of complementary DNA throughout the entire hydrogel plug (squares) and the position of the leading edge of fluorescence (circles) in the polymer plug are plotted as a function of electrophoresis time in Figure 4. The linear behavior of both the position of the edge and the average concentration indicate that, under these conditions, the rate of capture of DNA targets is limited by the speed at which DNA can be electrophoresed into the plug. The ability of this polymeric system to detect complementary DNA can be considered an integrative process, where sensitivity will depend on the concentration of Analytical Chemistry, Vol. 74, No. 6, March 15, 2002

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Figure 4. Position of the leading edge (circles) of TAMRA fluorescence and average concentration (squares) of TAMRA-tagged S2 complement in hydrogel plug plotted with respect to time. A solution of 150 nM S2 complement and 0.5 M NaCl in 1× TE buffer was electrophoresed into the polymer plug for 25 min at 25 V. Fluorescence intensity was averaged over the entire length and width of the DNA hydrogel plug.

Figure 3. Concentration of TAMRA-tagged S2 complement with respect to distance from the edge of the hydrogel plug after (a) 5, (b) 10, (c) 15, and (d) 20 min of electrophoresis. A solution of 150 nM S2 complement and 0.5 M NaCl in 1× TE buffer was electrophoresed into the polymer plug for 25 min at 25 V. Fluorescence intensity was averaged across the entire (north/south) width of the channel for each data point along the length of the channel.

DNA target, the concentration of DNA probe in the hydrogel, and the time allotted for electrophoresis. Selective Multicolor Detection of DNA Complements. Multianalyte detection is realized by using different color fluorescing tags or by spatially localizing plugs that contain different DNA probes. To establish proof of concept, three spatially separated hydrogel plugs containing different sequence DNA probes or no DNA whatsoever were photopolymerized in the same channel. Figure 5 demonstrates the selectivity of two different DNA polymer plugs to uniquely detect their own complements. In this experiment, the aperture was decreased to create three individual 130-µm plugs separated by solution. A detailed diagram of the microfluidic chip utilized in this experiment is presented in Figure 5a. As is noted in Figure 5b, the left plug contains DNA probes complementary to a fluorescein tagged target, S1, while the right plug contains DNA probes complementary to a TAMRA1440 Analytical Chemistry, Vol. 74, No. 6, March 15, 2002

Figure 5. Multicolor multianalyte selective detection of DNA in a mixed sample. (a) Schematic diagram of the microfluidic chip used in this experiment. (b) Microfluidic channel illuminated with white light with three separate hydrogel plugs containing S1, no DNA, and S2, respectively. The width of the polymer plugs is ∼130 µm. (c, d) Microfluidic channel and hydrogel plugs into which 6 µM fluoresceintagged S1 complement, 7.5 µM TAMRA-tagged S2 complement, and 0.5 M NaCl in 1× TE buffer were electrophoresced, followed by a rinse process and the electrophoresis of 0.5 M NaCl in 1× TE buffer through all three hydrogel plugs. (c) The channel is illuminated with a TAMRA filter cube. (d) The channel is illuminated with a fluorescein filter cube.

tagged target, S2. The two DNA-containing plugs are separated by a hydrogel plug that does not contain probe DNA. Initially, the entire chip was filled with a photopolymerization solution containing acrylamide-modified S2, and the S2 hydrogel plug was created by focusing UV light on the far right portion of the microfluidic channel. The E-F-G and H-I-J portions of the channel were rinsed with 0.5 M NaCl in 1× TE buffer and the F-I portion of the chip was then rinsed by electrophoresing the

0.5 M NaCl in 1× TE buffer through the F-I channel (+25 V from reservoir H to reservoir G). The H-I-J portion of the channel was then filled with a photopolymerization solution containing no DNA, and the center hydrogel plug was created by electrophorescing the polymerization solution through the S2 hydrogel plug and into the F-I channel (+25 V from reservoir H to reservoir G). UV light was then focused onto the center of the F-I channel. The rinse and buffer electrophoresis was repeated, and in the third step, a photopolymerization solution containing acrylamide-modified S1 was introduced into the H-I-J portion of the channel. The S1 hydrogel plug was formed by electrophorescing the polymerization solution through the S2 and control (non-DNA) hydrogel plugs, and UV light was focused on the far left portion of the microfluidic channel. A final rinse of the E-F-G and H-I-J portions of the channel with 0.5 M NaCl in 1× TE buffer completes the fabrication process. To demonstrate multianalyte detection, a solution containing complements to both S1 and S2 was introduced into the H-I-J portion of the channel and then electrophoresed through all three hydrogel plugs. After rinsing the E-F-G and H-I-J portions of the channel with 0.5 M NaCl in 1× TE buffer and electrophoresis of the buffer through the hydrogel plugs to remove unbound DNA complements (+25 V from reservoir H to reservoir G), green fluorescence is observed predominantly from the left plug,

indicating capture of the S1 complement (Figure 5c). Conversely, red fluorescence is observed solely from the right plug, indicating the presence of bound S2 complement (Figure 5d). In addition to improving the stability of the plugs, we are working to improve protocols for fabricating “barcode-like” linear arrays of polymeric plugs in microfluidic channels. Finally, we note that the microchannel plugs could be applied to other biological affinity reactions. ACKNOWLEDGMENT The authors thank T. Christian Boles of Mosaic Technologies for the gift of the acrylamide-modified DNA probes and helpful discussions and Dr. Laurie Locascio for the use of laboratory facilities. The authors acknowledge the financial support of the NRC/NIST postdoctoral Research Program and the Loyola College Faculty Summer Development Grant Program. K.G.O. acknowledges Dr. Kirsten Crossgrove for many helpful discussions in the creation of the polymerization and denaturation methodology.

Received for review November 19, 2001. Accepted January 6, 2002. AC0156969

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