Abstract:
A method and device for analyzing species in a sample includes immobilized ligands in a porous matrix disposed in a microchannel. A method of analysis is provided by loading a sample to the microchannel and establishing an analyte flow through the porous matrix. The analyte is detected as the species that binds to the ligand immobilized in the porous matrix. The present method may be adapted to identify multiple species present in a sample by immobilizing different ligands in a series in a single microfluidic channel by sequentially photopolymerization of various porous matrixes, each containing different ligands.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
       [0001]    This application claims benefit of the filing date of copending Provisional Patent Application No. 60/348,094, filed on Jan. 14, 2002. 
     
    
     
       FIELD OF THE INVENTION  
         [0002]    The present invention relates to a method and device for analyzing species in a sample and in particular the present invention relates to the use of a porous matrix disposed in a microfluidic channel for analyzing an analyte present in a sample.  
         BACKGROUND OF THE INVENTION  
         [0003]    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. Many microarray chips have DNA immobilized in a variety of polymeric surface pads in the form of a planar array to facilitate spatially localized detection of DNA hybridization.  
           [0004]    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. For example, microfluidic channel walls have been modified to facilitate the detection of proteins. In addition, 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.  
         SUMMARY OF THE INVENTION  
         [0005]    In general, in accordance with the invention, ligands acting as probes are immobilized in a porous matrix disposed in a microfluidic channel thereby forming a microfluidic device. This microfluidic device may be used for analyzing a sample containing an analyte by loading the sample to the microchannel and then flowing the analyte through the porous matrix.  
           [0006]    In one implementation of the present invention, 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 polycarbonate microfluidic channels with polyacrylamide matrix may be reused with a new analytical sample. The present polymeric structures, i.e., hydrogel plug, provide for immobilizing DNA in microfluidic structures.  
           [0007]    In one further implementation, a single microfluidic channel includes hydrogel plugs containing different DNA probe sequences, thereby enabling the selective detection of multiple DNA targets in one electrophoretic pathway.  
           [0008]    In another implementation, analyte flow through the porous matrix is generated by applying a pressure gradient to induce flow of fluid and analyte through the pores of the matrix, thereby bringing analyte into contact with the ligands immobilized in the matrix.  
           [0009]    In accordance with a further affect of the invention, a method is provided for selectively immobilizing hydrogel plugs of DNA probe/polyacrylamide copolymers in microfluidic channels. The hydrogel plugs, which are photopolymerized in the channels, 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.  
           [0010]    The present invention, in one form thereof, relates to a method for analyzing species in a sample. The method includes supplying a substrate having a microchannel formed therein where the microchannel has a geometry with at least one spatial dimension on the order of micrometers and has a porous matrix disposed in the microchannel. Ligands are immobilized in the porous matrix. A sample containing an analyte is loaded in the microchannel and an analyte flow is established through the porous matrix.  
           [0011]    Preferably, the ligand is selected from the group consisting of single stranded DNA, double stranded DNA, catalytic DNA, DNA aptamers, RNA, catalytic RNA, antibodies, antigens, and protein. Advantageously, the method includes the step of detecting the analyte which bonds to the ligand immobilized in the porous matrix.  
           [0012]    In a further, alternative embodiment, the ligand includes catalytic nucleic acid and the method further includes allowing the sample to flow through the porous matrix, where the cleaved portion of the catalytic nucleic acid binds to a second ligand immobilized in a second porous matrix.  
           [0013]    The present invention, in another form thereof, concerns a method for analyzing species in a sample. The method includes supplying a microchannel having at least some spatial dimension on the order of micrometers and having a porous matrix disposed in the microchannel. One of a plurality of different ligands are immobilized in a different linear section of the porous matrix whereby each different linear section of the porous matrix has immobilized a different ligand that binds a specific analyte. An analyte flow is establishes through the porous matrix.  
           [0014]    The present invention, in yet another form thereof, concerns a microfluidic device including a substrate with a microchannel formed therein where the microchannel has a geometry with at least one spatial dimension on the order of micrometers and has a porous matrix disposed in the microchannel. A ligand is immobilized in the porous matrix. In a preferred embodiment, an electrical power supply source is operatively associated with the microchannel for applying an electric current to the microchannel thereby establishing an electrophoretic pathway through the porous matrix. Alternatively, the analyte flow may be pressure-driven. In further advantageous embodiments, the microchannel is formed of a plastic or glass substrate and the porous matrix includes a ligand as an acrylamide-modified ligand in a polyacrylamide matrix.  
           [0015]    The invention, in yet another embodiment thereof, concerns a microfluidic device including a microchannel having a geometry with at least one spatial dimension on the order of micrometers and having a porous matrix disposed in the microchannel. A plurality of different ligands are immobilized in a different linear section of the porous matrix whereby each different ligand immobilized in a different linear section of the porous matrix and each different ligand binds a specific analyte when flowed through the porous matrix. In one preferred embodiment, the porous matrix includes a hydrogel plug and may comprises a ligand as a acrylamide-modified ligand in a polyacrylamide matrix.  
           [0016]    Further features and advantages of the present invention will be set forth in, or apparent from, the detailed description of preferred embodiments thereof which follows. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0017]    The invention will now be described in detail with respect to preferred embodiments with reference to the accompanying drawings, wherein:  
         [0018]    [0018]FIG. 1 is a schematic diagram of a side-by-side two-channel microchannel device according to the present invention;  
         [0019]    [0019]FIG. 2 is a cross section of the microchannel of FIG. 1 along line  2 - 2 ;  
         [0020]    [0020]FIG. 3( a ) to  3 ( d ) depict the concentration of TAMRA tagged DNA oligomer after various periods of electrophoresis, where FIG. 3( a ) is after 5 minutes, FIG. 3( b ) is after 10 minutes, FIG. 3( c ) is after 15 minutes, and FIG. 3( d ) is after 20 minutes of electrophoresis; and  
         [0021]    [0021]FIG. 4( a ) is a schematic of a microfluidic device used in the separation of a multiple analyte sample and FIG. 4( b ) is an enlargement of a portion of the microfluidic device of FIG. 4( a ) according to another embodiment of the present invention. 
     
    
     DESCRIPTION OF PREFERRED EMBODIMENTS  
       [0022]    Further description of the present invention is provided with reference to the drawings and in particular to FIGS. 1 and 2 which depicts a plan view of microfluidic device  10  with a plurality of microchannels pathways formed in a substrate  12 . The microchannels  11  can be formed in any suitable substrate known in the art. In one embodiment, an excimer laser system is used to form microchannels  11  in a polycarbonate substrate. The dimensions of the microchannels are approximately 50 μM wide and 90 μm deep with a slightly rounded bottom as is best shown in FIG. 2.  
         [0023]    The polycarbonate microchannel chip, i.e., substrate  12  is covered with an acrylic lid  13  containing a plurality of 2-mm diameter holes  14  to provide fluid access to the microchannel  11 . The two pieces, i.e., substrate  12  and lid  13 , were clamped together between glass slides and bonded by placing in a circulating air oven at 103° C. for 30 minutes.  
         [0024]    Hydrogel plug  15  is formed in microchannel  11  using a modified procedure similar to the one described by Rehman et al. (Rehman, F. N.; Audeh, M.; Abrams, E. S.; Hammond, P. W.; Kenney, M.; Boles, T. C.,  Nucleic Acids Research  1999, 27, 649-55) which is directed to fabricating DNA copolymers on optical fibers. Rehman et al. is herein incorporated by reference. Hydrogel plug  15  is a porous matrix into which is incorporated, ligands to be used as a probe for analyzing a sample containing an analyte.  
         [0025]    In forming hydrogel plugs  15 , the microchannel  11  is 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 0.00007% 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 microchannel  11  was illuminated with 515-560 nm light and the emission from the beads was detected at 590 nm. The movement of the Fluoresbrite beads was then monitored while adjusting the volumes of each of the fluid reservoirs and the volumes of each of the fluid reservoirs was adjusted to balance the fluid reservoirs and minimize fluid flow in the microchannel  11 . The microchannel  11  was then illuminated with 340-380 nm light focused on a portion of the microchannel  11  for five minutes to effect polymerization. An adjustable aperture in the microscope illumination path was used to define the size of the illuminated spot (typically between 500 μm 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 plug  15  and replaced with either a buffer solution containing 0.5 M NaCl and 1×TE (10 mM Tris-HCl, 1 mM EDTA, pH 7.4) buffer or the same buffer containing complementary DNA. The microfluidic device  10  was refrigerated and filled with 0.5 M NaCl and 1×TE (10 mM Tris-HCl, 1 mM EDTA, pH 7.4) buffer when not in use.  
         [0026]    Platinum electrodes  24   a,    26   a  are placed in contact with the solution in reservoirs  16   a  and  17   a,  respectively, and simultaneously electrodes  24   b,    26   b  are placed in contact with the solution in reservoirs  16   b  and  17   b  and all electrodes  24   a,    24   b,    26   a,    26   b  are connected to a high voltage power supply  18 . The current through the microchannel  11  is determined by measuring the voltage drop across a 100 KΩ resistor connected to the power supply in series with the microchannel  11 .  
         [0027]    The geometry of the microfluidic device  10  permits easy flushing and replacement of solutions in microchannel  11  on both sides of the hydrogel plug  15  after polymerization. In addition, the side-by-side microchannels  11   a,    11   b  facilitate the concurrent photopolymerization of two adjacent microchannels allowing comparison of two channels photopolymerized under the same conditions, but containing different DNA copolymers.  
         [0028]    It should be noted that chemical modifications of the microchannel walls is not required before photopolymerization to obtain stable hydrogel plugs. It is believed that the polymers are not chemically bound to the microchannel surface, but nonetheless are able to withstand pressures up to three psi and voltages as high as 100 V for short periods of time. The polymers are stable for extended periods of time to routine exposure to multiple 10-minute applications of 10-25 V. For example, one microfluidic device was utilized for a total of several hours over the course of two days. Advantageously, the exposure period of greater than 25 minutes at a voltage of 10-25 V should be avoided as such conditions could lead to polymer failure.  
         [0029]    The hydrogel filled microchannel  11  can be used to detect an analyte in a sample. For example, if the hydrogel plug  15  contains single strand DNA (ssDNA), it is possible to use the hydrogel plug  15  (i.e., polymer-filled microfluidic channel  11 ) to detect complementary DNA via hybridization, as depicted in FIG. 3.  
         [0030]    The reproducibility and regeneration of DNA detection of hydrogel plug  15  has been demonstrated. In an example, the hydrogel plug  15   a  in microchannel  11   a  contains an immobilized acrylamide-modified 20-base oligomer GCA CCT TGT CAT GTA CCA TC (Seq. ID No. 1) identified as S 1 , and microchannel  11   b  holds a hydrogel plug  15   b  containing a second, different immobilized acrylamide-modified 20-base oligomer AGG CCC GGG AAC GTA TTC AC (Seq. ID No. 2) identified as S 2 . A 12 μM solution of fluorescein-tagged S 1  complement and 0.5 M NaCl in 1×TE buffer was electrophoresed into both hydrogel plugs  15   a,    15   b  and rinsed with 0.5 M NaCl in 1×TE buffer to remove unhybridized DNA. The bound S 1  complement was denatured by the electrophoresis of 0.4 M NaOH/0.5 M NaCl in 1×TE buffer through both hydrogel plugs  15   a,    15   b.  The process was repeated in two instances, but in a third, the unbound DNA is removed from the noncomplementary hydrogel plug by inverting the polarity of the electric field for both microchannels  11   a,    11   b  and thereby reversing the movement of the unbound DNA out of the hydrogel plugs  15   a,    15   b.    
         [0031]    In all examples, the hydrogel plug in microchannel  11   a  contains an immobilized acrylamide-modified 20-base oligomer S 1 , and the microchannel  11   b  holds a hydrogel plug  15   b  containing a second, different immobilized acrylamide-modified 20-base oligomer S 2 . Initially the wells  16   a - 19   a  and  16   b - 19   b  and associated segments of the microchannel  11  were filled with 12 μM of the S 1  complement tagged with fluorescein in a solution containing 0.5 M NaCl and 1×TE (10 mM Tris-HCl, 1 mM EDTA, pH 7.4) buffer. The wells  20   a - 17   a  and  20   b - 17   b  and associated segments of the microchannels  11   a,    11   b  were filled with the 0.5 M NaCl-1×TE buffer alone. In the initial example, the complementary DNA solution was electrophoresed into the polymer plug for five minutes at an applied potential of 25 V. Once the hydrogel plugs  15   a,    15   b  appeared to be full of the complementary DNA, the wells  16   a - 19   a,    16   b - 19   b,    20   a - 17   a,   20   b - 17   b,  and the associated microchannels segments, were all rinsed with 0.5 M NaCl in 1×TE buffer and the clean buffer solution was then electrophoresed through both hydrogel plugs  15   a,    15   b  at 25 V for 5 minutes.  
         [0032]    Fluorescence images of the hydrogel plugs indicate that S 1  complement remains in the hydrogel plug  15   a  which contains the immobilized S 1  probe, while the S 1  complement is largely flushed from the hydrogel plug  15   b  which contains the noncomplementary S 2  probe. The remaining fluorescence at the edges of the hydrogel plug  15   b  appears slightly different than the fluorescein fluorescence of the hydrogel plug  15   a,  possibly as a result of riboflavin remaining in the hydrogel plug  15   b  from the polymerization process.  
         [0033]    The hybridization of complementary DNA targets with the probe DNA-containing hydrogel plug is reversible. Duplexes formed in the copolymer can be denatured either electrophoretically or chemically and the hybridization process repeated. For example, if the microchannels  15   a,    15   b  are rinsed with 1×TE buffer containing no NaCl and electrophoresis is re-initiated, a gradual, but incomplete, loss of complement from the hydrogel plugs occurs over a span of 10-15 minutes. 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.  
         [0034]    In the other examples referred to above, the process is essential the same, with one change, namely, the rinsing of the wells  16   a - 19   a,    16   b - 19   b,    20   a - 17   a,    20   b - 17   b  and associated segments of microchannels  15   a,    15   b  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 hydrogel plug. Treatment with aqueous NaOH is a commonly used denaturation procedure in Southern blot chemistry, and acrylamide hydrogels are known to be chemically stable under these conditions.  
         [0035]    The DNA hydrogel plug  15  also acts to scavenge complementary DNA and low concentrations of complementary DNA in solution can be accumulated and concentrated in the hydrogel plug  15 . The ability of the hydrogel plugs  15   a,    15   b  to concentrate DNA is demonstrated by the following example with data presented in FIGS.  3 ( a ) to  3 ( d ).  
         [0036]    In FIGS.  3 ( a ) to  3 ( d ), a solution containing 150 nm TAMRA-tagged S 2  complement in 0.5 M NaCl in 1×TE buffer was electrophoresed into an S 2 -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 TAMRA-tagged S 2  complement accumulated in the hydrogel plug over time. Concentration profiles of accumulating TAMRA-tagged S 2  complement with increasing time of electrophoresis are shown in FIGS.  3 ( a ) to  3 ( d ).  
         [0037]    Concentrations were determined by generating a calibration curve of fluorescence intensity vs. DNA concentration after measuring the fluorescent intensities of solutions of TAMRA-tagged S 2  complement of varying known concentrations. Then the fluorescence intensity of the hydrogel plug during the accumulation experiment was compared against the TAMRA-tagged S 2  complement calibration curve, resulting in concentration values for the TAMRA-tagged S 2  complement hybridizing in the hydrogel plug. The fluorescence was averaged across the entire width of the microchannel  11  for each data point along the length of the hydrogel plug  15 . The potential variation in TAMRA fluorescence intensity between solution and the hydrogel plug  15  environments was not considered in calculating concentrations.  
         [0038]    The sharp peak of TAMRA-tagged S 2  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 minutes of electrophoresis, the fluorescence intensity reaches a plateau at a distance of approximately 40 to 100 μm into the plug. The intensity of the plateau roughly corresponds to a concentration of 20 μM in the plug, some two orders of magnitude higher than the initial 150 nM concentration of the solution in the microchannel. Although the exact concentration of the acrylamide-modified ssDNA in the hydrogel plug were not analytically measured, based on the operating conditions, it is calculated that the concentration of ssDNA S 2  complement captured is 20 μM based on a hydrogel plug photopolymerization solution containing 15 μM of acrylamide-modified ssDNA and the hydrogel plug which has not been rinsed with 0.5 M NaCl in 1×TE buffer. 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.  
         [0039]    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 FIGS.  3 ( a )- 3 ( d ) 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 and the position of the leading edge of fluorescence in the polymer plug, plotted as a function of electrophoresis time, is generally linear. 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 DNA target, the concentration of DNA probe in the hydrogel, and time allotted for electrophoresis.  
         [0040]    In another embodiment of the present invention, FIG. 4( a ) and  4 ( b ) depict a single microchannel fluidic device  410  which can be used for multi-analyte detection. Detection of multi-analytes can be realized by using different color fluorescing tags or by spatially localizing hydrogel plugs that contain different DNA probes. Three spatially-separated hydrogel plugs linear sections,  415   a,    415   b,    415   c,  contain different sequence DNA probes or no DNA whatsoever where photopolymerized in the same microchannel  11  but located in a different linear section of the microchannel  411 . Hydrogel plug section  415   a  contains DNA probes complementary to a fluorescein tagged target, S 1 , while the hydrogel plug section  415   c  contains DNA probes complementary to a TAMRA tagged target, S 2  best shown in FIG. 4( b ). The two DNA-containing plugs are separated by hydrogel plug section  415   b  that does not contain probe DNA.  
         [0041]    Initially the entire chip or microfluidic device  410  was filled with a photopolymerization solution containing acrylamide-modified S 2 , and the hydrogel plug section  415   c  was created by focusing UV light on the far right portion of the microfluidic channel  411 . The wells sections  414 ,  417 ,  419  and wells sections  420 ,  421 ,  422  and associated sections of the microchannel  411  were rinsed with 0.5 M NaCl in 1×TE buffer and well section  417 - 421  of the microchannel  411  was rinsed by electrophorescing the 0.5 M NaCl in 1×TE buffer through the section  417 - 421  of the microchannel  411  with the application of +25 V from reservoir  420  to reservoir  419 . The wells sections  420 ,  421 ,  422  of the microchannel  411  were then filled with a photopolymerization solution containing no DNA, and the hydrogel plug section  415   b  was created by electrophorescing the polymerization solution through the hydrogel plug section  415   c  and into the wells sections  417 - 421  of microchannel  411  via a +25 V from reservoir  420  to reservoir  419 . UV light was then focused onto the center of the section  417 - 421  of microchannel  411 . The rinse and buffer electrophoresis was repeated, and in the third step a photopolymerization solution containing acrylamide-modified S 1  was introduced into the well sections  420 ,  421 ,  422  of the microchannel  411 .  
         [0042]    The hydrogel plug section  415   a  was formed by electrophorescing the polymerization solution through hydrogel plug  415   c  containing the S 2  and hydrogel plug  415   b  having no DNA and UV light was focused on the far left portion of the microfluidic channel  411 . A final rinse of the well sections  414 ,  417 ,  419  and well sections  420 ,  421 ,  422  of the microchannel  411  with 0.5 M NaCl in 1×TE buffer completes the fabrication process.  
         [0043]    To demonstrate multi-analyte detection, a solution containing complements to both S 1  and S 2  was introduced into the well section  420 ,  421 ,  422  of the microchannel  411  and was electrophoresed through all three hydrogel plugs sections  415   a,    415   b,    415   c.  After rinsing the well sections  414 ,  417 ,  419  and well sections  420 ,  421 ,  422  of the microchannel  411  with 0.5 M NaCl in 1×TE buffer and electrophoresis of the buffer through the hydrogel plug sections  415   a,    415   b,    415   c  to remove unbound DNA complements at +25 V from reservoir  420  to reservoir  419 , green fluorescence is observed predominantly from the hydrogel plug section  415   a  indicating capture of the S 1  complement. Conversely, red fluorescence is observed solely from the hydrogel plug section  415   c,  indicating the presence of bound S 2  complement.  
         [0044]    Although DNA has been used herein as an exemplary ligand for use in the present microfluidic device, numerous additional ligands may be employed for use in the present device. A potentially useful list of ligands that could be immobilized in hydrogels for a variety analysis including single stranded DNA for capturing DNA and RNA targets, double stranded DNA for determination of protein-DNA interactions, protein or enzymes for capturing target proteins for proteomic applications, DNA aptamers for capturing target proteins for proteomic applications, and antibodies or antigens for immunoassay applications. Further, catalytic DNA or RNA may be used for analysis of the metal ions, small molecules, metabolites, or proteins.  
         [0045]    In the case of catalytic nucleic acid applications, the catalytic DNA or RNA is immobilized in a first hydrogel and an analyte is electrophoresed or pumped through the first hydrogel where the catalytic DNA or RNA undergo self-cleavage. The cleaved strand, having been previously labeled with a fluorophore or suitable group, is then transported to a second hydrogel that contains an immobilized capture strand that is complimentary to the cleaved strand where it is captured. As a result, all cleaved strands are concentrated in the second gel and sensitivity is enhanced.  
         [0046]    In addition, multi-analyte detection is also possible using the present device where multiple catalytic DNA&#39;s or RNA&#39;s are immobilized in a first hydrogel and all are labeled with the same fluorophore. Spatially separated additional hydrogels contain appropriate complimentary sequences which capture cleaved strands. Spatial separation of the captured regions permits the same fluorescent tag to be used for all catalytic reactions.  
         [0047]    Further, although previously described herein the analyte is electrokinetically driven through the porous matrix is generated by analyte flow, application of a pressure gradient can be used to induce flow of fluid and analyte through the pores of the matrix, thereby bringing analyte into contact with the ligands immobilized in the matrix.  
         [0048]    It will now be apparent to one of ordinary skill in the art that the present invention offers advantages previously not found in the art for use in achieving rapid multiplexed analysis of biological species in applications such as genomics, proteomics, and drug discovery, via biological ligands immobilized in hydrous gel plugs that are contained in microfluidic channels. Further, different ligands can be immobilized in series in a single microfluidic channel by sequential photopolymerization of hydrogel plugs containing the different ligands.  
         [0049]    Further, the present invention offers the advantage over prior systems by ensuring that target molecules collide with a captured ligand as the targets are electrophoresed through the hydrogel plugs. The primary advantages of the microfluidic hydrogel plugs versus two-dimensional bio-array formats include a greatly increased capacity relative to two-dimensional formats where monolayers of captured ligands are typically used because they three-dimensional nature of the gel plugs. In addition, the three-dimensional nature of the plugs generally increases the probability that the targets will encounter a captured ligand and biologically bind. Further, mass transport of biological targets to capture ligands is greatly enhanced because all targets are electrophoretically driven through the gel plugs. Therefore, analysis times are greatly reduced. Because the gel plugs are confined to the reduced space of a microchannel, the driving of sample through the plugs results in a concentrating effect. In addition, hydrous plugs containing different ligands can be mobilized in series in microphoretic channels thereby allowing multiplexed detection of targets (i.e., analytes).  
         [0050]    Although the invention has been described above in relation to preferred embodiments thereof, it will be understood by those skilled in the art that variations and modifications can be made in these preferred embodiments without departing from the scope and spirit of the invention.  
     
       
       
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gcaccttgtc atgtaccatc                                                 20 

 
           
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aggcccggga acgtattcac                                              20