Abstract:
A capillary electrophoresis device and separation protocol uses a hydraulic resistance-providing structure (HRPS) in the main separation channel to separate the divide the main separate channel into an upstream portion and a downstream portion. The HRPS may take the form of a porous plug, or a solid plug provided with at least one shallow channel. A sample separates and migrates through the porous structure or the shallow channel, upon application of a voltage difference between the upstream and downstream sides. Among other things, the HRPS helps reduce electrokinetic flow in the presence of conductivity gradients and facilitates robust, high-gradient on-chip field amplified sample stacking. The HRPS also enables the use of a pressure-injection scheme for the introduction of a high conductivity gradient in a separation channel and thereby avoids flow instabilities associated with high conductivity gradient electrokinetics. The approach also allows for the suppression of electroosmotic flow (EOF) and benefits from the associated minimization of sample dispersion caused by non-uniform EOF mobilities. An injection procedure employing a single pressure-flow high-conductivity buffer injection step followed by standard high voltage control of electrophoretic fluxes of sample, may be employed.

Description:
GOVERNMENT RIGHTS  
       [0001]     A portion of the work associated with the present invention was funded by DARPA grant F30602-00-2-0609. The U.S. Government may have rights to the present invention. 
     
    
     FIELD OF THE INVENTION  
       [0002]     The present invention is directed to microfluidic devices for carrying out electrophoresis. More particular, the present invention is directed to devices and methods designed for Field Amplified Sample Stacking (FASS) applications and their integration with electrophoretic separations.  
       BACKGROUND  
       [0003]     On-chip electrophoresis devices offer reduced sample volumes, rapid analysis time, and ease of automation. One drawback of microchannels is that the depth dimensions of etched channels (typically 10-20 μm deep) provide a short line-of-sight-integration length for optical detectors, and this adversely affects their limit of detection (LOD). One way of improving LOD is to integrate an on-line preconcentration process for sample analytes. Sample preconcentration offers higher sensitivity assays, robust electrokinetic injection schemes, and the use of detection modes less sensitive than fluorescence, such as electrochemical detection. Field-amplified sample stacking (FASS) has been used with free-standing capillaries, and also microchips. FASS is one of the most important preconcentration methods for on-chip electrophoresis as it is easily implemented into on-chip capillary zone electrophoresis (CZE) systems and provides a single-step method of achieving high sensitivity. In the past, on-chip FASS, as a stand-alone method, has been limited to less than 10 2  fold increases in signal strength.  
         [0004]     In conventional on-chip FASS systems, a sample analyte is dissolved in a solution of low ionic conductivity, and a small volume of this solution is introduced into the microchannel system using various electrokinetic—or pressure—injection methods. U.S. Pat. No. 6,695,009, whose contents are incorporated by reference to the extent necessary to understand the present invention, shows one prior art approach to sample stacking.  
         [0005]      FIGS. 1   a  &amp;  1   b  show a schematic of on-chip FASS in the absence of electroosmotic flow (EOF), in a microchip  102  having a “double-T” construction The microchip is provided with first  104   a  and second  104   b  regions of high conductivity at opposite ends of the main separation channel and a low conductivity region  106  between the side channels. For the purposes of illustration, only sample ions (typically present in lowest concentration) are shown. First, as seen in  FIG. 1   a,  anionic  108   a  and cationic  108   b  sample ions are introduced into the horizontal separation channel within a region of low ionic conductivity. And as seen in  FIG. 1   b,  on application of an electric field, E (indicated by the arrow  110 ), along the separation channel, sample ions exit the low conductivity/high electric field region and enter the high conductivity/low electric field region. Sample concentration increases as sample ions cross the interface between the high and low conductivity buffers. Cations electromigrate in the direction of electric field and stack at the interface on the cathode side, while anions stack at the anodic interface.  
       SUMMARY OF THE INVENTION  
       [0006]     In one aspect, the present invention is directed to a capillary electrophoresis microchip having a hydraulic resistance-providing structure (HRPS) in a main separation channel thereof. The HRPS divides the main separation channel into upstream and downstream portions. In one embodiment, the HRPS is a porous polymer plug formed in the main separation channel. In another embodiment, the HRPS is a channeled plug provided with one or more shallow channels.  
         [0007]     In another aspect, the present invention is directed to a method of performing electrophoresis using such a microchip. A first buffer having a first conductivity can be introduced into both the upstream and downstream portions of the main separation channel, into the first side channel and into the second side channel. A second buffer having a second conductivity may then be introduced into the upstream portion and the first and second side channels, but not into the downstream portion, first conductivity being higher than the second conductivity. A sample is then introduced into the main separation channel and a separation voltage applied, which causes at least a part of the sample to migrate through said HRPS and into the downstream portion.  
         [0008]     In another aspect, the present invention is directed to making such microchips:  
         [0009]     In the case of the porous polymer plug, a monomer solution is introduced into main separation channel, a mask applied, and then uncovered portions of the monomer are activated using UV light.  
         [0010]     In the case of the channeled plug, the upper surface of the substrate is etched to form the upstream portion, etched to form the downstream portion, and etched to form one or more plug channels in the region between the upstream and downstream portions. The etching may be done in any sequence, including having the upstream and downstream portions etched at the same time. Regardless of the etch sequence, in the resulting device, the one or more plug channels connect the upstream portion with the downstream portion, thereby permitting fluid flow there between. In this embodiment, the channeled plug has unitary, one-piece construction with the substrate.  
         [0011]     In an alternate embodiment for forming the channeled plug, a plug is formed as a separate plug insert with bottom and side surfaces that conform to the contour of the main separation channel of a microchip, and an upper surface provided with one or more channels. The separate plug insert is then positioned and fixed in the main separation channel using an adhesive or the like.  
         [0012]     In another aspect, the present invention is directed to a method of reducing electrokinetic flow instabilities during electrophoresis of a sample across a conductivity gradient in a main separation channel of a microfluidic electrophoresis chip. The method calls for providing a high hydraulic resistance region in the main separation channel between an upstream portion and a downstream portion, introducing first and second buffers on different sides of the high hydraulic resistance region, introducing a sample into the upstream portion, and then applying a voltage to cause the sample to separate and migrate in the direction of the downstream portion.  
         [0013]     In yet another aspect, the present invention is directed to a method of performing electrophoresis on a sample present in a main separation channel of a microfluidic electrophoresis chip. This is done by first providing a high hydraulic resistance region in the main separation channel between an upstream portion and a downstream portion, subjecting the sample to an electric field so as to form a stacked sample on an upstream side of the hydraulic resistance region, applying a voltage difference between the upstream side and a downstream side of the HRPS that is sufficient to cause the stacked sample to separate and migrate through the HRPS; and detecting the sample after it has separated and migrated. In still another aspect, a system in accordance with the present invention employs a simple pressure flow control scheme that uses a single pressure-driven loading step for high conductivity buffer, followed by a single pressure-driven loading step for low conductivity buffer, followed by a single pressure-driven loading step for sample ions. These loading steps are then followed by standard high voltage electrokinetic injection process. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0014]      FIGS. 1   a  &amp;  1   b  illustrate field amplified sample stacking;  
         [0015]      FIG. 2   a  shows a microchip having a hydraulic resistance-providing structure (HRPS) in accordance with the present invention;  
         [0016]      FIGS. 2   b  &amp;  2   c  show alternate configurations for HRPS in a microchip in accordance with the present invention;  
         [0017]      FIG. 3   a  illustrates a method of introducing oil and monomer into a microchip to create a polymer plug;  
         [0018]      FIG. 3   b  shows a mask covering the substrate to form a polymer plug;  
         [0019]      FIG. 3   c  shows an arrangement for initiating the monomer with light;  
         [0020]      FIGS. 4   a - 4   d  illustrate a field amplified sample stacking/capillary electrophoresis (FASS/CE) assay protocol using a microchip in accordance with  FIG. 2   a.    
         [0021]      FIG. 5  shows an apparatus for separating and detecting samples;  
         [0022]      FIGS. 6   a,    6   b  and  6   c  show views of a second embodiment of a portion of a microchip in accordance with the present invention;  
         [0023]      FIG. 7   a  shows a section of the main separation channel having an HRPS in the form of an obstruction provided with at least one shallow channel; and  
         [0024]      FIGS. 7   b  &amp;  7   c  show cross-sections taken along lines of  3   b - 3   b  and  3   c - 3   c,  respectively, of  FIG. 3   a.    
     
    
     DETAILED DESCRIPTION  
       [0025]      FIG. 2   a  shows a microchip  200  in accordance with the present invention. The microchip  200  has a hydraulic resistance-providing structure (HRPS)  202  of length L 1  along the horizontal, main separation channel  204 . The HRPS  202  is positioned such that an ‘open’ channel extends on either side. Thus, the HRPS  202  has an upstream interface  202   a  facing an upstream portion  204   a  of the main separation channel  204  and a downstream interface  202   b  facing a downstream portion  204   b  of the main separation channel  204 . As seen in  FIG. 1 , the downstream portion  204   b  extends for some non-zero length L 2 .  
         [0026]     Connected to the main separation channel at a first channel center point is a first, or north, side channel  206 . A second, or south, side channel  208  is connected to the main separation channel  204  at a second channel center point. In a preferred embodiment, the first and second channel center points are spaced apart from each other by a distance d and so the microchip has a “double-T” construction.  
         [0027]     The ends of the various separation channels are provided with reservoirs  222 ,  224 ,  226  and  228  for the introduction of buffers, samples other fluids and materials. In this regard, the first side channel  206  is provided with north reservoir  222 ; the second side channel  208  is provided with south reservoir  224 , and the main separation channel  204  is provided with east reservoir  226  on the downstream side  204   b  and west reservoir  228  on the upstream side  204   a.    
         [0028]     The length L 1  of the HRPS  202  preferably is between 0.01 mm and 5 mm, more preferably between 0.1 mm and 1.0 mm and most preferably is about 0.5 mm. It is understood, however, that the HRPS  202  may be of some other length, instead. The HRPS is a distance d 1  from the center point between the two side channels and a distance d 2  from the nearest portion of the closest side channel, which in the construction shown is the first side channel  206 . In a preferred embodiment, d 1  is between 0.2 mm and 0.4 mm, and more preferably about 0.31 mm.  
         [0029]      FIG. 2   b  shows a double-T microchip  250  with dual HRPS&#39;s, one on either side of the side channels, and  FIG. 2   c  shows an X-type microchip  260  with a single HRPS. Other configurations for placement of one or more HRPS&#39;s are also possible.  
         [0030]     One function of the HRPS is to retard flow between the upstream  204   a  and downstream  204   b  portions of the main separation channel  204 . In the present invention, the HRPS is implemented in one of two general ways: (1) providing a porous polymer plug in the main separation channel  204 ; or (2) providing a solid obstruction in the main separation channel, the solid obstruction having at least one shallow channel which connects the upstream  204   a  and downstream  204   b  portions of the main separation channel. Both approaches result in a structure that retards or otherwise constricts the flow of liquid between the upstream  204   a  and downstream  204   b  portions.  
       POROUS POLYMER PLUG AS THE HRPS  
       [0031]     The present inventors have described implementation and experimentation of a device in accordance with the present invention having a porous polymer plug in: Jung, B., Bharadwaj, R. &amp; Santiago, J. G., “Thousand-Fold Signal Increase Using Field-Amplified Sample Stacking for On-Chip Electrophoresis”, Electrophoreses 2003, v. 24, No. 19-20, (Oct., 2003). The contents of this paper are incorporated by reference.  
         [0000]     Formation of Porous Polymer Plug  
         [0032]     The starting point for the polymer plug implementation was a commercially available microchip from Micralyne of Alberta, Canada (www.micralyne.com). The microchip has a double-T geometry, with a channel width of 50 μm and a channel depth everywhere at a maximum 20 μm.  
         [0033]     The porous polymer plug was fabricated using a photoinitiated polymerization process similar to that described in Yu, C., Xu, M. C., Svec, F., Frechet, J. M. J., “Preparation of Monolithic Polymers with Controlled Porous Properties for Microfluidic Chip Application”, J. Polymer Science Part A 2002, 40, 755-769, whose contents are incorporated by reference. Ethylene dimethacrylate (EDMA; Sartomer, PA), glycidyl methacrylate (GMA; Sartomer, PA), and azo-bisisobutyronitrile (AIBN; Aldrich, Wis.) were obtained. The monomer (EDMA 0.96 g, GMA 1.421 g), porogenic solvent (50/50 wt % methanol/ethanol 3.6 g), and photoinitiator (AIBN 24 mg) are mixed and then purged with nitrogen for 10 min before use. Prior to introducing the monomer, the microchip was prepared by first rinsing with 0.1 M NaOH for 10 minutes, and then rinsing with deionized water for 30 minutes using a syringe pump.  
         [0034]      FIGS. 3   a  - 3   c  show the process for forming a porous polymer plug-type HRPS 102 in accordance with one embodiment of the invention. The upstream interface  104   a  of the porous polymer plug-type HRPS is defined by an immiscible interface of oil and the monomer solution.  
         [0035]     In the microchip  300 , monomer solution  304  is introduced into the east reservoir  326  in a controlled manner, such as by a first syringe  306  driven by a first syringe pump under computer control. As the monomer solution  304  is being introduced, oil  302  is simultaneously introduced into the north  322  reservoir, also in a controlled manner, such as by a second syringe  308  driven by a second syringe pump under computer control. It is understood that instead of, or in addition to, the north reservoir  322 , the oil may be introduced into the south  324  and/or west  328  reservoirs, as well. Regardless of into which reservoir(s) the oil  302  is introduced, one may control the rates of introduction of the monomer  304  and the oil  302  such that the leading oil front  302   a  and the leading monomer front  304   a  move toward each other as indicated by the arrows in  FIG. 3   a,  and ultimately meet at the future upstream interface  314 . The immiscibility of oil and the monomer helps ensure that the boundary between them is well-defined.  
         [0036]     After the oil  302  and monomer  304  have been loaded into the channels and have met at the future upstream interface  314 , a mask  350  having a window  352  is placed over the microchip  300 . In a preferred embodiment, the mask  350  is a printed ink-on-mylar film shadow mask, and the window  352  permits exposure of only that portion of the monomer  304  to be polymerized into the porous polymer plug-type HRPS.  
         [0037]     As seen in  FIG. 3   c,  broadband light from a mercury arc lamp  364  is focused on the plane of microchip  300  via a UV transmitting filter cube  366  and an epifluorescent microscope  368 . In one embodiment, the microchip  300  is exposed for four hours, although other lengths of time may also be used. After the monomer has been photo-polymerized, the remaining monomer is removed from the system, preferably by rinsing the microchannel with methanol for 2 hours and then deionized water for 3 hours using a syringe pump.  
         [0038]     In the foregoing photo-polymerization example, due to blurring that results from using the broadband mercury arc lamp  364 , an oil-monomer interface was used to provide the porous polymer plug-type HRPS  202  with a more precise upstream interface  202   a  where the sample for separation is to be introduced, the downstream interface  202   b  not being as critical. In an alternate embodiment for forming the plug, one may use a laser instead of the mercury arc lamp  364  as the light source. In such an alternate embodiment, the monomer may be introduced throughout the length of the main separation channel, a mask placed over the microchip, and a laser used to perform the photo-polymerization, thereby dispensing with the need to first form the oil-monomer interface. Other methods may also be used to form the polymer plug-type HRPS  202 .  
         [0039]     The pore diameter distribution of the porous polymer structure can be analyzed by polymerizing monoliths off-chip. In one experiment, a small glass chamber was filled with the same monomer solution, and then exposed to similar polymerization conditions. After polymerization, the monoliths were removed from the glass chamber, washed with methanol and dried. The median pore diameter is about 4.6 μm, with at least 90% of the pores having a diameter between 1 nm and 10 μm. A void volume of the material is about 0.5, but preparations having void volumes on the order of between 0.05 and 0.9 can be prepared.  
         [0000]     Buffer &amp; Sample  
         [0040]     A low conductivity buffer, a high conductivity buffer and a fluorescent sample are first prepared. A 5 mM HEPES (Sigma, Mo.) buffer solution with a pH of 7.0 was used with a 0.4 wt % methyl cellulose (Aldrich, Wis.) solute to suppress electroosmotic flow (EOF). This serves as the “low conductivity buffer”. A high conductivity buffer (77.6 mS/cm) was prepared by dissolving a requisite amount of NaCl salt (J. T. Baker, N.J.) to the HEPES buffer. The sample solute comprises an aqueous solution of 1 μM bodipy dye (available from Molecular Probes, Oreg.) and 2 μM fluorescein dye (available from J. T. Baker, N.J.). All sample and buffer solutions were filtered with 0.2 μm syringe filter before use. The conductivity of buffer and sample solution were measured using a conductivity meter (available from Jenco Instruments, Calif.).  
         [0000]     Field Amplified Sample Stacking/Capillary Electrophoresis (FASS/CE) Assay Protocol  
         [0041]      FIGS. 4   a - 4   d  illustrate a preferred embodiment of a FASS/CE assay protocol in accordance with the present invention, in which a porous polymer plug-type HRPS  402  was used.  
         [0042]     Prior to introducing any buffer, a microchannel glass surface treatment was performed. This was done by rinsing the microchip with a dynamic coating reagent. Although a variety of coating reagents may be employed, the aforementioned 0.4% methyl cellulose solution was used in this role, and so was introduced into the entire microchip by flowing for 30 min. All buffers used in the experiment contain the same amount of methyl cellulose, to help suppress EOF throughout the microchip.  
         [0043]     As depicted in  FIG. 4   a,  first, high conductivity buffer  410  is introduced, via the east reservoir  426 , into the downstream portion  404   b  of the main separation channel, through the HRPS, and into the upstream portion  404   a,  and the side channels  406 ,  408 , as indicated by the arrows. In one embodiment, the high conductivity buffer  410  is introduced by injection with a computer-controlled syringe pump system  430 . The syringe pump introduced the high conductivity buffer at a flow rate of about 1.0 μl/min for approximately 1.0 minute. During this introduction, the porous polymer plug-type HRPS provides high hydraulic resistance to buffer flow.  
         [0044]     The hydraulic resistance per unit length can be quantified as the ratio of the local pressure gradient to the volume flow rate. A typical 50 micron wide by 20 micron deep channel has a hydraulic resistance per unit length of 4.41×10 16  Pa·s/m 4 . For an exemplary chip in accordance with the present invention, the porous region has a hydraulic resistance per unit length that is roughly 25 times larger, about 1.18×1018 Pa·s/m 4 , based on the equation:  
             R   =         Δ   ⁢           ⁢   P     QL     =       8   ⁢   τμ       ψ   ⁢           ⁢     Aa   2                   (     Eq   .           ⁢   1     )             
 
 where ΔP/L is pressure gradient; Q is flow rate; L is the length of the porous plug; porosity ψ=0.45; A is the cross-sectional area of the porous plug, the average pore diameter a=4.9 μm, tortuosity τ=1.45; the viscosity of the buffer μ=0.001 Pa·s, and assuming no electric field present. 
 
         [0045]     As depicted in  FIG. 4   b,  low conductivity buffer  412  is then introduced from the north reservoir  422  using syringe  432 . It is understood, however, that the low conductivity buffer could be introduced via the south  424  or west  428  reservoirs instead. In one embodiment, the low conductivity buffer  412  is introduced at a flow rate of about 0.1 μl/min for 0.5 min. Introducing the low conductivity buffer  412  at a lower pressure and for a lower time than that used to introduce the high conductivity buffer, helps reduce the amount of low conductivity buffer that passes through the porous polymer plug-type HRPS  402  from the upstream interface  402   a  to the downstream interface  402   b.  It therefore helps prevent mixing of the low conductivity buffer with the high conductivity buffer in the downstream portion  404   b  of the main separation channel. In this instance, the porous polymer plug-type HRPS provides high hydraulic resistance which minimizes the mixing of two buffers at the upstream HRPS/buffer interface  402   a,  as well. The result of this step is that high conductivity buffer  410  occupies the downstream portion  404   b  of the main separation channel  404  while low conductivity buffer  412  is present in the upstream portion  404   a  of the main separation channel  404  and also in the first  406  and second  408  side channels.  
         [0046]     Next, as seen in  FIG. 4   c,  an anionic sample  444  was then electrokinetically introduced into the double-T injector, via the south reservoir  424 . For this, the south reservoir was filled with the sample mixture of bodipy and fluorescein and electrically grounded. A positive voltage source  450  providing a voltage V 1 , which in one embodiment is 1 kV, was applied to the north reservoir  422  with the south reservoir  424  connected to ground  452  and the east  426  and west  428  reservoirs allowed to electrically float. This creates an electric field that caused negatively charged sample ions to electromigrate from the south reservoir  424  towards the north reservoir  422 , with at least a portion of the sample ending up in the main separation channel  404 , between the two side channels  406 ,  408 .  
         [0047]     Finally, as seen in  FIG. 4   d,  a positive voltage source  454 , providing in one embodiment, 3 kV, is applied at the east reservoir  426  while the west reservoir  428  is connected to ground  456 , thereby establishing an east-to-west electric field. This field initiates both sample stacking and electrophoretic separation of the negatively charged sample ions. The sample in the main separation channel  404  thus undergoes stacking and migration in the downstream direction through the porous polymer plug-type HRPS  402 , and separates into bands  480  which can then be detected in a manner known to those skilled in the art. Preferably, these bands are detected in the downstream portion  404   b,  as seen in  FIG. 4   d,  though the detection may also be performed while the bands are transiting through the HRPS  402 . In a preferred embodiment, the separated sample peaks were detected using an epifluorescent microscope and a CCD camera with a viewing region positioned 10 mm downstream of the injection region.  
         [0048]     While specific values are presented in the foregoing description, it is understood that a wide variety of values may be used.  
         [0049]     For example, it is understood that the terms “low hydraulic resistance” and “high hydraulic resistance” are relative terms. In general, a “high hydraulic resistance” may be anywhere from 1×10 16  Pa·s/m 4  to 1×10 19  Pa·s/m 4 , depending on the hydraulic resistance of the channel where no plug is present. In general, however, the region of high hydraulic resistance preferably has a hydraulic resistance that is 10-100 times as great as the low hydraulic resistance region.  
         [0050]     Furthermore, the terms “low conductivity” and “high conductivity”, as applied to buffers, are relative terms. Thus, a low conductivity buffer may have a conductivity between 1 uS/cm and 1 mS/cm, while a high conductivity buffer has a conductivity that is about 10-10,000 times higher.  
         [0051]     As to the voltage applied to effect stacking and separation, it is possible to have this depend on the length of the high hydraulic resistance region. Thus, for instance, one may apply a voltage difference of between 100-100,000 volts, if the length of the high hydraulic resistance region is between 1 and 100 cm, and a voltage difference of between 1-100 volts, if the length of the high hydraulic resistance region is between 0.05 and 1 cm. Preferably, though, the applied voltage is sufficient to cause the sample to enter a region adjacent to the upstream side of the porous plug at a rate between 1 and 100 nl/min.  
         [0000]     Detection System  
         [0052]      FIG. 5  shows a schematic of an experimental FASS/CE microchip setup  500 . The detection/visualization system  502  includes an intensified CCD camera  504  (Roper Scientific, IPentaMAX, N.J.) connected to a computer  506  for processing and display. The CCD camera  504  receives light from an inverted epifluorescent microscope  508  (Olympus, IX70, N.Y.) comprising a 10× objective  510  (numerical aperture (N.A.) of 0.3, Olympus, N.Y.) and a XF100-3 filter cube  512  (Omega Optical, Vt.) with peak excitation and emission wavelength ranges of 450-500 nm and 500-575 nm. A mercury lamp  514 , whose beam is directed via the filter cube  512  before impinging on the separated samples, is used to cause the dyes to fluoresce. The setup  500  also includes the microchip  520  itself, a multi-valve syringe pump  522  (Harvard Apparatus, Pump 33, Mass.), for pressure-injection control, and a multi-port high voltage power supply  524  (Micralyne, Alberta, Canada). The syringe pump  522  and the power supply  524  are under the control of computer  526 . Various pressure/flow and electrical connections to the microchip are shown as solid  530  and dashed  532  lines, respectively, and are known to those skilled in the art.  
       CHANNELED PLUG AS THE HRPS  
       [0053]      FIG. 6   a  shows a channeled plug  602  having an upper surface  603  provided with three linear, shallow plug channels  607 . The channeled plug  602  preferably is solid in that buffers and the like do not normally pass through the plug material itself, but rather only through the channels  607 . Thus, the channeled plug  602  is relatively non-porous, in contrast to the porous polymer plug  402  discussed above. Preferably, the channeled plug  602  is formed of the same material as the substrate in which the main separation channel is formed.  
         [0054]     It is understood that the upper surface  603  of the channeled plug  602 , as well as the rest of the main separation channel  604 , are under a glass surface  632 , as is typical with microchips. It is also understood that a different number, such as 1, 2, 4 or even more, plug channels may be provided. It is further understood that the plug channels do not necessarily have to be linear or have the same cross-sectional area, though both are preferable.  
         [0055]     The plug channels  607  connect the upstream side  604   a  of a main separation channel  604  with the downstream side  604   b.  The plug channels  607  are configured and dimensioned to permit a fluid to pass between the upstream  604   a  and downstream  604   b  portions of the main separation channel  604 . During the pressure injection protocol, the smaller cross-sectional area of the plug channels  607 , relative to that of the main separation channel  604 , provides hydraulic resistance to fluid flow. Detection of a migrating sample can take place while the sample still occupies channels  607 , or after the sample has exited the channels  607 .  
         [0056]     The plug channels  607  have a plug channel depth h 1  that is less than a depth h 2  of the main separation channel. The plug channel depth h 1  is nominally between 100 nm and 2 μm although it may take on other heights, as well. Furthermore, the plug channel depth h 1  preferably is no greater than 1/10 the depth h 2  of the main separation channel. The plug channels have a plug channel width w 1  that is less than a width w 2  of the main separation channel. The plug channel width w 1  is nominally between 1 μm and 10 μm. Furthermore, the plug channel width w 1  is no greater than ⅕ the width w 2  of the main separation channel. And while the channels  607  formed in the upper surface of the plug  603  preferably have a rectangular cross-section, they may instead take on other cross-sectional shapes.  
         [0057]     In one embodiment, the plug has unitary one-piece construction with the substrate. In such case, the channels  607  and the upstream and downstream portions are formed of one continuous piece of substrate material, and the substrate is subjected to etching and/or machining to create the various formations therein.  
         [0058]     In an all-etch process, a first portion of the substrate is etched to form an upstream portion of the main separation channel, a second portion of the substrate is etched to form a downstream portion of the main separation channel, and one or more shallow channels are etched in a third portion of the substrate, the one or more shallow channels in the resulting structure connecting the upstream and downstream portions. The various etching is performed under appropriate conditions so that the etched shallow channel depth h 1  is less than a depth h 2  of either the upstream portion or the downstream portion. Preferably, the upstream and downstream channels are etched simultaneously, and then the shallow channels are etched. However, the present invention contemplates that these three portions of the substrate can be etched in any order in either two or three separate steps.  
         [0059]      FIG. 7   a  shows an example of a mask  700  that can be used to prepare for simultaneously etching both the upstream and downstream portions of a main separation channel. The mask  700  has a first opening  704   a  that corresponds to the region where at least the upstream portion will be formed and a second opening  704   b  that corresponds to the region where at least the downstream portion will be formed. The mask  700  has a channel portion  702  that separates the first  704   a  and second  704   b  openings. The mask  700  also has a pair of alignment marks  738   a,    738   b  to facilitate positioning the openings in the proper locations.  
         [0060]      FIG. 7   b  shows an example of a mask  750  that can be used to prepare for etching the channels  607  of the channeled plug  602 . The mask  750  has a plurality of slots  757  that correspond to the positions where the channels  607  are to be formed. The mask  750  also has a pair of alignment marks  788   a,    788   b  that match the location of alignment marks on mask  700 . This results in the main separation channel having an elevated portion provided with the plug channels  
         [0061]     Preferably, mask  700  is used to etch the upstream  604   a  and downstream  604   b  portions in a first etching step, and then mask  750  is used to etch the channels  607  in a second etching step.  
         [0062]      FIG. 8   a  depicts an alternative embodiment for preparing a microchip in accordance with the present invention, a plug insert  803  is first formed. The plug insert  803  has a lower surface that conforms to the cross-sectional, typically D-shaped, contour of the main separation channel  804  of a microchip. The upper surface of the plug insert  803  is provided with one or more channels, whose shape and dimensions are described above, the channels being formed by etching or machining. Regardless of how it is formed, as depicted by the arrow in  FIG. 8   a,  the plug insert  803  ultimately is placed in the main separation channel  803  and fixed thereto by means of an adhesive or the like.  
         [0063]     As seen in  FIG. 8   b,  an alternative plug insert  853  has plurality of channels  854  formed within, and along, the body of the insert  853  in a longitudinal direction. In such case, during the pressure injection protocol, the buffers and other materials pass though the body of the plug insert  854 , and sample detection occurs only after the sample has exited the plug insert  854  on the downstream side of the main separation channel.  
         [0064]     From the foregoing, it is evident that the term ‘plug’, as used herein, covers a structure that (a) is formed, in situ, in a main separation channel (such as the porous polymer plug), (b) is formed as a separate component, and then inserted into the main separation channel (such as the plug insert), or (c) has unitary construction with the main separation channel (such as being formed by etching a region of the substrate located between what are, or will become, the upstream and downstream sides).  
         [0065]     It is further understood that one uses the channeled plug-type HRPS in a manner similar to that of the porous polymer plug-type HRPS, described above. Thus, a substantially similar pressure-injection protocol may be employed with channeled plug-type HRPS. Generally speaking, the HRPS  202 , however implemented, provides a region of high hydraulic resistance to pressure driven flow that still allows electrophoretic migration to take place. The above-described pressure-injection protocol takes advantage of this, resulting in two consequences.  
         [0066]     First, the pressure-injection protocol results in a device having a high conductivity gradient within the separation channel while still having suppressed electroosmotic flow, EOF suppression being realized in the above-described embodiment by the use of methyl cellulose. Suppressing the EOF helps reduce sample dispersion during the simultaneous FASS/CE process.  
         [0067]     Second, the pressure-injection protocol helps reduce electrokinetic instabilities. As is known to those skilled in the art, electrokinetic instabilities are associated with high conductivity gradient regions near channel intersections where conductivity gradients and electric fields are three-dimensional. Such electrokinetic instabilities can cause excessive dispersion of the buffer-buffer interface, thereby limiting the performance of FASS with high stacking ratios. The pressure-injection scheme allows for the establishment of an initial conductivity gradient within the separation channel, followed by sample introduction into one side channel, and application of a voltage V 1  across both two side channels, thereby creating an electric field and causing the sample to enter into the main separation channel. In particular, the protocol allows for a voltage V 1  creates an electric field sufficiently large to introduce a portion of the sample into the main separation channel, yet not so large as to induce electrokinetic instabilities at the upstream interface  402   a  of the HRPS  402 .  
         [0068]     Finally, while the present invention has been described with respect to one or more preferred embodiments, it should be kept in mind that variations from this are also contemplated to be within the scope of the invention, as claimed below.