Microfluidic matrix localization apparatus and methods

Abstract

Multiphasic microfluidic apparatus for performing product fluid manipulation and separation in a single continuous unit are provided. Related methods, kits, and compositions are also provided.

What is claimed is:  
     
         1 . A microfluidic apparatus comprising: 
 a microfluidic substrate comprising a first channel intersecting a second channel, wherein the first channel contains a first fluidic phase and the second channel contains a second fluidic phase, wherein the first and second fluidic phases cross and are substantially unmixed at the intersection between the channels.    
     
     
         2 . The microfluidic apparatus of  claim 1 , wherein the intersection between the first and second channel is unvalved or electrically gated.  
     
     
         3 . The microfluidic apparatus of  claim 1 , wherein the first fluidic phase provides for electoosmotic movement of a component in the first phase and the second fluidic phase provides for electrophoretic movement of the component in the second phase.  
     
     
         4 . The microfluidic apparatus of  claim 1 , wherein the first or second fluidic phase is electrically loaded in the first or second channel.  
     
     
         5 . A microfluidic apparatus comprising: 
 a microfluidic substrate comprising a first channel intersecting a second channel, wherein the first channel comprises an electroosmotic fluidic phase and the second channel comprises an electrophoretic fluidic phase and the intersection between the first and second channel is unvalved or electrically gated.    
     
     
         6 . The microfluidic apparatus of  claim 5 , wherein the electrophoretic fluid phase comprises a sieving matrix.  
     
     
         7 . The microfluidic apparatus of  claim 1  or  5 , the apparatus further comprising a multiport electrical controller which is in fluid communication with the first and second channels.  
     
     
         8 . The microfluidic apparatus of  claim 7 , wherein the electrical controller provides electroosmotic control of a component in the first channel and electrophoretic control of the component in the second channel.  
     
     
         9 . The microfluidic apparatus of  claim 7 , wherein the electrical controller provides crossed electroosmotic and electrophoretic control of one or more components in the first and second channels.  
     
     
         10 . The microfluidic apparatus of  claim 5 , wherein the intersection of the first and second channels is substantially free of the electrophoretic phase.  
     
     
         11 . The microfluidic apparatus of  claim 10 , wherein molecular components stack against the electrophoretic phase in the second channel at an edge of the intersection between the first and second channels.  
     
     
         12 . The microfluidic apparatus of  claim 1  or  5 , wherein an interior portion of the first or second channel has at least one cross sectional dimension between about 0.1 μm and 500 μm.  
     
     
         13 . The microfluidic apparatus of  claim 5 , wherein the electrophoretic phase or the electroosmotic phase are electrically loaded in the first or second channel.  
     
     
         14 . The microfluidic apparatus of  claim 1  or  5 , further comprising a third channel in fluid communication with the first or second channel.  
     
     
         15 . The microfluidic apparatus of  claim 1  or  5 , further comprising a high resistance channel region for joule heating.  
     
     
         16 . The microfluidic apparatus of  claim 1  or  5 , wherein the first channel comprises a resistance region for joule heating and wherein the apparatus comprises PCR reagents in the first channel.  
     
     
         17 . The microfluidic apparatus of  claim 1  or  5 , wherein the microfluidic substrate comprises a pressure port in fluid communication with the first or second channel.  
     
     
         18 . The microfluidic apparatus of  claim 1  or  5 , further comprising a microfluidic substrate holder for mounting the substrate.  
     
     
         19 . The microfluidic apparatus of  claim 1  or  5 , further comprising a component detection region in a channel wall and a detector mounted to view a component.  
     
     
         20 . The microfluidic apparatus of  claim 1  or  5 , further comprising a component detection region in a channel wall, a detector mounted to view a component, a recording device operably linked to the detector, and a digital computer operably linked to the recording device.  
     
     
         21 . A microfluidic apparatus comprising at least two intersecting channels in fluid communication, wherein the two intersecting channels are substantially filled with an electrophoretic phase and wherein the electrophoretic phase comprises at least two reactants dispersed in the electrophoretic phase.  
     
     
         22 . The microfluidic apparatus of  claim 21 , wherein the electrophoretic phase is a sieving matrix.  
     
     
         23 . The microfluidic apparatus of  claim 21 , wherein the apparatus comprises an electrical controller which directs electrophoretic movement of the at least two reactants in the electrophoretic phase.  
     
     
         24 . The microfluidic apparatus of  claim 21 , wherein the apparatus comprises a reaction region.  
     
     
         25 . The microfluidic apparatus of  claim 21 , wherein the apparatus comprises a high-resistance channel region for heating the at least two reactants.  
     
     
         26 . The microfluidic apparatus of  claim 21 , wherein the at least two reactants are heterogeneously dispersed throughout at least a portion of the electrophoretic phase.  
     
     
         27 . The microfluidic apparatus of  claim 21 , wherein the at least two reactants are homogeneously dispersed throughout the electrophoretic phase.  
     
     
         28 . The microfluidic apparatus of  claim 21 , wherein one of the at least two reactants is homogeneously dispersed throughout the electrophoretic phase and one of the reactants is heterogeneously dispersed in at least a portion of the electrophoretic phase.  
     
     
         29 . The microfluidic apparatus of  claim 21 , wherein the channels comprise a region with an interior dimension of between about 0.1 μm and 500 μm.  
     
     
         30 . The microfluidic apparatus of  claim 21 , wherein the reactants comprise PCR reagents.  
     
     
         31 . A method of making a microfluidic substrate comprising: 
 fabricating at least a first and second channel in the microfluidic substrate, wherein an interior portion of the first or second channel has at least one cross sectional dimension between about 0.1 μm and 500 μm and wherein the first and second channels intersect; and,    selectively fixing an electrophoretic phase in a selected region of the first or second channel.    
     
     
         32 . The method of  claim 31 , wherein the intersection of the first and second channels is unvalved or electrically gated.  
     
     
         33 . The method of  claim 31 , wherein the electrophoretic phase is electrically loaded.  
     
     
         34 . The method of  claim 31 , wherein the electrophoretic phase is loaded under pressure.  
     
     
         35 . The method of  claim 31 , wherein the electrophoretic phase is a sieving matrix.  
     
     
         36 . The method of  claim 31 , wherein the electrophoretic phase is selectively fixed by photopolymerizing the electrophoretic phase in the selected region.  
     
     
         37 . A method of making a microfluidic substrate comprising an electrophoretic phase in a selected region of a microfluidic channel in the microfluidic substrate, the method comprising: 
 providing a microfluidic substrate comprising at least one microfluidic channel intersection;    flowing a component of the electrophoretic phase into at least the selected region of at least one of the intersecting channels; and    fixing the component in the selected region, thereby providing the electrophoretic phase in the selected region.    
     
     
         38 . The method of  claim 37  wherein the electrophoretic phase is a sieving matrix.  
     
     
         39 . The method of  claim 31  or  claim 37 , wherein the electrophoretic phase is fixed by replacing a portion of the electrophoretic phase with a selected fluid.  
     
     
         40 . The method of  claim 39 , wherein replacement of the electrophoretic phase with the selected fluid is performed by introducing the selected fluid into a region of the first or second channel under pressure.  
     
     
         41 . The method of  claim 31  or  37 , wherein the electrophoretic phase is photopolymerizable, and fixing the component in the selected region comprises exposing the selected region to light, thereby polymerizing the component to provide the electrophoretic phase in the selected region.  
     
     
         42 . The method of  claim 31  or  37 , the method comprising flowing a cross-linking or activating compound into the selected region whereby the cross-linking or activating compound results in polymerization of the component to provide the electrophoretic phase in the selected region.  
     
     
         43 . The method of  claim 42 , wherein the electrophoretic phase is polyacrylamide and the activating or cross linking compound comprises TEMED.  
     
     
         44 . The method of  claim 31  or  37 , wherein the component of the electrophoretic phase is flowed into multiple regions of one or more channels in the microfluidic substrate including the selected region and is selectively fixed in place in the selected region.  
     
     
         45 . The method of  claim 44 , the method comprising flowing a solubilization compound into an unselected region of a channel, thereby dissolving the electrophoretic phase in the unselected region.  
     
     
         46 . The method of  claim 45 , wherein the solubilization compound comprises sodium periodate.  
     
     
         47 . The method of  claim 37 , the method comprising dispersing reactant components in the component of the electrophoretic phase and electrophoresing the reactant components into the selected region, whereby the reactant components are concentrated in the selected region to provide concentrated reaction components.  
     
     
         48 . The method of  claim 47 , wherein the concentrated reaction components are reacted.  
     
     
         49 . The method of  claim 48 , wherein the reaction components are reacted by heating the components.  
     
     
         50 . The method of  claim 48 , wherein the reaction components comprise PCR reagents and the method comprises cycled heating of the components.  
     
     
         51 . A microfluidic apparatus for performing PCR comprising a first microchannel having disposed therein a mixture comprising a sieving matrix and a plurality of PCR reaction components.  
     
     
         52 . The apparatus of  claim 51 , further comprising means for heating the mixture in the channel.  
     
     
         53 . The apparatus of  claim 51 , further comprising a second microchannel intersecting the first microchannel, wherein the first microchannel comprises a resistance region for joule heating and the second channel comprises a detection region for detecting a PCR product.  
     
     
         54 . The apparatus of  claim 53 , the apparatus further comprising a fluorescence detector positioned to view the detection region.  
     
     
         55 . The apparatus of  claim 51 , the wherein the plurality of PCR reaction components comprises a plurality of: a thermostable DNA polymerase, a plurality of nucleotides, a nucleic acid template, and at least one primer which hybridizes to the nucleic acid template.  
     
     
         56 . The apparatus of  claim 55 , wherein the plurality of PCR reaction components comprises a thermostable DNA polymerase, a plurality of nucleotides, a nucleic acid template, at least one primer which hybridizes to the nucleic acid template and Mg ++ .  
     
     
         57 . The apparatus of  claim 55 , wherein the nucleic acid template is a DNA template.  
     
     
         58 . The apparatus of  claim 55 , the apparatus further comprising an electrokinetic direction system for electrophoretic movement of charged molecules in the microchannel.  
     
     
         59 . The apparatus of  claim 58 , wherein the electrokinetic direction system is used for joule heating of the PCR reaction mixture.  
     
     
         60 . The apparatus of  claim 51 , wherein the sieving matrix is selected from the group consisting of agarose, linear polyacrylamide, methylcellulose, polyethylene oxide and hydroxy ethyl cellulose.  
     
     
         61 . A method of performing PCR comprising: 
 mixing components of a PCR reaction mixture with a sieving matrix to    provide a PCR sieving matrix mixture; and,    thermocycling the resulting PCR sieving matrix mixture to produce a PCR product.    
     
     
         62 . The method of  claim 61 , wherein the components of the PCR reaction mixture are mixed with the sieving matrix in a microfluidic channel.  
     
     
         63 . The method of  claim 62 , further comprising joule thermocycling of the PCR reaction mixture in the channel to produce the PCR product.  
     
     
         64 . The method of  claim 62 , further comprising electrophoresing the PCR product in the microfluidic channel.  
     
     
         65 . The method of  claim 62 , wherein the PCR product is electrokinetically transported into a second microfluidic channel which intersects the first microfluidic channel.  
     
     
         66 . The method of  claim 61 , wherein the sieving matrix is selected from the group consisting of agarose, linear polyacrylamide, methylcellulose, polyethylene oxide and hydroxy ethyl cellulose.