In-situ generated microfluidic assay structures, related kits, and methods of use thereof

Abstract

In situ-generated microfluidic capture structures incorporating a solidified polymer network, methods of preparation and use, compositions and kits therefor are described. Microfluidic capture structures may be advantageously used for assays performed within the microfluidic environment, providing flexibility in assaying micro-objects such as biological cells. Assay reagents and analytes may be incorporated within the microfluidic capture structures.

What is claimed: 
     
         1 . A method of assaying a micro-object in a microfluidic device, wherein the microfluidic device comprises:
 an enclosure comprising a substrate and microfluidic circuit material, the enclosure defining a flow region and a chamber fluidically connected to the flow region;   a first in situ-generated capture structure disposed at a first location within the chamber and comprising a first solidified polymer network, wherein the first solidified polymer network comprises a first assay reagent; and   a second in situ-generated capture structure disposed at a second location within the chamber and comprising a second solidified polymer network, wherein the second solidified polymer network comprises a second assay reagent;   wherein the first assay reagent is different from the second assay reagent, and further wherein the first location and the second location are physically distinguishable;   
       wherein the method comprises:
 disposing a micro-object within the microfluidic device in a region proximal to the first in situ-generated capture structure and the second in situ-generated capture structure; 
 contacting the first assay reagent and the second assay reagent with the micro-object or a biological product thereof; and 
 detecting a first interaction of the first assay reagent with the micro-object or the biological product thereof and/or a second interaction of the second assay reagent with the micro-object or the biological product thereof. 
 
     
     
         2 . The method of  claim 1 , wherein detecting the first interaction and/or detecting the second interaction comprises detecting a first signal from the first location and/or a second signal from the second location. 
     
     
         3 . The method of  claim 1 , wherein the chamber comprises a sequestration pen comprising an isolation region and a connection region, the connection region having a proximal opening to the flow region and a distal opening to the isolation region; and further wherein the first in situ-generated capture structure and the second in situ-generated capture structure are disposed within the sequestration pen. 
     
     
         4 . The method of  claim 1 ,
 wherein detecting the first interaction comprises detecting the first interaction between the first assay reagent with a first biological product; and wherein detecting the second interaction comprises detecting the second interaction between the second assay reagent with a second biological product; or   wherein detecting the first interaction comprises detecting the first interaction between the first assay reagent with the biological product; and wherein detecting the second interaction comprises detecting the second interaction between the second assay reagent with the micro-object.   
     
     
         5 . The method of  claim 1 ,
 wherein detecting the first interaction comprises detecting a first characteristic of the biological product; and   wherein detecting the second interaction comprises detecting a second characteristic of the biological product.   
     
     
         6 . The method of  claim 1 ,
 wherein detecting the first interaction comprises introducing a first detection reagent comprising a first detectable label to a region adjacent to the first in situ-generated capture structures; and   wherein detecting the second interaction comprises introducing a second detection reagent comprising a second detectable label to a region adjacent to the second in situ-generated capture structures.   
     
     
         7 . The method of  claim 6 , wherein the first detectable label and the second detectable label are independently a fluorescent label, a colorimetric label, or a luminescent label. 
     
     
         8 . The method of  claim 6 , wherein the first detectable label and the second detectable label are spectrally distinct. 
     
     
         9 . The method of  claim 1 , wherein the first in situ-generated capture structure and/or the second in situ-generated capture structure permits export of the micro-object from the microfluidic device. 
     
     
         10 . The method of  claim 1 , wherein the first assay reagent and/or the second assay reagent independently comprises a protein, a nucleic acid, an organic molecule, a saccharide, a combination thereof. 
     
     
         11 . The method of  claim 1 , wherein the first solidified polymer network and/or the second solidified polymer network comprises a synthetic polymer, a modified synthetic polymer, a biological polymer, or any combination thereof. 
     
     
         12 . The method of  claim 1 , wherein the first solidified polymer network and/or the second solidified polymer network comprises polyethylene glycol, modified polyethylene glycol, polylactic acid (PLA), modified polylactic acid, polyglycolic acid (PGA), modified polyglycolic acid, polyacrylamide (PAM), modified polyacrylamide, poly-N-isopropylacrylamide (PNIPAm), modified poly-N-isopropylacrylamide, polyvinyl alcohol (PVA), modified polyvinyl alcohol, polyacrylic acid (PAA), modified polyacrylic acid, polycaprolactone (PCL), modified polycaprolactone, fibronectin, modified fibronectin, collagen, modified collagen, gelatin, modified gelatin, laminin, modified laminin, polysaccharide, modified polysaccharide, or a co-polymer in any combination. 
     
     
         13 . The method of  claim 1 , wherein the first solidified polymer network and/or the second solidified polymer network comprises a modified polyethylene glycol polymer. 
     
     
         14 . The method of  claim 13 , wherein the modified polyethylene glycol polymer is a polyethylene glycol diacrylate (PEGDA). 
     
     
         15 . The method of  claim 1 , wherein the first in situ-generated capture structure and/or the second in situ-generated capture structure is porous to a flow of a fluidic medium. 
     
     
         16 . The method of  claim 1 , wherein the microfluidic device further comprises a substrate configured to generate dielectrophoresis (DEP) forces within the enclosure. 
     
     
         17 . A microfluidic device comprising:
 an enclosure comprising a substrate and microfluidic circuit material, the enclosure defining a flow region and a chamber fluidically connected to the flow region;   a first in situ-generated capture structure disposed at a first location within the chamber and comprising a first solidified polymer network, wherein the first solidified polymer network comprises a first functionalized site configured to bind a first assay reagent or assay analyte; and   a second in situ-generated capture structure disposed at a second location within the chamber and comprising a second solidified polymer network, wherein the second solidified polymer network comprises a second functionalized site configured to bind a second assay reagent or assay analyte;   wherein the first assay reagent or assay analyte is different from the second assay reagent or assay analyte, and further wherein the first location and the second location are physically distinguishable.   
     
     
         18 . The microfluidic device of  claim 17 , wherein the chamber comprises a sequestration pen comprising an isolation region and a connection region, the connection region having a proximal opening to the flow region and a distal opening to the isolation region, and further wherein the first in situ-generated capture structure and the second in situ-generated capture structure are disposed within the sequestration pen. 
     
     
         19 . The microfluidic device of  claim 17 , wherein the first in situ-generated capture structure and/or the second in situ-generated capture structure permits export of a micro-object from the microfluidic device. 
     
     
         20 . The microfluidic device of  claim 17 , wherein the first solidified polymer network comprises the first assay reagent or assay analyte, and wherein the second solidified polymer network comprises the second assay reagent or assay analyte. 
     
     
         21 . The microfluidic device  claim 17  wherein the first functionalized site comprises a first reactive moiety configured to bind the first assay analyte or the assay reagent; and/or the second functionalized site comprises a second reactive moiety configured to bind the second assay analyte or the assay reagent. 
     
     
         22 . The microfluidic device of  claim 21 , wherein the first reactive moiety and/or the second reactive moiety independently comprises an antibody, an antigen, a biotin, a streptavidin, an avidin, an alkynyl moiety, an azido moiety, a chelating moiety, an oligonucleotide hybridization sequence, a cell recognition motif, or a combination thereof. 
     
     
         23 . The microfluidic device of  claim 17 , wherein the first assay reagent or assay analyte and/or the second assay reagent or assay analyte independently comprises a protein, a nucleic acid, an organic molecule, a saccharide, a combination thereof. 
     
     
         24 . The microfluidic device of  claim 17 , wherein the first solidified polymer network and/or the second solidified polymer network comprises a synthetic polymer, a modified synthetic polymer, a biological polymer, or any combination thereof. 
     
     
         25 . The microfluidic device of  claim 17 , wherein the first solidified polymer network and/or the second solidified polymer network comprises polyethylene glycol, modified polyethylene glycol, polylactic acid (PLA), modified polylactic acid, polyglycolic acid (PGA), modified polyglycolic acid, polyacrylamide (PAM), modified polyacrylamide, poly-N-isopropylacrylamide (PNIPAm), modified poly-N-isopropylacrylamide, polyvinyl alcohol (PVA), modified polyvinyl alcohol, polyacrylic acid (PAA), modified polyacrylic acid, polycaprolactone (PCL), modified polycaprolactone, fibronectin, modified fibronectin, collagen, modified collagen, gelatin, modified gelatin, laminin, modified laminin, polysaccharide, modified polysaccharide, or a co-polymer in any combination. 
     
     
         26 . The microfluidic device of  claim 17 , wherein the first solidified polymer network and/or the second solidified polymer network comprises a modified polyethylene glycol polymer. 
     
     
         27 . The microfluidic device of  claim 26 , wherein the modified polyethylene glycol polymer is a polyethylene glycol diacrylate (PEGDA). 
     
     
         28 . The microfluidic device of  claim 17 , wherein the first in situ-generated capture structure and/or the second in situ-generated capture structure is porous to a flow of a fluidic medium. 
     
     
         29 . The microfluidic device of  claim 17 , wherein the microfluidic device further comprises a substrate configured to generate dielectrophoresis (DEP) forces within the enclosure. 
     
     
         30 . A method of preparing a microfluidic device comprising a first in situ-generated capture structure and a second in situ-generated capture structure, the method comprising:
 providing the microfluidic device, wherein the microfluidic device comprises an enclosure comprising a substrate and microfluidic circuit material, the enclosure defining a flow region and a chamber fluidically connected to the flow region;   introducing a first flowable functionalized pre-polymer into the flow region;   activating solidification of the first flowable functionalized pre-polymer at a first location of the chamber, thereby forming the first in situ-generated capture structure therein; wherein the first in-situ generated capture structure comprises a first solidified polymer network comprising one or more functionalized sites;   introducing a second flowable functionalized pre-polymer into the flow region; and   activating solidification of the second flowable functionalized pre-polymer at a second location of the chamber, thereby forming the second in situ-generated capture structure therein; wherein the second in-situ generated capture structure comprises a second solidified polymer network comprising one or more functionalized sites;   wherein the first location and the second location are physically distinguishable.   
     
     
         31 . The method of  claim 30 , wherein
 the one or more functionalized sites of the first solidified polymer network comprises a first reactive moiety configured to react with a first assay analyte or assay reagent via a non-covalent binding, a covalent binding, or an association; and/or   the one or more functionalized sites of the second solidified polymer network comprises a second reactive moiety configured to react with a second assay analyte or assay reagent via a non-covalent binding, a covalent binding, or an association.   
     
     
         32 . The method of  claim 31 , wherein the first reactive moiety and/or the second reactive moiety comprises an antibody, an antigen, a biotin, a streptavidin, an avidin, an alkynyl moiety, an azido moiety, a chelating moiety, an oligonucleotide hybridization sequence, a cell recognition motif, or a combination thereof.