Single cell whole genome amplification via micropillar arrays under flow conditions

Abstract

The present invention relates to, inter alia, a microfluidic device for performing single cell genomic DNA isolation and amplification under flow. The microfluidic device comprises a solid substrate having one or more microfluidic channel system formed therein. Each microfluidic channel system of the microfluidic device comprises: (a) an intake region comprising a single microchannel; (b) a plurality of cell segregation microchannels; (c) a cell capture site located downstream of each cell segregation microchannel; and (d) a DNA capture array positioned downstream of the cell capture site and comprising a plurality of micropillars. Also disclosed is a whole genome amplification system that includes the microfluidic device of the present disclosure, as well as a method for conducting single cell DNA analysis via on-chip whole genome amplification while under flow, and a method for multiple displacement amplification (MDA) reactions of one or more nucleic acid sequence isolated single cells.

1 . A microfluidic device for performing single cell genomic DNA isolation and amplification under flow, said microfluidic device comprising a solid substrate having one or more microfluidic channel system formed therein, wherein each microfluidic channel system comprises:
 (a) an intake region comprising a single microchannel configured for receiving a plurality of cells and transporting them downstream to a cell capture staging region;   (b) a plurality of cell segregation microchannels extending downstream from the cell capture staging region and configured for moving the cells further downstream;   (c) a cell capture site located downstream of each cell segregation microchannel and comprising a structural barrier effective for physically capturing a single cell and arresting any further movement of the single cell through the microfluidic channel system; and   (d) a DNA capture array positioned downstream of the cell capture site and comprising a plurality of micropillars configured and arranged in a manner effective for physically entangling and immobilizing thereon genomic DNA isolated from the captured single cell for use as DNA templates for one or more rounds of amplification of the isolated genomic DNA, said DNA capture array terminating in a collection region for collecting DNA amplification products of the isolated genomic DNA.   
     
     
         2 . The microfluidic device according to  claim 1  further comprising:
 an input port comprising an opening extending into the solid substrate and being in fluidic connection to the intake region of the microfluidic channel system, said input port being configured for introducing cells into the microfluidic channel system. 
 
     
     
         3 . The microfluidic device according to  claim 1  further comprising:
 an output reservoir comprising an opening extending out of the solid substrate and being in fluidic connection to the collection region of the DNA capture array, said output reservoir being configured for collecting DNA amplification products from the microfluidic channel system. 
 
     
     
         4 . The microfluidic device according to  claim 3  further comprising:
 a bypass channel region comprising one or more bypass microchannel extending downstream from the cell capture staging region and connecting directly to the output reservoir, said bypass microchannel being configured to transport and expel non-arrested cells and other debris from the microfluidic device without passing through the DNA capture array. 
 
     
     
         5 . The microfluidic device according to  claim 4 , wherein the bypass channel region comprises a first bypass microchannel running alongside a first side of the DNA capture array and a second bypass microchannel running alongside a second and opposite side of the DNA capture array. 
     
     
         6 . The microfluidic device according to  claim 1 , wherein the DNA capture array further comprises a physical border comprising side walls surrounding the plurality of micropillars so as to prevent any non-captured cells from becoming lodged in the micropillars once a single cell is arrested at the cell capture site. 
     
     
         7 . The microfluidic device according to  claim 1 , wherein the solid substrate comprises between 1 and 10 separate microfluidic channel systems formed therein. 
     
     
         8 . The microfluidic device according to  claim 1 , wherein each microfluidic channel system includes 2 or more cell segregation microchannels extending downstream from each cell capture staging region. 
     
     
         9 . The microfluidic device according to  claim 1 , wherein the single microchannel of the intake region has a width of between about 10 μm and about 2500 μm, a height of between about 0.1 μm and about 1000 μm, and a length of between about 50 μm and about 30 cm. 
     
     
         10 . The microfluidic device according to  claim 1 , wherein the single microchannel of the intake region has a width of between about 50 μm and about 250 μm, a height of between about 8 μm and about 20 μm, and a length of between about 1 cm and about 5 cm. 
     
     
         11 . The microfluidic device according to  claim 1 , wherein the micropillars have a diameter ranging from between about 0.5 μm and about 15 μm. 
     
     
         12 . The microfluidic device according to  claim 1 , wherein the micropillars have a diameter ranging from between about 1.5 μm and about 2 μm. 
     
     
         13 . The microfluidic device according to  claim 1 , wherein the micropillars are arranged in a gradient so that the spacing between the micropillars narrows in a downstream manner. 
     
     
         14 . The microfluidic device according to  claim 13 , wherein said gradient of micropillars comprises 1-3 distinct regions located downstream of one another, each distinct region having its own uniform spacing of micropillars. 
     
     
         15 . The microfluidic device according to  claim 1 , wherein the solid substrate is made from a material selected from the group consisting of polydimethylsiloxane (PDMS), glass, metals, and plastics. 
     
     
         16 . The microfluidic device according to  claim 1 , wherein the micropillars are made from a material selected from the group consisting of polydimethylsiloxane (PDMS), glass, and plastics. 
     
     
         17 - 22 . (canceled)