Multifunctional microfluidic device for capturing target cells and analyzing genomic DNA isolated from the target cells while under flow conditions
The present invention relates to, inter alia, a microfluidic device for capturing target cells and analyzing genomic DNA isolated from the target cells while under flow conditions. The microfluidic device includes a cell microchannel and a nucleic acid microchannel that intersect in an orthogonal manner, thereby forming a cell capture intersection region. The microfluidic device also includes a cell capture array and a nucleic acid entanglement array. The cell capture array includes a plurality of cell capturing micropillars and is located in the cell capture intersection region. The nucleic acid entanglement array includes a plurality of nucleic acid entanglement micropillars that function to physically entangle and maintain thereon genomic DNA isolated from the one or more target cell, and is located in a portion of the nucleic acid microchannel that is adjacent to and downstream of the cell capture intersection region. Methods of using the microfluidic device are also disclosed.
1 . A method of amplifying nucleic acids, the method comprising:
flowing a sample through a cell channel within a microfluidic device to a cell capture array disposed at an intersection of the cell channel and a nucleic acid channel within the microfluidic device to immobilize a cell from the sample on a plurality of structures of the cell capture array while flowing a remainder of the sample downstream of the cell capture array in the cell channel;
lysing the cell to release nucleic acids from the cell and flow the nucleic acids from the cell capture array to an entanglement array within the nucleic acid channel to capture the nucleic acids within the microfluidic device on the entanglement array, wherein the entanglement array comprises a plurality of nucleic acid entanglement micropillars configured and arranged in a manner effective to physically entangle and maintain thereon genomic DNA isolated from the cell;
flowing amplification reagents into the nucleic acid channel; and
copying the captured nucleic acids within the microfluidic device while the nucleic acids are captured and maintained on the plurality of nucleic acid entanglement micropillars under flow at the entanglement array using the amplification reagents.
2 . The method of claim 1 , wherein the nucleic acids include genomic DNA (gDNA), the method further comprising eluting the gDNA from the nucleic acid channel.
3 . The method of claim 1 , wherein the cell is a target cell, and the remainder of the sample includes a mixture of non-target cells.
4 . The method of claim 3 , further comprising immobilizing the target cell with a capture ligand specific to a cell type of the target cell.
5 . The method of claim 1 , wherein the amplification reagents comprise one or more primers and a polymerase.
6 . The method of claim 1 , further comprising extracting amplification products from an outlet of the nucleic acid channel.
7 . The device of claim 1 , wherein the plurality of structures are functionalized with antibodies or aptamers.
8 . The method of claim 5 , wherein copying the nucleic acids at the entanglement array comprises a multiple displacement amplification (MDA) process.
9 . The method of claim 8 , wherein the polymerase is φ29 and the one or more primers are selected from one or more of SEQ ID NOS: 4-123.
10 . The method of claim 1 , wherein the cell capture array and the entanglement array are separated, and flowing the nucleic acids from the cell capture array to the entanglement array comprises moving the nucleic acids from the cell capture array to the entanglement array.
11 . The method of claim 1 , wherein the cell capture array has attached thereto cell-capture ligands that bind to cells of a specific cell type.
12 . The method of claim 11 , wherein the cells of the specific cell type are cancer cells.
13 . The method of claim 12 , wherein the cell-capture ligands comprise aptamers.
14 . The method of claim 13 , wherein the nucleic acids include genomic DNA (gDNA), the method further comprising collecting amplification products from an outlet of the nucleic acid channel.
15 . The method of claim 14 , wherein the gDNA remains entangled at the entanglement array during flow of the amplification reagents through the nucleic acid channel.
16 . The method of claim 11 , wherein the copying step includes amplifying one or more individual genes of interest while the gDNA is maintained under flow conditions.
17 . The method of claim 16 , further comprising amplifying the individual genes of interest consecutively.
18 . The method of claim 17 , further comprising collecting amplification products separately.