Microfluidic device for extracting, isolating, and analyzing DNA from cells

Patent metadata

Field	Value
Publication number	US-9926552-B2
Application number	US-201214124479-A
Country	US
Kind code	B2
Filing date	Jun 6, 2012
Priority date	Jun 6, 2011
Publication date	Mar 27, 2018
Grant date	Mar 27, 2018

Abstract

The present invention relates to a microfluidic device for extracting and isolating DNA from cells. The device includes a support having an inlet port for receiving a sample containing a cell, an outlet port for dispensing DNA isolated from the cell, and a microfluidic channel disposed within the support and extending from the inlet port to the outlet port. The microfluidic channel includes a micropillar array, an inflow channel disposed between the inlet port and the micropillar array, and an outflow channel disposed between the micropillar array and the outlet port. The micropillar array includes micropillars spatially configured to entrap, by size exclusion, the cell, to immobilize DNA released from the cell, and to maintain the immobilized DNA in elongated or non-elongated form when hydrodynamic force is applied to the microfluidic channel. Systems and methods of making and using the device are also provided herein.

First claim

What is claimed is:

1. A method of extracting and isolating DNA from at least one cell, said method comprising the steps of:
(a) providing a microfluidic device for extracting and isolating DNA from at least one cell, said device comprising a support having:
(i) an inlet port for receiving a sample containing at least one cell;
(ii) an outlet port for dispensing DNA isolated from the at least one cell; and
(iii) a microfluidic channel disposed within the support and extending from the inlet port to the outlet port,

wherein said microfluidic channel comprises a micropillar array, an inflow channel disposed between the inlet port and the micropillar array, and an outflow channel disposed between the micropillar array and the outlet port,
wherein said micropillar array comprises micropillars spatially configured to entrap, by size exclusion, the at least one cell, to immobilize DNA released from the at least one cell, and to maintain the immobilized DNA in elongated or non-elongated form when hydrodynamic force is applied to the microfluidic channel, and
wherein said micropillar array comprises a first region having micropillars effectively spaced to entrap the at least one cell and a second region having micropillars effectively spaced to immobilize the released DNA and maintain the immobilized DNA in elongated or non-elongated form, wherein the micropillars of the second region are more closely spaced than the micropillars of the first region; and
(b) flowing a sample comprising at least one cell through the microfluidic channel of the microfluidic device, thereby causing the DNA to be released from the at least one cell and immobilized within the micropillar array of the microfluidic device, wherein said flowing comprises:
(i) introducing the sample into the inlet port of the device under sufficient hydrodynamic flow to entrap, by size exclusion, the at least one cell within the micropillar array; and
(ii) lysing the entrapped cell using chemical lysis with an ionic surfactant under sufficient hydrodynamic flow to release DNA contained in the cell without causing DNA shearing, thereby causing the released DNA to be immobilized within the micropillar array of the microfluidic device.

2. The method according to claim 1 , wherein said flowing comprises applying sufficient hydrodynamic flow to elongate the immobilized DNA within the micropillar array without shearing.

3. The method according to claim 1 further comprising:
analyzing the immobilized DNA while the DNA is maintained within the micropillar array.

4. The method according to claim 3 , wherein said analyzing the immobilized DNA comprises involves using techniques selected from the group consisting of fluorescence microscopy, optical microscopy, and hybridization assays.

5. The method according to claim 1 further comprising:
removing the immobilized DNA from the micropillar array to yield isolated DNA.

6. The method according to claim 5 , wherein said removing comprises subjecting the immobilized DNA to hydrodynamic force sufficient to pass the DNA through the micropillar array and into the outflow channel.

7. The method according to claim 5 , wherein said removing comprises subjecting the immobilized DNA to enzymatic digestion sufficient to fragment the DNA so that it passes through the micropillar array and into the outflow channel.

8. The method according to claim 5 , wherein said removing comprises sonicating the microfluidic device and subjecting the immobilized DNA to hydrodynamic force sufficient to pass the DNA through the micropillar array and into the outflow channel.

9. The method according to claim 5 further comprising:
analyzing the isolated DNA.

10. The method according to claim 9 , wherein said analyzing the isolated DNA comprises conducting techniques selected from the group consisting of nucleic acid amplification, absorbance, fluorospectrometry, spectrophotometry, gel electrophoresis, nanofluidics assays, protein expression assays, microarray assays, qPCR, fluorescence microscopy, optical microscopy, and hybridization assays.

11. The method according to claim 5 further comprising:
prior to said removing, purifying the immobilized DNA to detach proteins or other biomaterials bound or in contact with the immobilized DNA or released from the cell.

12. The method according to claim 11 , wherein said purifying comprises contacting a proteinase buffer to the immobilized DNA.

13. The method according to claim 1 , wherein the at least one cell is selected from the group consisting of a stem cell, bacterial cell, cancer cell, leucocyte, plant cell, and fungal cell.

14. The method according to claim 1 , wherein the sample comprises a population of cells.

15. The method according to claim 1 , wherein the sample is a single cell.

16. The method according to claim 1 , wherein the DNA comprises genomic DNA.

17. A method of extracting, isolating, and analyzing a single DNA molecule from at least one cell, said method comprising the steps of:
(a) providing a microfluidic-nanofluidic system for extracting, isolating, and analyzing DNA from at least one cell, said system comprising:
(i) a microfluidic device for extracting and isolating DNA from at least one cell, said device comprising a support having an inlet port for receiving a sample containing at least one cell, an outlet port for dispensing DNA isolated from the at least one cell, and a microfluidic channel disposed within the support and extending from the inlet port to the outlet port, wherein said microfluidic channel comprises a micropillar array, an inflow channel disposed between the inlet port and the micropillar array, and an outflow channel disposed between the micropillar array and the outlet port, and wherein said micropillar array comprises micropillars spatially configured to entrap, by size exclusion, the at least one cell, to immobilize DNA released from the at least one cell, and to maintain the immobilized DNA in elongated or non-elongated form when hydrodynamic force is applied to the microfluidic channel;
(ii) a nanofluidic device in fluid communication with the outlet port of the microfluidic device so as to receive DNA released from the at least one cell, wherein said nanofluidic device functions to isolate a single DNA molecule from the released DNA; and
(iii) an apparatus for analyzing single DNA molecules released from the at least one cell;

(b) flowing a sample comprising at least one cell through the microfluidic channel of the microfluidic device, thereby causing the DNA to be released from the at least one cell and temporarily immobilized within the micropillar array of the microfluidic device, wherein said flowing comprises:
(i) introducing the sample into the inlet port of the microfluidic device under sufficient hydrodynamic flow to entrap, by size exclusion, the at least one cell within the micropillar array; and
(ii) lysing the entrapped cell using chemical lysis with an ionic surfactant under sufficient hydrodynamic flow to release DNA contained in the cell without causing DNA shearing, thereby causing the released DNA to be immobilized within the micropillar array of the microfluidic device;

(c) passing the DNA through the nanofluidic device to isolate a single DNA molecule from the released and de-immobilized DNA; and
(d) using the apparatus to analyze the isolated single DNA molecule.

18. The method according to claim 1 , wherein the micropillar array forms a chamber having a maximum width that is greater than, less than, or equivalent to the maximum width of each of the inflow

Assignees

• Craighead Harold G
• Topolancik Juraj
• Tian Harvey
• Wallin Christopher
• Univ Cornell

Inventors

• Craighead Harold G
• Topolancik Juraj
• Tian Harvey
• Wallin Christopher

Classifications

• Y10T29/49826

  Assembling or joining · CPC title

• B01L3/502761

  specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads or physically stretching molecules · CPC title

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12N15/1017 Primary

  by filtration, e.g. using filters, frits, membranes · CPC title

• C12M47/10 Primary

  Separation or concentration of fermentation products (bioreactors combined with means for distillation or extraction of liquid fuel C12M43/02) · CPC title