# US 2014/0194313 A1 · Microfluidic device for extracting, isolating, and analyzing dna from cells > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2014/0194313 A1 - Document ID: US20140194313A1 - Status: Published application - Kind: A1 - Application number: 14/124,479 - Filed: 2012-06-06 - Published: 2014-07-10 - Earliest claimed: 2011-06-06 - Assignees: Cornell University - Inventors: Harold G. Craighead; Juraj Topolancik; Harvey Tian; Christopher Wallin - Web page: https://patentgrep.com/patent/US20140194313A1 ## CPC - C12N 15/1017: {by filtration, e.g. using filters, frits, membranes} - B01L 3/502761: {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} - C12M 47/10: {Separation or concentration of fermentation products (bioreactors combined with means for distillation or extraction of liquid fuel C12M43/02)} - C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) - B01L 2200/0663: Stretching or orienting elongated molecules or particles - B01L 2400/0415: electrical forces, e.g. electrokinetic - B01L 2400/0487: fluid pressure, pneumatics - B01L 2400/086: using baffles or other fixed flow obstructions - Y10T 29/49826: Assembling or joining - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE - C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS} - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION ## 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. ## Claims ### Claim 1 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. ### Claim 2 (depends on 1) The device according to claim 1, 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. ### Claim 3 (depends on 1) The device 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 channel and the outflow channel. ### Claim 4 (depends on 3) The device according to claim 3, wherein the chamber has a length sufficient to contain elongated chromosomal DNA released from the at least one cell. ### Claim 5 (depends on 3) The device according to claim 3, wherein the chamber further comprises: at least one linear array of micropillars substantially spanning the width of the chamber, thereby providing support for an immobilized and elongated DNA suspended within the chamber. ### Claim 6 (depends on 1) The device according to claim 1, wherein the inflow channel, the outflow channel, or both the inflow channel and the outflow channel have at least one micropillar disposed therein. ### Claim 7 (depends on 1) The device according to claim 1, wherein the micropillars are spaced in a random manner, a uniform manner, and/or in a desired spacing pattern. ### Claim 8 (depends on 1) The device according to claim 1, wherein the support comprises polydimethylsiloxane (PDMS), polystyrene, epoxy, polymethylmethacrylate (PMMA), and silica. ### Claim 9 (depends on 1) The device according to claim 1, wherein the micropillar array comprises micropillars that are coated with at least one binding agent that has affinity to at least a portion of the surface of the at least one cell. ### Claim 10 (depends on 9) The device according to claim 9, wherein said at least one binding agent is selected from the group consisting of an antibody and an aptamer. ### Claim 11 (depends on 1) The device according to claim 1 further comprising: a hydrodynamic flow controller in functional communication with the outlet port and effective to generate hydrodynamic flow of a fluid from the inlet port to the outlet port. ### Claim 12 (depends on 1) A method of extracting and isolating DNA from at least one cell, said method comprising the steps of: providing a microfluidic device according to claim 1; and 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. ### Claim 13 (depends on 12) The method according to claim 12, wherein the flowing step comprises applying sufficient hydrodynamic flow to elongate the immobilized DNA within the micropillar array. ### Claim 14 (depends on 12) The method according to claim 12, wherein the flowing step comprises: 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 lysing the entrapped cell under sufficient hydrodynamic flow to release DNA contained in the cell, thereby causing the released DNA to be immobilized within the micropillar array of the microfluidic device. ### Claim 15 (depends on 12) The method according to claim 12 further comprising: analyzing the immobilized DNA while the DNA is maintained within the micropillar array. ### Claim 16 (depends on 15) The method according to claim 15, wherein the analyzing step comprises fluorescence microscopy, optical microscopy, and hybridization assays. ### Claim 17 (depends on 12) The method according to claim 12 further comprising: removing the immobilized DNA from the micropillar array to yield isolated DNA. ### Claim 18 (depends on 17) The method according to claim 17, wherein the removing step comprises subjecting the immobilized DNA to hydrodynamic force sufficient to pass the DNA through the micropillar array and into the outflow channel. ### Claim 19 (depends on 17) The method according to claim 17, wherein the removing step 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. ### Claim 20 (depends on 17) The method according to claim 17, wherein the removing step 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. ### Claim 21 (depends on 17) The method according to claim 17 further comprising: analyzing the isolated DNA. ### Claim 22 (depends on 21) The method according to claim 21, wherein the analyzing step comprises conducting techniques selected from the group consisting of nucleic acid amplification, absorbance, fluorospectrometry, spectrophotometry, gel electrophoresis, nanofluidics, protein assays, microarrays, qPCR, fluorescence microscopy, optical microscopy, and hybridization assays. ### Claim 23 (depends on 17) The method according to claim 17 further comprising: prior to said removing step, purifying the immobilized DNA to detach proteins or other biomaterials bound or in contact with the immobilized DNA or released from the cell. ### Claim 24 (depends on 23) The method according to claim 23, wherein the purifying step comprising contacting a proteinase buffer to the immobilized DNA. ### Claim 25 (depends on 12) The method according to claim 12, wherein the at least one cell is selected from the group consisting of a stem cell, bacterial cell, cancer cell, leucocyte, plant cell, fungal cell, and the like. ### Claim 26 (depends on 12) The method according to claim 12, wherein the sample comprises a population of cells. ### Claim 27 (depends on 12) The method according to claim 12, wherein the sample comprises a single cell. ### Claim 28 (depends on 12) The method according to claim 12, wherein the DNA comprises genomic DNA. ### Claim 29 (depends on 1) A microfluidic system for extracting, isolating, and analyzing DNA from at least one cell, said system comprising: a microfluidic device according to claim 1; and an apparatus for analyzing DNA immobilized on the micropillar array of the microfluidic device. ### Claim 30 (canceled) ### Claim 31 (canceled) ### Claim 32 (depends on 1) A microfluidic-nanofluidic system for extracting, isolating, and analyzing DNA from at least one cell, said system comprising: a microfluidic device according to claim 1; 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 an apparatus for analyzing single DNA molecules released from the at least one cell. ### Claim at position 33 33-35. (canceled) ### Claim 36 (depends on 32) A method of extracting, isolating, and analyzing a single DNA molecule from at least one cell, said method comprising the steps of: providing a microfluidic-nanofluidic system according to claim 32; 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; passing the DNA through the nanofluidic device to isolate a single DNA molecule from the released and de-immobilized DNA; and using the apparatus to analyze the isolated single DNA molecule. ### Claim at position 35 37-52. (canceled) ### Claim 53 A method of making a microfluidic device for extracting and isolating DNA from at least one cell, said method comprising the steps of: providing a support; and forming within said support 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. ### Claim at position 37 54-65. (canceled)