Systems and methods for on-chip analysis of nucleic acids and for multiplexed analysis of cells

Patent metadata

Field	Value
Publication number	US-2022016629-A1
Application number	US-201917298507-A
Country	US
Kind code	A1
Filing date	Nov 29, 2019
Priority date	Nov 28, 2018
Publication date	Jan 20, 2022
Grant date	—

Abstract

A microfluidic chip for on-chip detection of the presence or absence of a target nucleic acid region in an isolated nucleic acid sample is disclosed. The microfluidic chip includes a nucleic acid entanglement array, an isolated nucleic acid sample immobilized in the nucleic acid entanglement array, and at least one probe specific to a target nucleic acid region. Systems and methods of using the microfluidic chip are disclosed. An integrated microfluidic cell processing system is disclosed, which includes: a multiplexed microfluidic flow directing system having a plurality of reconfigurable microfluidic layers that form a plurality of reconfigurable microfluidic channels, where the multiplexed microfluidic flow directing system function to assist in directing flow of materials into, through, and out of the integrated cell processing system; and at least one microfluidic chip functionally integrated into at least one layer of the multiplexed microfluidic flow directing system, and operates under continuous flow conditions.

First claim

What is claimed is:

1 . A microfluidic chip for on-chip detection of a presence or absence of a target nucleic acid region in an isolated nucleic acid sample, said microfluidic chip comprising:
(a) a nucleic acid entanglement array comprising a plurality of nucleic acid entanglement micropillars configured and arranged in a manner effective to physically entangle and maintain thereon an isolated nucleic acid sample; (b) the isolated nucleic acid sample immobilized in the nucleic acid entanglement array; and (c) at least one probe specific to a target nucleic acid region, said at least one probe being specifically bound to the target nucleic acid region and detectable when said isolated nucleic acid sample includes the target nucleic acid region, thereby enabling on-chip analysis of the presence or absence of the target nucleic acid region in the isolated nucleic acid sample.

2 . The microfluidic chip according to claim 1 , wherein the isolated nucleic acid sample comprises genomic DNA (gDNA), extrachromosomal DNA, chromatin, plasmid DNA, a nucleic acid aptamer, an oligonucleotide, and a nucleic acid biomarker.

3 . The microfluidic chip according to claim 1 , wherein the isolated nucleic acid sample comprises single-stranded DNA, double-stranded DNA, or a combination of both single- and double-stranded DNA.

4 . The microfluidic chip according to claim 1 , wherein the target nucleic acid region comprises a gene, mutation, or nucleotide sequence from a eukaryotic cell.

5 . The microfluidic chip according to claim 1 , wherein the target nucleic acid region comprises a gene, mutation, or nucleotide sequence from an animal.

6 . The microfluidic chip according to claim 5 , wherein the target nucleic acid region comprises a gene, mutation, or nucleotide sequence from a human or non-human animal.

7 . The microfluidic chip according to claim 1 , wherein the target nucleic acid region comprises a gene, mutation, or nucleotide sequence that is related to or that is a marker for presence or risk of a disease or abnormal condition of a multicellular organism.

8 . The microfluidic chip according to claim 7 , wherein the disease or abnormal condition is a human disease selected from the group consisting of cancer, Down's syndrome, Huntington's disease, heart disease, thalassemia, cystic fibrosis, tay-sachs disease, sickle cell anemia, marfan syndrome, fragile X syndrome, hemochromatosis, and the like, or a canine disease selected from the group consisting of hip dysplasia, urinary bladder stones, epilepsy, heart disease, degenerative myelopathy, brachycephalic syndrome, and the like.

9 . The microfluidic chip according to claim 8 , wherein the disease is cancer and the mutation is a therapeutically-actionable (TA) mutation.

10 . The microfluidic chip according to claim 1 , wherein the isolated nucleic acid sample is isolated from one or more cell.

11 . The microfluidic chip according to claim 1 , wherein the target nucleic acid region is specific for a pathogen, antibiotic resistant strain of bacteria, food contaminant, foodborne illness, paternity determination, DNA fingerprinting, or individual identity.

12 . The microfluidic chip according to claim 1 , wherein the at least one probe comprises a collection of multiple probes with each probe being specific to a unique target nucleic acid region or to another probe which is specific to a unique target nucleic acid region.

13 . The microfluidic chip according to claim 12 , wherein the collection of multiple probes comprises a panel of probes effective to produce a genetic profile for a disease or abnormal condition in a multicellular organism.

14 . The microfluidic chip according to claim 1 , wherein the probe is a fluorescence probe.

15 . The microfluidic chip according to claim 1 , wherein the probe is selected from the group consisting of molecular beacons, peptide nucleic acid probes, aptamers, and protein probes.

16 . A microfluidic system for on-chip detection of a presence or absence of a target nucleic acid region in an isolated nucleic acid sample, said system comprising:
(a) an input controller component for introducing fluids, cells, reagents, and probes into the system under flow conditions; (b) a collection reservoir for collecting materials exported out of the system; (c) at least one microfluidic chip according to any one of claims 1 - 15 , wherein said at least one microfluidic chip is detachably connected to the fluid controller by a fluidic input port and detachably connected to the collection reservoir by an output port; and (d) a detection component effective for on-chip detection of the presence or absence of the target nucleic acid region in the isolated nucleic acid sample immobilized on the at least one microfluidic chip.

17 . The microfluidic system according to claim 16 further comprising:
a multiplexed microfluidic flow directing system comprising a plurality of reconfigurable microfluidic layers that form a plurality of reconfigurable microfluidic channels, wherein said multiplexed microfluidic flow directing system functions to assist in directing flow of materials into the microfluidic system, through the microfluidic system, and out of the microfluidic system.

18 . The microfluidic system according to claim 17 , wherein the at least one microfluidic chip is functionally integrated with the multiplexed microfluidic flow directing system.

19 . The microfluidic system according to claim 18 , wherein the at least one microfluidic chip is integrated with the multiplexed microfluidic flow directing system in a manner so that flow of materials into the microfluidic system is directed into and through the at least one microfluidic chip before being exported from the microfluidic system.

20 . The microfluidic system according to claim 17 , wherein the multiplexed microfluidic flow directing system is configured to receive and export one or more sample in manual and/or automated fashion.

21 . The microfluidic system according to claim 20 , wherein the multiplexed microfluidic flow directing system is configured to receive the one or more sample via one or more pipette.

22 . The microfluidic system according to claim 17 , wherein the multiplexed microfluidic flow directing system is configured to assist in sorting materials for export from the microfluidic system for further collection, purification, and/or analysis.

23 . A method for on-chip detection of a presence or absence of a target nucleic acid region in an isolated nucleic acid sample, said method comprising the steps of:
providing a microfluidic system according to claim 16 ; and operating the microfluidic system in a manner sufficient to conduct on-chip detection of the presence or absence of a target nucleic acid region in an isolated nucleic acid sample.

24 . The method according to claim 23 , wherein the step of operating the microfluidic system comprises the steps of:
introducing at least one cell of interest into the microfluidic system using the input controller component under conditions sufficient to (i) capture the at least one cell of interest; (ii) extract gDNA from the captured at least one cell of interest; and (iii) isolate and immobilize the gDNA in the nucleic acid entanglement micropillars of the microfluidic chip; providing at least one probe specific to the target nucleic acid region and introducing the at least one probe into the microfluidic system

Assignees

• Univ Cornell

Inventors

• Craighead Harold G
• Tian Harvey C
• Lin David M
• Bisogni Adam J

Classifications

• B01D15/1885

  placed in parallel · CPC title

• B01L2300/0636

  Integrated biosensor, microarrays · CPC title

• B01D15/24

  relating to the treatment of the fractions to be distributed · CPC title

• B01L3/5025

  for parallel transport of multiple samples · CPC title

• B01D15/3804

  Affinity chromatography · CPC title