Multiomic analysis of cell analytes using microfluidic systems

Patent metadata

Field	Value
Publication number	US-12338487-B2
Application number	US-201917298533-A
Country	US
Kind code	B2
Filing date	Nov 29, 2019
Priority date	Nov 28, 2018
Publication date	Jun 24, 2025
Grant date	Jun 24, 2025

Abstract

A method for isolating one or more distinct analyte component from a cell sample is disclosed, as well as processes for testing and analyzing the distinct analyte components. The distinct analyte components include: (i) a total protein fraction; (ii) a plasma membrane protein fraction; (iii) a total RNA fraction; (iv) a cytosolic RNA fraction; (v) a cytosolic protein fraction; (vi) a nuclear RNA fraction; (vii) a nuclear protein fraction; (viii) a chromatin fraction comprising genomic DNA (gDNA); (ix) a gDNA markers fraction. This method involves the use of microfluidic device having a cell capture component and a nucleic acid entanglement component, where the cell capture component includes a cell capture array having a plurality of cell capture micropillars, where the nucleic acid entanglement component includes a nucleic acid entanglement array having a plurality of nucleic acid entanglement micropillars, and where the microfluidic device operates under continuous flow conditions.

First claim

What is claimed is:

1. A method for isolating one or more distinct analyte component from a cell sample, said method comprising the acts of:
introducing, under continuous flow conditions, a cell sample comprising at least one cell into a microfluidic device comprising an array of cell capture micropillars and a plurality of nucleic acid entanglement micropillars
capturing the at least one cell in the array of cell capture micropillars while the microfluidic device is subjected to a continuous flow rate;
treating the at least one captured cell with a sequential workflow procedure under conditions effective to separate at least one analyte therefrom, wherein said analyte is selected from the group consisting of: (i) a total protein fraction; (ii) a plasma membrane protein fraction; (iii) a total RNA fraction; (iv) a cytosolic RNA fraction; (v) a cytosolic protein fraction; (vi) a nuclear RNA fraction; (vii) a nuclear protein fraction; (viii) a chromatin fraction comprising genomic DNA (gDNA) regions of open chromatin; (ix) a gDNA markers fraction comprising epigenetic and regulatory markers of gDNA; (x) an amplified gDNA fraction; and (xi) a methylated gDNA fraction, wherein said gDNA is single-stranded gDNA, double-stranded gDNA, or a combination of single-and double-stranded gDNA; and
isolating the analyte in a manner suitable for further testing and/or analysis thereof.

2. The method according to claim 1 , wherein the sequential workflow procedure comprises implementing a separation protocol to separate a plurality of distinct classes of analyte component from the cell sample, and wherein the sequential workflow procedure requires that only one distinct analyte component can be isolated per class.

3. The method according to claim 2 , wherein the separation protocol is for separating the total protein fraction from the cell sample and comprises flowing a total protein treatment solution through the microfluidic device under continuous flow conditions effective to release the total protein fraction from the one or more captured cell, thereby causing the total protein fraction to flow out of the microfluidic device.

4. The method according to claim 2 , wherein the separation protocol is for separating the plasma membrane protein fraction from the cell sample and comprises flowing a plasma membrane protein treatment solution through the microfluidic device under continuous flow conditions effective to cleave proteins from plasma membranes of the one or more captured cell without lysing the plasma membranes, thereby causing the plasma membrane protein fraction to flow out of the microfluidic device.

5. The method according to claim 2 , wherein the separation protocol is for separating the total RNA fraction from the cell sample and comprises flowing a total RNA treatment solution through the microfluidic device under continuous flow conditions effective to release the total RNA fraction from the one or more captured cell, thereby causing the total RNA fraction to flow out of the microfluidic device.

6. The method according to claim 2 , wherein the separation protocol is for separating the cytosolic RNA fraction from the cell sample and comprises flowing a cytosolic RNA treatment solution through the microfluidic device under continuous flow conditions effective to release the cytosolic RNA fraction from the one or more captured cell, thereby causing the cytosolic RNA fraction to flow out of the microfluidic device.

7. The method according to claim 2 , wherein the separation protocol is for separating the cytosolic protein fraction from the cell sample and comprises flowing a cytosolic protein treatment solution through the microfluidic device under continuous flow conditions effective to release the cytosolic protein fraction from the one or more captured cell, thereby causing the cytosolic protein fraction to flow out of the microfluidic device.

8. The method according to claim 2 , wherein the separation protocol is for separating the nuclear RNA fraction from the cell sample and comprises flowing a nuclear RNA treatment solution through the microfluidic device under continuous flow conditions effective to release the nuclear RNA fraction from the one or more captured cell, thereby causing the nuclear RNA fraction to flow out of the microfluidic device.

9. The method according to claim 2 , wherein the separation protocol is for separating the nuclear protein fraction from the cell sample and comprises flowing a nuclear protein treatment solution through the microfluidic device under continuous flow conditions effective to release the nuclear protein fraction from the one or more captured cell, thereby causing the nuclear protein fraction to flow out of the microfluidic device.

10. The method according to claim 2 , wherein the separation protocol is for separating the chromatin fraction from the cell sample and comprises flowing a chromatin treatment solution through the microfluidic device under continuous flow conditions effective to release the chromatin fraction from the one or more captured cell, thereby causing the chromatin fraction to flow out of the microfluidic device.

11. The method according to claim 2 , wherein the separation protocol is for separating the gDNA markers fraction from the cell sample and comprises flowing a gDNA markers treatment solution through the microfluidic device under continuous flow conditions effective to release the gDNA markers fraction from the one or more captured cell, thereby causing the gDNA markers fraction to flow out of the microfluidic device.

12. The method according to claim 2 , wherein the separation protocol is for separating the amplified gDNA fraction from the cell sample and comprises flowing an amplified gDNA treatment solution through the microfluidic device under continuous flow conditions effective to release the amplified gDNA fraction from the one or more captured cell, thereby causing the amplified gDNA fraction to flow out of the microfluidic device.

13. The method according to claim 2 , wherein the separation protocol is for separating the methylated gDNA fraction from the cell sample and comprises flowing a methylated gDNA treatment solution through the microfluidic device under continuous flow conditions effective to release the methylated gDNA fraction from the one or more captured cell, thereby causing the methylated gDNA fraction to flow out of the microfluidic device.

14. The method according to claim 2 , wherein said act of treating comprises implementing a combination of the separation protocols to isolate one or more of the distinct analyte components for purposes of single or multiomic analysis thereof.

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

16. The method according to claim 1 , wherein the cell sample is selected from the group consisting of a cancer cell, a primary cell type isolated from tissue of a human, an animal, or a plant, and an immortalized cell line.

17. The method according to claim 1 , wherein the flow rate ranges from between 0.001 nL/minute and 100 uL/minute.

18. The method according to claim 1 , further comprising analyzing the at least one analyte.

19. The method according to claim 18 , wherein the at least one analyte is a plasma membrane protein fraction, a total protein fraction, a cytosolic protein fraction, and/or a nuclear protein fraction that is subjected to further testing and/or analysis selected from the group consisting of mass spectrometry, immuno-based detection assays, and aptamer-based detectio

Assignees

• Univ Cornell

Inventors

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

Classifications

• C12M23/16

  Microfluidic devices; Capillary tubes (integrated microfluidic structures B01L3/5027; microreactors B01J19/0093) · CPC title

• B01L3/5027

  by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip · CPC title

• C12N15/1006

  by means of a solid support carrier, e.g. particles, polymers · CPC title

• B01L2400/086

  using baffles or other fixed flow obstructions · CPC title

• B01L2300/0816

  Cards, e.g. flat sample carriers usually with flow in two horizontal directions · CPC title