Multiomic analysis of cell analytes using microfluidic systems

Patent metadata

Field	Value
Publication number	US-2022010360-A1
Application number	US-201917298533-A
Country	US
Kind code	A1
Filing date	Nov 29, 2019
Priority date	Nov 28, 2018
Publication date	Jan 13, 2022
Grant date	—

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 steps of:
(a) providing a microfluidic device comprising a cell capture component and a nucleic acid entanglement component, wherein said cell capture component comprises a cell capture array comprising a plurality of cell capture micropillars, wherein said nucleic acid entanglement component comprises a nucleic acid entanglement array comprising a plurality of nucleic acid entanglement micropillars, and wherein the microfluidic device operates under continuous flow conditions; (b) capturing one or more cell in the cell capture array of the microfluidic device while being subjected to a continuous flow rate, said capturing step comprising introducing the one or more cell sample into the microfluidic device under sufficient hydrodynamic flow to entrap, by size exclusion, the one or more cell within the cell capture component; (c) treating the one or more captured cell with a sequential workflow procedure under conditions effective to separate one or more distinct analyte component therefrom, wherein said one or more distinct analyte component 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; and
(d) isolating the one or more distinct analyte component 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 up to six distinct classes of analyte component from the cell sample, wherein the distinct classes comprise:
Class 1: the total protein and plasma membrane protein fractions; Class 2: the total protein, total RNA, cytosolic RNA, and cytosolic protein fractions; Class 3: the total protein, total RNA, nuclear RNA, and nuclear protein fractions; Class 4: the chromatin fraction; Class 5: the gDNA markers and amplified gDNA fractions; and Class 6: the gDNA markers and methylated gDNA fractions,
wherein the sequential workflow procedure operates under the following rules:
Rule A: only one distinct analyte component can be isolated per class;
Rule B: isolation of one or more of the classes may start with any of the classes;
Rule C: if more than one class is to be isolated, the order of isolation must proceed progressively along a classification gradient that includes, in sequence, Class 1, Class 2, Class 3, Class 4, Class 5, and Class 6, where Class 1 represents the start of the classification gradient and Class 6 represents the end of the classification gradient;
Rule D: if more than one class is to be isolated, the classes to be isolated will begin with an initial class comprising the class closest to the start of the classification gradient and terminate with a terminal class comprising the class closest to the end of the classification gradient; and
Rule E: if more than one class is to be isolated, any intermediate class falling between the initial class and the terminal class of the classification gradient may either be omitted or included from the isolating process.

3 . The method according to claim 2 , wherein the separation protocol is for separating the total protein fraction from the cell sample and comprises the step of:
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 3 , wherein the total protein treatment solution comprises a detergent, a salt, and a chelating agent.

5 . The method according to claim 4 , wherein the total protein treatment solution comprises a buffer containing Tris (10-50 mM, pH 7-8), NaCl (10-500 mM), and/or KCl (10-500 mM), MgCl2 (0-5 mM), CaCl2 (0-5 mM), detergent (NP40 (0-1%), sodium deoxycholate (0-1%), or SDS (0-1%)), and EDTA (1 uM-10 mM).

6 . The method according to claim 2 , wherein the separation protocol is for separating the plasma membrane protein fraction from the cell sample and comprises the step of:
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.

7 . The method according to claim 6 , wherein the plasma membrane protein treatment solution comprises a protease a salt, and a chelating agent.

8 . The method according to claim 6 , wherein the plasma membrane protein treatment solution comprises trypsin (0.01-0.5%) in phosphate buffered saline, with or without EDTA (10 uM-2 mM), at pH 7-8.

9 . The method according to claim 6 , wherein the flowing of the plasma membrane protein treatment solution is for a period of time under flow ranging from 1-30 minutes.

10 . The method according to claim 6 , wherein the stable incubation temperature ranges from 20-40 degrees Celsius.

11 . The method according to claim 6 , wherein the plasma membrane protein treatment solution comprises trypsin (0.01-0.5%) with or without EDTA (10 uM-2 mM), at pH 7-8, under flow ranging from 1-30 minutes, wherein the stable incubation temperature ranges from 20-40 degrees Celsius.

12 . The method according to claim 2 , wherein the separation protocol is for separating the total RNA fraction from the cell sample and comprises the step of:
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.

13 . The method according to claim 12 , wherein the total RNA treatment solution comprises a salt, a detergent, and an RNase inhibitor.

14 . The method according to claim 12 , wherein the total RNA treatment solution comprises a buffer containing Tris (pH 7-8, 1-50 mM), NaCl (25-500 mM), KCl (25-500 mM), detergent (e.g. Triton X-100, 0.25-2%), and RNase inhibitor.

15 . The method according to claim 2 , wherein the separation protocol is for separating the cytosolic RNA fraction from the cell sample and comprises the step of:
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.

16 . The method according to claim 15 , wherein the cytosolic RNA treatment solution comprises a detergent, a salt, an RNase inhibitor.

17 . The method according to claim 15 , wherein the cytosolic RNA treatment solution comprises a buffer containing Tris (pH 7-8, 1-25 mM), NaCl (10-300 mM) and/or KCl (10-300 mM), detergent (e.g. Triton X-100, Tween 20, or NP40, or digitonin 0.1-2%), with RNase inhibitors.

18 . The method according to

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

• B01L2200/0668

  Trapping microscopic beads · CPC title

• C12Q1/6806 Primary

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

• B01L2400/086

  using baffles or other fixed flow obstructions · CPC title