Single-particle analysis of particle populations

Patent metadata

Field	Value
Publication number	US-9840732-B2
Application number	US-201313899397-A
Country	US
Kind code	B2
Filing date	May 21, 2013
Priority date	May 21, 2012
Publication date	Dec 12, 2017
Grant date	Dec 12, 2017

Abstract

In certain embodiments, the invention provides methods and devices for assaying single particles in a population of particles, wherein at least two parameters are measured for each particle. One or more parameters can be measured while the particles are in the separate reaction volumes. Alternatively or in addition, one or more parameters can be measured in a later analytic step, e.g., where reactions are carried out in the separate reaction volumes and the reaction products are recovered and analyzed. In particular embodiments, one or more parameter measurements are carried out “in parallel,” i.e., essentially simultaneously in the separate reaction volumes.

First claim

What is claimed is:

1. A method of producing a data set comprising a plurality of parameters for single particles in a plurality of particles, wherein each of said single particles comprises one or more target nucleic acids, said method comprising:
capturing particles of said plurality of particles in separate compartments of a microfluidic device, thereby producing a plurality of separate reaction volumes in the separate compartments of the microfluidic device such that at least 35% of said separate reaction volumes comprise only a single particle of said plurality of particles, the separate compartments being arranged as an array defined by rows and columns;
determining which of said separate reaction volumes comprise only a single particle of said plurality of particles by microscopy and/or using a stain, dye, or label;
performing a plurality of reactions in each of said separate reaction volumes, thereby producing a plurality of reaction products in each of said separate reaction volumes, wherein at least one reaction of the plurality of reactions in each of said separate reaction volumes comprises a nucleic acid amplification reaction that produces at least one amplicon in each of said separate reaction volumes,
wherein the at least one amplicon in each of said separate reaction volumes is produced using amplification primers, wherein each of at least two of the amplification primers comprises a barcode nucleotide sequence, the at least one amplicon comprising a combination of the barcode nucleotide sequence from each of the at least two of the amplification primers,
wherein the combination of the barcode nucleotide sequence from each of the at least two amplification primers in the at least one amplicon in each of said separate reaction volumes identifies the row and column of a compartment of the separate compartments of the microfluidic device and thereby identifies the source of the at least one amplicon;
recovering the reaction products, comprising the at least one amplicon from each of said separate reaction volumes by pooling the reaction products from each of the separate reaction compartments, thereby producing pooled reaction products;
obtaining analysis results by analyzing the pooled reaction products;
generating analysis results for the single particles in the plurality of particles by disregarding the analysis results from said separate reaction volumes that contain no particle of said plurality of particles or contain more than one single particle of said plurality of particles; and
producing the data set comprising the plurality of parameters for the single particles in the plurality of particles by associating the analysis results for the single particles in the plurality of particles with each of said separate reaction volumes that comprise only a single particle of the plurality of particles.

2. The method of claim 1 , wherein said at least 35% of said separate reaction volumes that comprise only a single particle of said plurality of particles is at least 50% of the total number of said separate reaction volumes.

3. The method of claim 1 , wherein said at least 35% of said separate reaction volumes that comprise only a single particle of said plurality of particles is at least 65% of the total number of said separate reaction volumes.

4. The method of claim 1 , wherein said at least 35% of said separate reaction volumes that comprise only a single particle of said plurality of particles is at least 85% of the total number of said separate reaction volumes.

5. The method of claim 1 , wherein the particles are captured in said separate reaction volumes prior to adding one or more reagents for performing said plurality of reactions in each of said separate reaction volumes.

6. The method of claim 1 , wherein the particles are cells.

7. The method of claim 1 , wherein the particles are nucleic acids.

8. The method of claim 1 , wherein fewer than 40,000 of said particles are employed in the method.

9. The method of claim 1 , wherein fewer than 10,000 of said particles are employed in the method.

10. The method of claim 1 , wherein the number of said particles distributed in said separate reaction volumes is greater than 100.

11. The method of claim 1 , wherein a limiting dilution of said particles is carried out before said capturing particles of said plurality of particles in separate compartments of the microfluidic device.

12. The method of claim 11 , wherein said limiting dilution of said particles is carried out by:
preparing a series of dilutions of a suspension of said plurality of particles;
distributing the particles from each dilution of said series of dilutions into the separate compartments of a microfluidic device;
determining which of said separate compartments comprise only a single particle of said plurality of particles; and
selecting the dilution that produces the highest number of said separate compartments that comprise only a single particle of said plurality of particles for use in said capturing single particles of said plurality of particles in separate compartments of the microfluidic device.

13. The method of claim 12 , wherein the number of particles in each of said separate compartments is determined by brightfield microscopy or by fluorescence microscopy when the particles are labeled with a fluorescent dye or label.

14. The method of claim 12 , wherein a stain, dye, or label is employed for determining which of said separate compartments comprise only a single particle of said plurality of particles.

15. The method of claim 14 , wherein the particles are cells, and the stain, dye, or label is a membrane-permeant stain, dye, or label.

16. The method of claim 14 , wherein the particles are cells, and said determining which of said separate compartments comprise only a single particle of said plurality of particles is by using a cell membrane-permeant nucleic acid dye.

17. The method of claim 14 , wherein the particles are cells, and the stain, dye, or label is a cell-surface stain, dye, or label.

18. The method of claim 14 , wherein the particles are cells, and said determining which of said separate compartments comprise only a single particle of said plurality of particles is by using a labeled antibody specific for a cell-surface marker.

19. The method of claim 1 , wherein said capturing particles of said plurality of particles in separate compartments of the microfluidic device comprises mechanically capturing the particles of said plurality of particles at a plurality of capture sites in the separate compartments of the microfluidic device.

20. The method of claim 19 , wherein each of said plurality of capture sites comprises:
a capture feature with a size holding only one single particle of said plurality of particles;
and a drain feature, wherein, when the capture feature is not occupied by the single particle, the drain feature permits a flow of fluid to pass through the capture site of said plurality of capture sites.

21. The method of claim 19 , wherein the microfluidic device comprises a focusing feature to focus said plurality of particles to each of said plurality of capture sites.

22. The method of claim 19 , wherein said capturing particles of said plurality of particles in separate compartments of the microfluidic device comprises passing a solution comprising said plurality of particles through the separate compartments of the microfluidic device.

23. The method of claim 1 , wherein sa

Assignees

• Fluidigm Corp

Inventors

• Anderson Megan
• Chen Peilin
• Fowler Brian
• Kaper Fiona
• Lebofsky Ronald
• May Andrew

Classifications

• B01D15/08 Primary

  Selective adsorption, e.g. chromatography · CPC title

• C12Q2563/159

  Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components · CPC title

• C12Q2565/629

  being a microfluidic device · CPC title

• C12Q1/6806 Primary

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

• C12Q2563/179

  the label being a nucleic acid · CPC title