Methods, systems and devices for multiple single-cell capturing and processing using microfluidics

• US 9,429,500 B2
• Filed: 02/28/2013
• Issued: 08/30/2016
• Est. Priority Date: 02/29/2012
• Status: Active Grant

• Current Assignee: Standard Biotools Incorporated
• Original Assignee: Fluidigm Corporation
• Inventors: Fowler, Brian, Kimball, Jake, Maung, Myo Thu, May, Andrew, Norris, Michael C., Toppani, Dominique G., Unger, Marc A., Wang, Jing, West, Jason A. A.
• Primary Examiner(s): WARDEN, JILL ALICE
• Assistant Examiner(s): Kingan, Timothy G
• Application Number:
  US13/781,292
• Publication Number:
  US 20130302883A1
• Time in Patent Office: 1,279 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2400/0409 centrifugal forces
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/043 magnetic forces
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/086 using baffles or other fixed flow obstructions
• CPC Class Codes: B01L 2400/088 by specific surface properties
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2565/601 being a microscope, e.g. atomic force microscopy [AFM]
• CPC Class Codes: C12Q 2565/629 being a microfluidic device
• CPC Class Codes: G01N 1/28 : Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Qmounting specimens on microscopic slides G02B21/34; means for supporting the objects or the materials to be analysed in electron microscopes H01J37/20 ; laboratory gas handling apparatus B01L5/00
• CPC Class Codes: G01N 1/34 : Purifying; Cleaning processes or apparatus for extracting or separating nucleic acids from biological samples C12N15/1003
• CPC Class Codes: G01N 15/1484 : microstructural devices

Abstract

Methods, systems, and devices are described for multiple single-cell capturing and processing utilizing microfluidics. Tools and techniques are provided for capturing, partitioning, and/or manipulating individual cells from a larger population of cells along with generating genetic information and/or reactions related to each individual cell. Different capture configurations may be utilized to capture individual cells and then processing each individual cell in a multi-chamber reaction configuration. Some embodiments may provide for specific target amplification, whole genome amplification, whole transcriptome amplification, real-time PCR preparation, copy number variation, preamplification, mRNA sequencing, and/or haplotyping of the multiple individual cells that have been partitioned from the larger population of cells. Some embodiments may provide for other applications. Some embodiments may be configured for imaging the individual cells or associated reaction products as part of the processing. Reaction products may be harvested and/or further analyzed in some cases.

Claims

1. A microfluidic device for multiple single-cell capturing and processing, the microfluidic device comprising:

a) a plurality of capture configurations coupled in series, wherein each respective capture configuration is configured to capture a single cell,

(i) wherein each capture configuration comprises

a single input channel,

a single output channel,

a capture nest comprising one or more physical barriers configured to capture a single cell,

a drain channel, and

a pair of bypass channels, and

(ii) wherein in each capture configuration

the capture nest is in fluidic communication with the drain channel,

the capture nest is situated near a junction of the input channel and the pair of bypass channels,

the drain channel is in fluidic communication with the input channel and the output channel such that solution that flows from the input channel through the drain channel exits the capture configuration through the output channel without flowing through the bypass channels,

the bypass channels are in fluidic communication with the input channel and the output channel such that solution that flows from the input channel through the bypass channels exits the capture configuration through the output channel,

the capture nest is configured to capture a single cell from a plurality of cells flowing through the input channel, and

the output channel is configured to allow cells to pass through said output channel to the next capture configuration in the series,

wherein the only fluidic paths through which solution can flow into or out of the capture nest, drain and bypass channel portions of the capture configuration-comprises flow through the single input channel or a the single output channel; and

(b) a plurality of multi-chamber reaction configurations,

wherein each respective multi-chamber reaction configuration is in fluidic communication with an output channel or input channel of a respective capture configuration from the plurality of capture configurations and said multi-chamber reaction configurations are configured for single-cell processing.

2. The microfluidic device of claim 1, wherein the bypass channels are symmetrically configured.

3. The microfluidic device of claim 1, wherein the input channel is further configured as a focusing channel.

4. The microfluidic device of claim 1, wherein the plurality of multi-chamber reaction configurations are further configured for thermal cycling while one or more valves of a respective multi-chamber reaction configuration is actuated.

5. The microfluidic device of claim 1, further comprising:

one or more imaging features, wherein each respective imaging feature allows for imaging of captured single cells at a respective capture nest site.

6. The microfluidic device of claim 1, further comprising:

a plurality of harvest wells, wherein each respective harvest well is coupled with a respective multi-chamber reaction configuration and configured to deliver reaction products for further analysis.

7. The microfluidic device of claim 1, further comprising:

a genomic analysis configuration coupled with each respective multi-chamber reaction configuration to further analyze the reaction products from each respective multi-chamber reaction configuration.

8. The microfluidic device of claim 1 wherein the multi-chamber reaction configurations each comprise

a first reaction chamber coupled to the capture configuration,

a second reaction chamber coupled to the first reaction chamber through two non-overlapping fluid flow paths,

valves positioned to fluidically isolate the first and second reaction chambers from each other,

a valve positioned to fluidically isolate the capture configuration from the multi-chamber reaction configuration.

9. The microfluidic device of claim 8 wherein the second reaction chamber is adjacent to the first reaction chamber.

10. The microfluidic device of claim 8 wherein the multi-chamber reaction configurations each comprise a third reaction chamber that is coupled to the second reaction chamber through two nonoverlapping fluid flow paths, and valves positioned to fluidically isolate the second and third capture configurations from each other.

11. The system of claim 9 wherein the multi-chamber reaction configurations each comprise a third reaction chamber that is coupled to the second reaction chamber through two nonoverlapping fluid flow paths, and valves positioned to fluidically isolate the second and third capture configurations from each other.

12. The microfluidic device of claim 1, wherein the drain channel is sized to allow liquid but not cells to flow though.

13. The microfluidic device of claim 2, wherein the symmetrically configured pair of bypass channels comprise a first wing configuration and a second wing configuration.