Rapid and Continuous Analyte Processing in Droplet Microfluidic Devices

• US 20110059556A1
• Filed: 09/03/2010
• Published: 03/10/2011
• Est. Priority Date: 09/04/2009
• Status: Active Grant

• Current Assignee: The Research Foundation for The State University of New York (State University of New York)
• Original Assignee: The Research Foundation for The State University of New York (State University of New York)
• Inventors: Kimmerling, Robert, Bakowski, Tomasz, Strey, Helmut
• Granted Patent: US 9,625,454 B2
• Time in Patent Office: Days
• US Class Current: 436/518
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2200/0673 Handling of plugs of fluid surrounded by immiscible fluid
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2400/043 magnetic forces
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/50273 characterised by the means or forces applied to move the fluids micropumps F04B19/006, of the membrane type F04B43/043
• 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 3/502776 specially adapted for focusing or laminating flows
• CPC Class Codes: B01L 3/502784 specially adapted for droplet or plug flow, e.g. digital microfluidics automatic analysis using a stream of discrete samples in a tube system G01N35/08
• 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: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2563/159 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 Class Codes: C12Q 2565/629 being a microfluidic device
• CPC Class Codes: G01N 33/54313 the carrier being characterised by its particulate form
• CPC Class Codes: G01N 33/54326 Magnetic particles
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing

Abstract

The compositions and methods described herein are designed to introduce functionalized microparticles into droplets that can be manipulated in microfluidic devices by fields, including electric (dielectrophoretic) or magnetic fields, and extracted by splitting a droplet to separate the portion of the droplet that contains the majority of the microparticles from the part that is largely devoid of the microparticles. Within the device, channels are variously configured at Y- or T junctions that facilitate continuous, serial isolation and dilution of analytes in solution. The devices can be limited in the sense that they can be designed to output purified analytes that are then further analyzed in separate machines or they can include additional channels through which purified analytes can be further processed and analyzed.

Claims

1. A microfluidic device for separating an analyte from a complex mixture, the device comprising:

(a) a substrate comprising a series of contiguous channels and;

(b) a field generator,

wherein the series of contiguous channels comprises a first channel having an initial segment configured to receive a tagged droplet comprising an analyte bound to a functionalized particle, a middle segment through which the tagged droplet travels, and a terminal segment that bifurcates into a second channel and a third channel,

wherein the field generator is positioned adjacent to the first channel and marginalizes the analyte toward a side of the tagged droplet such that, upon reaching the bifurcation, a portion of the tagged droplet that includes the majority of the analyte enters the second channel and the remainder of the tagged droplet enters the third channel, thereby producing, in the second channel, a smaller droplet that contains the majority of the analyte while excluding at least some of the complex mixture.

2. The device of claim 1, wherein the field generator is a magnet and the functionalized particle comprises a magnetized microsphere;

the field generator is an electrode emitting an oscillating electric field gradient and the functionalized particle comprises a dielectric microsphere;

or the field generator is a focused light or laser beam and the functionalized particle comprises a dielectric microsphere.

3. The device of claim 1, wherein the functionalized particle comprises a tag.

4. The device of claim 3, wherein the tag is an oligo(dT) sequence or a sequence-specific oligonucleotide.

5. The device of claim 3, wherein the tag is a chelator or protein.

6. The device of claim 5, wherein the protein is an antibody, an analyte-binding fragment or variant thereof, or a protein scaffold.

7. The device of claim 1, wherein the substrate comprises silicon, glass, or molded plastic.

8. The device of claim 7, wherein the substrate comprises polydimethylsiloxane.

9. The device of claim 1, wherein the field generator is a magnet and the device further comprises a shield that suppresses the effect of the magnetic field in certain areas of the device.

10. The device of claim 9, wherein the shield comprises nickel, iron, copper, molybdenum, or mumetal.

11. The device of claim 1, wherein one or more of the droplets are aqueous and one or more of the channels contain a non-aqueous sheath fluid.

12. The device of claim 1, wherein one or more of the droplets are non-aqueous and one or more of the channels contain an aqueous sheath fluid.

18. A method of analyzing an analyte in a sample, the method comprising introducing the sample into the microfluidic device of claim 1.

13. A microfluidic device comprising:

a microchannel for encapsulating a single cell comprising an analyte in a droplet; and

a plurality of channels configured to form one or more of a T-, W-, X-, or Y-shaped junction for facilitating continuous, serial separation and dilution of the analyte.

14. The microfluidic device of claim 13, further comprising a field generator.

15. A microfluidic device configured to perform single cell mRNA analysis, the device comprising a series of contiguous channels in a substrate, the channels defining five functional zones:

a first zone configured to encase single cells in droplets with an enzyme that generates a cell lysate;

a second zone configured to add microparticles functionalized to specifically bind mRNA to the cell lysate, thereby generating microparticle-associated mRNA;

a third zone configured to separate the microparticle-associated mRNA from a portion of the remainder of the cell lysate;

a fourth zone configured to add reverse transcriptase to the microparticle-associated mRNA, thereby generating cDNA; and

a fifth zone configured to amplify the cDNA with sequence-specific primers.

16. The microfluidic device of claim 15, wherein the first zone comprises:

an opening for receiving cells in a suspension;

a microchannel, extending from the opening, that is configured to guide the cells in the suspension into single file;

a first channel, extending from the microchannel, having an initial segment and a terminal segment, wherein the initial segment, by virtue of being larger than the microchannel, permits each cell to become encapsulated in a single aqueous droplet upon emerging from the microchannel, thereby forming a cell-containing droplet;

a second channel having an initial segment and a terminal segment, wherein the initial segment comprises an opening for receiving an aqueous droplet comprising a lysis buffer and the terminal segment of the second channel merges with the terminal segment of the first channel to create a junction; and

a third channel extending from the junction of the first channel and the second channel, into which the cell-containing droplet and the droplet comprising a lysis buffer enter and coalesce, thereby generating a droplet comprising a single lysed cell.

17. The microfluidic device of claim 16, further comprising a pair of electrodes positioned adjacent to the third channel, wherein the electrodes generate an electric field sufficient to destabilize the cell-containing droplet or the droplet comprising a lysis buffer and thereby facilitate the coalescence of the droplets.

19. A method of manipulating an analyte within a sample, the method comprising:

providing the sample;

encapsulating the sample within a droplet comprising a functionalized particle, wherein the functionalized particle comprises a field-responsive element and a tag that specifically binds the analyte, thereby generating an analyte-tagged droplet;

marginalizing the analyte by exposing the analyte-tagged droplet to a field generator;

cleaving the analyte-tagged droplet so the portion of the tagged droplet that includes the majority of the marginalized analyte becomes contained within a smaller droplet; and

diluting the analyte by fusing the smaller droplet with a droplet comprising a buffer or reagent, thereby generating an analyte-diluted droplet.

20. The method of claim 19, wherein the analyte-tagged droplet is one of a plurality and the method is carried out by continuously and rapidly moving the plurality of droplets through the channels of a microfluidic device.

21. The method of claim 19, wherein the analyte is a biomolecule selected from the group consisting of a nucleic acid, a protein, a sugar, and a fat.

22. The method of claim 19, wherein the sample is a single, lysed biological cell.

23. The method of claim 22, wherein the single, lysed biological cell is prepared by:

encapsulating a single biological cell in a droplet, thereby generating a cell-containing droplet; and

fusing the cell-containing droplet with a droplet comprising a lysis buffer, thereby generating a droplet comprising a single, lysed biological cell.

24. The method of claim 19, further comprising the steps of

exposing the analyte-diluted droplet to a field generator, wherein the field generator marginalizes the analyte toward a side of the analyte-diluted droplet;

and

cleaving the analyte-diluted droplet so the portion of the dilution droplet that includes the majority of the analyte becomes contained within a smaller analyte-diluted droplet.

25. A kit comprising instructions for use, a microfluidic device comprising a plurality of channels configured to form junctions for facilitating continuous, serial separation and dilution of an analyte, and one or more of the following items:

(a) a fluid for encapsulating a sample comprising the analyte in a droplet;

(b) a fluid for sheathing the droplet;

(c) a lysis buffer;

(d) a functionalized particle;

(e) a dilution buffer; and

(f) a solution comprising a reagent.

26. The kit of claim 25, wherein the junctions comprise one or more of a T-shaped, W-shaped, X-shaped, or Y-shaped junction.

27. The kit of claim 25, wherein the microfluidic device further comprises a microchannel for encapsulating a single cell comprising an analyte in a droplet.

28. The kit of claim 25, wherein the microfluidic device further comprises a field generator.

29. The kit of claim 25, wherein the fluid for encapsulating the sample is an aqueous fluid and the fluid for sheathing the droplet is a non-aqueous fluid.

30. The kit of any of claim 25, wherein the reagent is an enzyme or a reagent required for carrying out PCR.