Microfluidic devices and methods of use in the formation and control of nanoreactors

• US 9,328,344 B2
• Filed: 02/05/2013
• Issued: 05/03/2016
• Est. Priority Date: 01/11/2006
• Status: Active Grant

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Raindance Technologies Incorporated (Bio-Rad Laboratories Incorporated)
• Inventors: Link, Darren, Boitard, Laurent, Branciforte, Jeffrey, Charles, Yves, Feke, Gilbert, Lu, John Q., Marran, David, Tabatabai, Ahmadali, Weiner, Michael, Hinz, Wolfgang, Rothberg, Jonathan M.
• Primary Examiner(s): Siefke, Sam P
• Assistant Examiner(s): Wecker, Jennifer
• Application Number:
  US13/759,660
• Publication Number:
  US 20130217583A1
• Time in Patent Office: 1,183 Days
• Field of Search: 506/2, 506/9, 506/16, 506/30, 506/37, 506/40
• US Class Current: 1/1
• CPC Class Codes: B01F 23/41 Emulsifying
• CPC Class Codes: B01F 33/3021 the components to be mixed being combined in a single independent droplet, e.g. these droplets being divided by a non-miscible fluid or consisting of independent droplets
• CPC Class Codes: B01F 33/3031 using electro-hydrodynamic [EHD] or electro-kinetic [EKI] phenomena to mix or move the fluids
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00286 Reactor vessels with top and bottom openings
• CPC Class Codes: B01J 2219/00459 Beads
• CPC Class Codes: B01J 2219/00466 in a slurry
• CPC Class Codes: B01J 2219/00468 by manipulation of individual beads
• CPC Class Codes: B01J 2219/00479 Means for mixing reactants or products in the reaction vessels
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00576 fluorophore
• CPC Class Codes: B01J 2219/00599 Solution-phase processes
• CPC Class Codes: B01J 2219/00702 Processes involving means for analysing and characterising the products
• CPC Class Codes: B01J 2219/0072 Organic compounds
• CPC Class Codes: B01J 2219/00743 Cells
• CPC Class Codes: B01L 2200/027 for microfluidic devices
• CPC Class Codes: B01L 2200/0621 Control of the sequence of chambers filled or emptied
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2200/0652 Sorting or classification of particles or molecules
• CPC Class Codes: B01L 2200/0673 Handling of plugs of fluid surrounded by immiscible fluid
• CPC Class Codes: B01L 2300/0816 : Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0864 : comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/0867 : Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2300/0896 : Nanoscaled
• CPC Class Codes: B01L 2400/0415 : electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/0424 : Dielectrophoretic forces
• CPC Class Codes: B01L 2400/0487 : fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/084 : Passive control of flow resistance
• CPC Class Codes: B01L 3/5027 : 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 B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• 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 3/502746 : characterised by the means for controlling flow resistance, e.g. flow controllers, baffles B01L3/502738 takes precedence
• 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 3/502784 : 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 3/502792 : for moving individual droplets on a plate, e.g. by locally altering surface tension
• CPC Class Codes: B01L 3/565 : Seals in general F16L
• 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: B82Y 30/00 : Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: B82Y 5/00 : Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C07K 1/047 : Simultaneous synthesis of different peptide species; Peptide libraries
• CPC Class Codes: C12N 15/1075 : by coupling phenotype to genotype, not provided for in other groups of this subclass
• CPC Class Codes: C12Q 1/6804 : Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6816 : characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6825 : Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6834 : Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/686 : Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 : Methods for sequencing
• CPC Class Codes: C12Q 1/6874 : involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6876 : Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2525/161 : incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2561/119 : Fluorescence polarisation
• CPC Class Codes: C12Q 2563/103 : luminescence
• CPC Class Codes: C12Q 2563/107 : fluorescence
• 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 2565/537 : characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: C12Q 2565/629 : being a microfluidic device
• CPC Class Codes: C12Q 2600/16 : Primer sets for multiplex assays
• CPC Class Codes: C40B 30/04 : by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• CPC Class Codes: C40B 40/04 : Libraries containing only organic compounds
• CPC Class Codes: C40B 50/08 : Liquid phase synthesis, i.e. wherein all library building blocks are in liquid phase or in solution during library creation; Particular methods of cleavage from the liquid support
• CPC Class Codes: C40B 60/08 : Integrated apparatus specially adapted for both creating and screening libraries
• CPC Class Codes: C40B 60/12 : for screening libraries
• CPC Class Codes: C40B 70/00 : Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or bar codes
• CPC Class Codes: G01N 21/6428 : Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6445 : Measuring fluorescence polarisation
• CPC Class Codes: G01N 21/6458 : Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G01N 2500/10 : involving cells
• CPC Class Codes: G01N 33/536 : with immune complex formed in liquid phase
• CPC Class Codes: G01N 33/542 : with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/543 : with an insoluble carrier for immobilising immunochemicals
• CPC Class Codes: G01N 33/6845 : Methods of identifying protein-protein interactions in protein mixtures
• CPC Class Codes: G01N 33/6854 : Immunoglobulins
• CPC Class Codes: G01N 35/08 : using a stream of discrete samples flowing along a tube system, e.g. flow injection analysis

Abstract

The present invention provides novel microfluidic devices and methods that are useful for performing high-throughput screening assays and combinatorial chemistry. Such methods can include labeling a library of compounds by emulsifying aqueous solutions of the compounds and aqueous solutions of unique liquid labels on a microfluidic device, which includes a plurality of electrically addressable, channel bearing fluidic modules integrally arranged on a microfabricated substrate such that a continuous channel is provided for flow of immiscible fluids, whereby each compound is labeled with a unique liquid label, pooling the labeled emulsions, coalescing the labeled emulsions with emulsions containing a specific cell or enzyme, thereby forming a nanoreactor, screening the nanoreactors for a desirable reaction between the contents of the nanoreactor, and decoding the liquid label, thereby identifying a single compound from a library of compounds.

Claims

1. A method for detecting a polymerase chain reaction (PCR) product in an aqueous droplet, the method comprising:

forming a plurality of aqueous droplets in a microfluidic chip;

moving the plurality of aqueous droplets to a separate off-chip reservoir outside of the microfluidic chip, wherein the plurality of aqueous droplets are pooled within the off-chip reservoir and at least one of the aqueous droplets comprises a nucleic acid and reagents for a PCR reaction;

conducting a plurality of PCR reactions simultaneously while the aqueous droplets are in the off-chip reservoir;

moving the plurality of aqueous droplets that have been subject to the plurality of PCR reactions from the off-chip reservoir and into a channel of a second microfluidic device that is operably associated with a detector;

adding an immiscible carrier fluid between the aqueous droplets in the channel of the second microfluidic device, thereby increasing the spacing between the aqueous droplets in the channel of the second microfluidic device; and

detecting a product of the PCR reaction via the detector in one or more of the aqueous droplets in the channel.

2. The method of claim 1, wherein the aqueous droplets are surrounded by the immiscible carrier fluid.

3. The method of claim 1, wherein the immiscible carrier fluid is injected from a direction substantially perpendicular to the channel.

4. The method of claim 1, wherein the aqueous droplets have the same composition.

5. The method of claim 1, wherein the aqueous droplets have different compositions.

6. The method of claim 1, wherein the immiscible carrier fluid is an oil.

7. The method of claim 6, wherein the oil comprises a surfactant.

8. The method of claim 7, wherein the surfactant is a fluorosurfactant.

9. The method of claim 1, wherein the immiscible carrier fluid is a fluorinated oil.

10. The method of claim 1, wherein the immiscible carrier fluid is injected using a syringe or pump.

11. The method of claim 1, wherein the immiscible carrier fluid is injected using positive or negative pressure source.

12. The method of claim 1, wherein the product of the PCR reaction detected in the one or more aqueous droplets comprises a plurality of fluorescent reporter molecules.

13. The method of claim 12, wherein the fluorescent reporter molecules are separated by a polymerase from quencher molecules during the PCR reaction.

14. The method of claim 1, wherein the nucleic acid is an antibiotic resistant gene.

15. The method of claim 1, wherein the at least one of the aqueous droplets further comprises a label.

16. The method of claim 15, wherein the label is an organic dye.

17. The method of claim 15, wherein the product of the PCR reaction comprises the label.

18. The method of claim 1, further comprising cloning the PCR product.

19. The method of claim 18, wherein the PCR product is cloned into a vector.

20. The method of claim 19, wherein the vector comprises a second antibiotic resistant gene.

21. The method of claim 18, wherein the PCR product is cloned into a host.

22. The method of claim 21, wherein the host is a bacterium.