Compartmentalized screening by microfluidic control

• US 9,498,759 B2
• Filed: 10/12/2005
• Issued: 11/22/2016
• Est. Priority Date: 10/12/2004
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation), UK Research and Innovation
• Original Assignee: Medical Research Council (Government of United Kingdom), President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Griffiths, Andrew, Weitz, David, Ahn, Keunho, Link, Darren R., Bibette, Jerome
• Primary Examiner(s): Crawford, Erik B
• Application Number:
  US11/665,145
• Publication Number:
  US 20090005254A1
• Time in Patent Office: 4,059 Days
• Field of Search: 506/2, 506/4, 506/79, 506/16, 506/18, 506/38
• US Class Current: 1/1
• CPC Class Codes: B01F 23/41 Emulsifying
• CPC Class Codes: B01F 23/4143 Microemulsions
• CPC Class Codes: B01F 23/4145 Emulsions of oils, e.g. fuel, and water
• CPC Class Codes: B01F 25/23 Mixing by intersecting jets
• CPC Class Codes: B01F 25/433 Mixing tubes wherein the shape of the tube influences the mixing, e.g. mixing tubes with varying cross-section or provided with inwardly extending profiles
• CPC Class Codes: B01F 25/4331 Mixers with bended, curved, coiled, wounded mixing tubes or comprising elements for bending the flow
• CPC Class Codes: B01F 25/4338 Mixers with a succession of converging-diverging cross-sections, i.e. undulating cross-section
• CPC Class Codes: B01F 33/3011 using a sheathing stream of a fluid surrounding a central stream of a different fluid, e.g. for reducing the cross-section of the central stream or to produce droplets from the central stream
• 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 2219/00576 fluorophore
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00599 Solution-phase processes
• CPC Class Codes: B01J 2219/00657 One-dimensional arrays
• CPC Class Codes: B01J 2219/00664 Three-dimensional arrays
• CPC Class Codes: B01J 2219/00666 One-dimensional arrays within three-dimensional arrays
• CPC Class Codes: B01J 2219/00702 Processes involving means for analysing and characterising the products
• 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 2400/0406 : capillary forces
• CPC Class Codes: B01L 2400/0415 : electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/0487 : fluid pressure, pneumatics
• 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: C12Q 1/42 : involving phosphatase
• 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 2565/119 : based on extraction of label to an organic phase, i.e. partitioning of label between different organic phases
• 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: G01N 15/14 : Optical investigation techniques, e.g. flow cytometry
• CPC Class Codes: G01N 15/149 : specially adapted for sorting particles, e.g. by their size or optical properties
• CPC Class Codes: G01N 2021/6439 : with indicators, stains, dyes, tags, labels, marks
• 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 2333/916 : acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
• CPC Class Codes: G01N 2500/04 : Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
• CPC Class Codes: G01N 2500/10 : involving cells
• CPC Class Codes: G01N 33/5008 : for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/5044 : involving specific cell types
• CPC Class Codes: G01N 33/5432 : Liposomes or microcapsules
• CPC Class Codes: G01N 33/573 : for enzymes or isoenzymes

Abstract

The invention describes a method for the identification of compounds which bind to a target component of a biochemical system or modulate the activity of the target, comprising the steps of: a) compartmentalizing the compounds into microcapsules together with the target, such that only a subset of the repertoire is represented in multiple copies in any one microcapsule; and b) identifying the compound which binds to or modulates the activity of the target; wherein at least one step is performed under microfluidic control. The invention enables the screening of large repertoires of molecules which can serve as leads for drug development.

Claims

1. A method for identifying a compound(s) in a repertoire of compounds, which compound(s) possess(es) a desired activity, comprising the steps of:

(a) obtaining the repertoire of compounds in an aqueous fluid;

(b) forming a plurality of microcapsules by partitioning the aqueous fluid with two counter propagating streams of immiscible fluid as the aqueous fluid is flowing through a microfluidic channel and passes through a constriction, wherein each microcapsule comprises a subset of the repertoire of compounds; and

(c) identifying at least one compound in at least one of the microcapsules possessing the desired activity from the repertoire of compounds, wherein one or both of the steps (b) and (c) are performed under microfluidic control.

2. The method of claim 1, further comprising detecting a signal from the plurality of microcapsules.

3. The method of claim 1, further comprising attaching the repertoire comprising at least 100 compounds to microbeads.

4. The method of claim 3, wherein each microcapsule or microbead comprises a detectable tag.

5. The method of claim 3, wherein each microcapsule comprises multiple copies of a single compound.

6. The method of claim 3, wherein the compounds of the repertoire are attached to the microbeads through one or more cleavable linkers.

7. The method of claim 1, wherein each microcapsule comprises the subset of the repertoire comprising at least 100 compounds and a target.

8. The method of claim 7, wherein the target is attached to a microbead.

9. The method of claim 7, wherein the target is an enzyme, a receptor, or a cell.

10. The method of claim 9, wherein the cell is from a single cell organism.

11. The method of claim 9, wherein the cell is from a multicellular organism.

12. The method of claim 1, wherein each of the compounds comprises a detectable tag.

13. The method of claim 7, further comprising incubating the microcapsules prior to the identifying step.

14. The method of claim 1, wherein the two counter propagating streams of immiscible fluid comprise a fluorocarbon oil.

15. The method of claim 1, wherein the repertoire of compounds comprises at least 100 compounds.

16. A method for identifying a compound(s) in a repertoire of compounds, which compound(s) possess(es) a desired activity on a target cell, comprising the steps of:

(a) obtaining the repertoire of compounds in an aqueous fluid;

(b) forming a plurality of microcapsules by partitioning the aqueous fluid with two counter propagating streams of immiscible fluid as the aqueous fluid is flowing through a microfluidic channel, wherein each microcapsule comprises a subset of the repertoire of compounds and the target cell; and

(c) identifying the microcapsules that contain the compound(s) having the desired activity on the target cell from the repertoire of compounds.

17. The method of claim 16, wherein each microcapsule comprises the subset of the repertoire of compounds and a plurality of the target cells.

18. The method of claim 16, wherein the target cell in each microcapsule is different.

19. The method of claim 16, wherein each microcapsule comprises the subset of the repertoire of compounds, the target cell, and a gene.

20. The method of claim 19, wherein at least one compound of the subset of the repertoire of compounds expresses or inhibits expression of the gene.

21. The method of claim 20, wherein the gene binds to the target cell to generate a detectable characteristic.

22. The method of claim 21, further comprising screening and sorting the target cells.

23. The method of claim 16, further comprising sorting the microcapsules that contain the compound(s) having the desired activity.

24. The method of claim 16, wherein in the forming step, the aqueous fluid is partitioned with two counter propagating streams of immiscible fluid as the aqueous fluid is flowing through a microfluidic channel and passes through a constriction.