Compartmentalised screening by microfluidic control

• US 10,371,699 B2
• Filed: 10/21/2016
• Issued: 08/06/2019
• 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: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Griffiths, Andrew David, Weitz, David A., Link, Darren Roy, Ahn, Keunho, Bibette, Jerome
• Primary Examiner(s): Crawford, Erik B
• Application Number:
  US15/331,445
• Publication Number:
  US 20170102381A1
• Time in Patent Office: 1,019 Days
• Field of Search: None
• 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 screening a repertoire of compounds for a compound having a desired activity, comprising the steps of:

(a) providing an aqueous fluid comprising the repertoire of compounds;

(b) compartmentalising the repertoire of compounds into microcapsules by partitioning the aqueous fluid with an immiscible fluid as the aqueous fluid is flowing through a microfluidic channel, such that only a subset of the repertoire is represented in multiple copies in any one microcapsule and wherein each microcapsule comprises a target cell;

(c) conducting a reaction involving a molecule associated with the target cell, wherein the compound has an effect on the reaction; and

(d) detecting a product of the reaction to identify a microcapsule containing the compound having the desired activity.

2. The method of claim 1, wherein the target cell is compartmentalized together with the repertoire of compounds in the microcapsules.

3. The method of claim 1, further comprising fusing the microcapsules with second microcapsules comprising the target cells.

4. The method of claim 1, wherein the repertoire of compounds are attached to microbeads.

5. The method of claim 4, wherein each microbead comprises a detectable tag.

6. The method of claim 4, wherein the repertoire of compounds are attached to microbeads through one or more cleavable linkers.

7. The method of claim 4, further comprising releasing the compounds from the microbeads.

8. The method of claim 4, wherein each microbead comprises one of the subsets of the repertoire of compounds represented in multiple copies.

9. The method of claim 1, wherein the molecule associated with the target cell is an enzyme or a receptor.

10. The method of claim 9, further comprising incubating the microcapsules.

11. The method of claim 1, wherein the target cell is from a single cell organism.

12. The method of claim 1, wherein the target cell is from a multicellular organism.

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

14. The method of claim 1, wherein the immiscible fluid comprises a fluorocarbon oil.

15. The method of claim 1, wherein the molecule associated with the target cell is a gene, and wherein at least one compound from the repertoire of compounds expresses or inhibits expression of a gene.