Method of synthesis and testing of combinatorial libraries using microcapsules

• US 10,052,605 B2
• Filed: 04/29/2010
• Issued: 08/21/2018
• Est. Priority Date: 03/31/2003
• Status: Active Grant

• Current Assignee: UK Research and Innovation
• Original Assignee: Medical Research Council (Government of United Kingdom)
• Inventors: Griffiths, Andrew David, Abell, Chris, Hollfelder, Florian, Mastrobattista, Enrico
• Primary Examiner(s): Bunker, Amy M
• Application Number:
  US12/770,006
• Publication Number:
  US 20100210479A1
• Time in Patent Office: 3,036 Days
• Field of Search: 506 23, 506 27
• 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 19/0093 Microreactors, e.g. miniaturised or microfabricated reactors laboratory containers with capillary fluid transport in microfabricated channels or chambers B01L3/5027
• CPC Class Codes: B01J 2219/00497 Features relating to the solid phase supports
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00576 fluorophore
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00599 Solution-phase processes
• CPC Class Codes: B01J 2219/00707 separated from the reactor apparatus
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01J 2219/00725 Peptides
• CPC Class Codes: B01J 2219/00743 Cells
• CPC Class Codes: B01J 2219/00835 Comprising catalytically active material
• CPC Class Codes: B01J 2219/0086 Dimensions of the flow channels
• CPC Class Codes: B01J 2219/00894 More than two inlets
• CPC Class Codes: B01J 2219/00903 Segmented flow
• CPC Class Codes: B82Y 10/00 Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• 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: C40B 20/04 : Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding processes
• 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 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 40/10 : Libraries containing peptides or polypeptides, or derivatives thereof
• 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 33/5432 : Liposomes or microcapsules

Abstract

Methods for use in the synthesis and identification of molecules which bind to a target component of a biological system or modulate the activity of a target are described.

Claims

1. A method for monitoring a reaction, the method comprising:

(a) forming a plurality of aqueous droplets comprising a first aqueous fluid surrounded by an a perfluorocarbon oil comprising a fluorosurfactant, each aqueous droplet comprising a single cell, comprising a gene that encodes an a biological enzyme and a plurality of molecules that modulate an activity of the biological enzyme;

(b) flowing the plurality of aqueous droplets through a microfluidic channel;

conducting in at least one of the plurality of aqueous droplets a first reaction involving the gene and a molecule from among the plurality of molecules while the plurality of aqueous droplets are flowing through the microfluidic channels to produce the biological enzyme in at least one of the cells;

(c) flowing a second aqueous fluid comprising a fluorogenic substrate that is specific for the biological enzyme through a second microfluidic channel to a junction between the first and second microfluidic channels to introduce the fluorogenic substrate into the plurality of aqueous droplets to form a plurality of merged aqueous droplets, thereby causing an enzyme-catalyzed reaction involving the fluorogenic substrate to produce a reaction product in the at least one of the cells; and

(d) optically detecting the merged aqueous droplets that contain cells comprising the reaction product by flow cytometry.

2. The method according to claim 1, wherein the optical detecting step detects a fluorescent signal.

3. The method according to claim 1, further comprising sorting the plurality of aqueous droplets.

4. The method according to claim 1, wherein the plurality of molecules and the fluorogenic substrate are attached to microbeads.

5. The method according to claim 4, wherein the plurality of molecules and the fluorogenic substrate are covalently attached to the microbeads.

6. The method according to claim 4, wherein the plurality of molecules and the fluorogenic substrate are non-covalently attached to the microbeads.

7. The method according to claim 1, wherein the plurality of molecules are attached to a plurality of microbeads.

8. The method according to claim 7, wherein the plurality of molecules is attached to each of the plurality of microbeads via a linker.

9. The method according to claim 8, wherein the plurality of molecules are attached to each of the plurality of microbeads via a cleavable linker.

10. The method of claim 7, wherein the plurality of microbeads further comprises an optical tag.