Compartmentalised combinatorial chemistry by microfluidic control

• US 9,839,890 B2
• Filed: 10/12/2005
• Issued: 12/12/2017
• Est. Priority Date: 03/31/2004
• Status: Active Grant

• Current Assignee: Medical Research Council (Government of United Kingdom), President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: National Science Foundation (Government of the United States of America), Medical Research Council (Government of United Kingdom), President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Griffiths, Andrew, Weitz, David, Link, Darren, Ahn, Keunho, Bibette, Jerome
• Primary Examiner(s): Bunker, Amy M
• Application Number:
  US11/665,099
• Publication Number:
  US 20090197772A1
• Time in Patent Office: 4,444 Days
• Field of Search: 506 9, 506 23, 506 7, 435 6
• CPC Class Codes: B01F 23/41 Emulsifying
• 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 33/3021 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 13/025 Applications of microcapsules not provided for in other subclasses
• 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 2219/00497 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 2219/00853 Employing electrode arrangements
• CPC Class Codes: B01J 2219/0086 Dimensions of the flow channels
• CPC Class Codes: B01J 2219/00889 : Mixing micromixers B01F33/30
• CPC Class Codes: B01L 2200/0605 : Metering of fluids
• 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 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 2400/084 : Passive control of flow resistance
• CPC Class Codes: B01L 3/5025 : for parallel transport of multiple samples
• 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 3/50273 : 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: 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: C07K 1/047 : Simultaneous synthesis of different peptide species; Peptide libraries
• CPC Class Codes: C12N 15/1096 : cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-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 2600/16 : Primer sets for multiplex assays
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 40/08 : Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
• 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 2035/1034 : Transferring microquantities of liquid
• CPC Class Codes: H05K 999/99 : dummy group

Abstract

The invention describes a method for the synthesis of compounds comprising the steps of: (a) compartmentalising two or more sets of primary compounds into microcapsules; such that a proportion of the microcapsules contains two or more compounds; and (b) forming secondary compounds in the microcapsules by chemical reactions between primary compounds from different sets; wherein one or both of steps (a) and (b) is performed under microfluidic control; preferably electronic microfluidic control, The invention further allows for the identification of compounds which bind to a target component of a biochemical system or modulate the activity of the target, and which is co-compartmentalised into the microcapsules.

Claims

1. A method for preparing secondary compounds comprising the steps of:

(a) attaching a first set of primary compounds to one or more microbeads in a first aqueous fluid;

(b) attaching a second set of primary compounds to one or more microbeads in a second aqueous fluid, wherein the second set of primary compounds is different from the first set of primary compounds;

(c) forming a plurality of first water-in-fluorocarbon oil microcapsules by partitioning the first aqueous fluid with an immiscible fluorocarbon oil comprising a fluorinated surfactant, as the first aqueous fluid is flowing through a microfluidic channel, wherein the first aqueous fluid of each first water-in-fluorocarbon oil microcapsule comprises one or more first sets of primary compounds attached to one or more microbeads;

(d) forming a plurality of second water-in-fluorocarbon oil microcapsules by partitioning the second aqueous fluid with the immiscible fluorocarbon oil comprising a fluorinated surfactant, as the second aqueous fluid is flowing through a microfluidic channel, wherein each aqueous fluid portion of the second water-in-fluororcarbon oil microcapsule comprises a second set of primary compounds attached to one or more microbeads;

(e) merging the plurality of first water-in-fluorocarbon oil microcapsules with the plurality of second water-in-fluorocarbon oil microcapsules in presence of an applied electric field to form a plurality of merged water-in-fluorocarbon oil microcapsules in the microfluidic channel, wherein the immiscible fluorocarbon oil in the plurality of first water-in-fluorocarbon oil microcapsules and the plurality of second water-in-fluorocarbon oil microcapsules comprises a fluorinated surfactant and the aqueous fluid portion of each merged water-in-fluorocarbon oil microcapsule comprises a first set of primary compounds attached to one or more microbeads and a second set of primary compounds attached to one or more microbeads;

(f) releasing, in the merged water-in-fluorocarbon oil microcapsules, the first set of primary compounds from the one or more microbeads and the second set of primary compounds from the one or more microbeads;

(g) forming secondary compounds in the merged water-in-fluorocarbon oil microcapsules by chemical reactions between the released first set of primary compounds and the released second set of primary compounds, wherein the secondary compounds are present in the aqueous fluid portion of each of the plurality of merged water-in-fluorocarbon oil microcapsules;

(h) fusing each merged water-in-fluorocarbon oil microcapsule with a third water-in-fluorocarbon oil microcapsule containing a target enzyme to form second merged water-in-fluorocarbon oil microcapsules containing the secondary compounds and the target enzyme, wherein one or more of steps (c) through (h) are performed under microfluidic control; and

(i) determining subsets of the first set of primary compounds or the second set of primary compounds which react to form secondary compounds that bind to or modulate the activity of the target enzyme.

2. The method according to claim 1, further comprising:

sorting the merged water-in-fluorocarbon oil microcapsules based upon identification of the secondary compounds that bind to or modulate the activity of the target.

3. The method of claim 1, wherein the first set of primary compounds and/or the second set of primary compounds are nucleic acids.

4. The method of claim 3, wherein each first water-in-fluorocarbon oil microcapsule comprises a single nucleic acid from each of the first set of primary compounds and the second set of primary compounds.

5. The method of claim 3, wherein said chemical reaction is a nucleic acid polymerization reaction.

6. The method of claim 1, wherein each of the first water-in-fluorocarbon oil microcapsules further comprises a cell.

7. The method of claim 6, wherein the target enzyme is associated with the cell.

8. The method of claim 7, wherein the target enzyme is in the cell.

9. The method of claim 1, further comprising merging each of the second merged water-in-fluorocarbon oil microcapsules containing the secondary compounds and the target enzyme with a fourth water-in-fluorocarbon oil microcapsule containing a fluorogenic substrate for the target enzyme.

10. The method of claim 1, wherein either the first set of primary compounds or the second set of primary compounds comprises a fluorogenic substrate for the target enzyme.