Selection of compartmentalized screening method

• US 9,273,308 B2
• Filed: 09/27/2012
• Issued: 03/01/2016
• Est. Priority Date: 05/11/2006
• Status: Active Grant

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Raindance Technologies Incorporated (Bio-Rad Laboratories Incorporated)
• Inventors: Link, Darren R., Weiner, Michael, Marran, David, Rothberg, Jonathan M.
• Primary Examiner(s): Kwak, Dean
• Application Number:
  US13/629,227
• Publication Number:
  US 20130090248A1
• Time in Patent Office: 1,251 Days
• Field of Search: 422/50, 422/98, 422/82.08, 422500-505, 422/507, 422/508, 422/514, 422/515, 422/516, 436/180
• US Class Current: 1/1
• CPC Class Codes: B01F 23/41 Emulsifying
• 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: 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: B01L 2200/027 for microfluidic devices
• CPC Class Codes: B01L 2200/0636 Focussing flows, e.g. to laminate flows
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2200/0673 Handling of plugs of fluid surrounded by immiscible fluid
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0861 Configuration of multiple channels and/or chambers in a single devices
• 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 2300/165 Specific details about hydrophobic, oleophobic surfaces
• 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 2400/086 using baffles or other fixed flow obstructions
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• 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 3/502784 : 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 3/565 : Seals in general F16L
• CPC Class Codes: B01L 7/525 : with physical movement of samples between temperature zones
• CPC Class Codes: B01L 9/527 : for microfluidic devices, e.g. used for lab-on-a-chip
• CPC Class Codes: B03C 5/005 : Dielectrophoresis, i.e. dielectric particles migrating towards the region of highest field strength
• CPC Class Codes: B03C 5/026 : using open-gradient differential dielectric separation, i.e. using electrodes of special shapes for non-uniform field creation, e.g. Fluid Integrated Circuit [FIC]
• CPC Class Codes: C12N 15/1068 : Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
• CPC Class Codes: C12N 15/1086 : Preparation or screening of expression libraries, e.g. reporter assays
• CPC Class Codes: C12Q 1/00 : Measuring or testing processes involving enzymes, nucleic acids or microorganisms measuring or testing apparatus with condition measuring or sensing means, e.g. colony counters, C12M1/34; Compositions therefor; Processes of preparing such compositions
• CPC Class Codes: C12Q 1/6806 : Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6837 : using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6844 : Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6846 : Common amplification features
• CPC Class Codes: C12Q 1/6855 : Ligating adaptors
• 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 2525/191 : incorporating an adaptor
• CPC Class Codes: C12Q 2531/113 : PCR
• CPC Class Codes: C12Q 2535/101 : Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2535/122 : Massive parallel sequencing
• CPC Class Codes: C12Q 2561/101 : Taqman
• CPC Class Codes: C12Q 2563/149 : Particles, e.g. beads
• 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 2565/518 : characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C12Q 2565/601 : being a microscope, e.g. atomic force microscopy [AFM]
• CPC Class Codes: C12Q 2565/628 : being a surface plasmon resonance spectrometer
• CPC Class Codes: C12Q 2565/629 : being a microfluidic device
• CPC Class Codes: G01N 15/147 : the analysis being performed on a sample stream
• CPC Class Codes: G01N 2021/0346 : Capillary cells; Microcells
• CPC Class Codes: G01N 2035/00237 : Handling microquantities of analyte, e.g. microvalves, capillary networks
• CPC Class Codes: G01N 2035/00326 : Analysers with modular structure
• CPC Class Codes: G01N 21/05 : Flow-through cuvettes G01N21/09 takes precedence; handling fluid samples G01N1/10
• CPC Class Codes: G01N 21/64 : Fluorescence; Phosphorescence
• CPC Class Codes: G01N 2201/024 : Modular construction
• CPC Class Codes: G01N 27/3275 : Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
• CPC Class Codes: G01N 35/08 : using a stream of discrete samples flowing along a tube system, e.g. flow injection analysis
• CPC Class Codes: Y10T 137/87571 : Multiple inlet with single outlet
• CPC Class Codes: Y10T 137/87619 : With selectively operated flow control means in inlet
• CPC Class Codes: Y10T 137/87652 : With means to promote mixing or combining of plural fluids

Abstract

The present invention provides novel microfluidic substrates and methods that are useful for performing biological, chemical and diagnostic assays. The substrates can include a plurality of electrically addressable, channel bearing fluidic modules integrally arranged such that a continuous channel is provided for flow of immiscible fluids.

Claims

1. A method for analyzing an amplification reaction product, the method comprising:

providing a plurality of droplets separated by an oil in a reservoir that is external to a microfluidic device, wherein a plurality of the droplets comprise nucleic acid molecules and reagents in an aqueous fluid for a nucleic acid amplification reaction;

conducting the nucleic acid amplification reaction simultaneously in the droplets while the droplets are in the reservoir;

subsequent to the conducting step, moving the droplets from the reservoir into a channel of the microfluidic device; and

flowing the droplets in the microfluidic device past an optical detector, wherein an optical signal in at least one of the droplets is detected, the optical signal indicating presence of a nucleic acid amplification product in at least one of the at least one droplets.

2. The method accordingly to claim 1, wherein the droplets and the immiscible fluid have different densities.

3. The method according to claim 2, wherein the density differences assist droplet collection by separating at least a portion of the immiscible fluid from the droplets.

4. The method according to claim 1, wherein the droplets are monodisperse within the channel.

5. The method according to claim 1, wherein the oil is a fluorinated oil.

6. The method according to claim 1, wherein the oil comprises a surfactant.

7. The method according to claim 6, wherein the surfactant is a fluorosurfactant.

8. The method according to claim 1, wherein the amplification reaction is a polymerase chain reaction.

9. The method according to claim 1, wherein after detecting, the method further comprising sorting the droplets.

10. The method according to claim 9, further comprising collecting at least one of the sorted droplets.

11. The method according to claim 1, wherein the optical detector comprises a laser.

12. The method according to claim 1, wherein the moving step further comprises spacing the droplets.

13. The method according to claim 12, wherein spacing comprises adding an immiscible fluid to the channel so as to increase spacing between droplets prior to the droplets moving past the detector.

14. The method according to claim 1, wherein the droplets pass through the channel and are detected by the detector one at a time.

15. The method according to claim 14, wherein the width of the channel is arranged to position the droplets so that they are detected by the detector one at a time.

16. The method according to claim 1, wherein the at least one droplet is a merged, fused, or coalesced droplet.

17. The method according to claim 1, further comprising the step of merging a reagent into the droplets prior to detecting the reaction product.

18. The method according to claim 1, wherein the reservoir is in fluid communication with the channel.

19. The method according to claim 1, wherein said moving step comprises aspirating.

20. The method according to claim 1, wherein said providing step comprises aspirating.

21. The method of claim 1, wherein the reservoir is a syringe, cartridge, vial, tube, or well plate.

22. The method of claim 1, wherein the reservoir is a collection module.

23. The method of claim 22, wherein the collection module is on a microfabricated substrate.