Sorting, amplification, detection, and identification of nucleic acid subsequences in a complex mixture

• US 8,338,166 B2
• Filed: 01/04/2007
• Issued: 12/25/2012
• Est. Priority Date: 01/04/2007
• Status: Active Grant

• Current Assignee: Lawrence Livermore National Security LLC (Government of the United States of America)
• Original Assignee: Lawrence Livermore National Security LLC (Government of the United States of America)
• Inventors: Beer, Neil R., Hindson, Benjamin J., Fitch, Joseph P., Colson, Billy W. Jr.
• Primary Examiner(s): BOWERS, NATHAN ANDREW
• Application Number:
  US11/650,363
• Publication Number:
  US 20080166793A1
• Time in Patent Office: 2,182 Days
• Field of Search: 435/283.1, 435/288.3, 435/305.1, 435/305.2, 435/288.5, 435/288.4, 435/287.2
• US Class Current: 435/288.3
• 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 2200/12 Specific details about manufacturing devices
• 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 2300/087 Multiple sequential chambers
• CPC Class Codes: B01L 2300/0887 Laminated structure
• CPC Class Codes: B01L 2300/1822 using Peltier elements
• CPC Class Codes: B01L 2300/1827 using resistive heater
• CPC Class Codes: B01L 2300/1861 using radiation
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 3/502753 characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation separation in general B01D; microapparatus for analysis using electrophoresis G01N27/44791; sample preparation G01N1/28
• 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: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: B01L 7/525 with physical movement of samples between temperature zones
• CPC Class Codes: C12Q 1/04 Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor C12Q1/6897 takes precedence
• CPC Class Codes: C12Q 1/6888 for detection or identification of organisms
• CPC Class Codes: G01N 33/5432 : Liposomes or microcapsules
• CPC Class Codes: G01N 33/569 : for microorganisms, e.g. protozoa, bacteria, viruses
• CPC Class Codes: G01N 33/587 : Nanoparticles

Abstract

A system for identifying all of the known and unknown pathogenic or non-pathogenic organisms in a sample. A droplet generator creates droplets from the sample. The droplets constitute sub-nanoliter volume reactors containing the organism sized particles. A lysis device performs lysis of the organisms to release the nucleic acids. An amplifier amplifies the nucleic acids. A fractionater releases the nucleic acids from the droplets. A parallel analyzer identifies all of the known and unknown pathogenic or non-pathogenic organisms in the sample.

Claims

1. An apparatus for identifying the known and unknown pathogenic or non-pathogenic organisms in a sample wherein the organisms include nucleic acids, comprising:

a microfluidic flow channel,

an emulsifier source that directs an emulsifier into said microfluidic flow channel,

a droplet generator that directs the sample into said emulsifier and creates discrete droplets from the sample wherein said droplets constitute sub-nanoliter volume reactors containing the organisms;

a lysis device for performing lysis of the organisms to release the nucleic acids;

an amplifier for amplifying the nucleic acids;

a fractionater that releases the nucleic acids from said droplets; and

a parallel analyzer for identifying all of the known and unknown pathogenic or non-pathogenic organisms in the sample.

2. The apparatus for identifying all of the known and unknown pathogenic or non-pathogenic organisms in a sample of claim 1 wherein said droplet generator comprises a mechanical orifice and the sample is forced through said mechanical orifice into said emulsifier in said microfluidic flow channel creating said discrete droplets and wherein said discrete droplets have emulsified shells.

3. The apparatus for identifying all of the known and unknown pathogenic or non-pathogenic organisms in a sample of claim 1 wherein said lysis device comprises an optical window.

4. The apparatus for identifying all of the known and unknown pathogenic or non-pathogenic organisms in a sample of claim 1 wherein said lysis device comprises a laser for directing electromagnetic radiation through an optical window.

5. The apparatus for identifying all of the known and unknown pathogenic or non-pathogenic organisms in a sample of claim 1 wherein said lysis device comprises an ultrasound-generating piezoelectric actuator.

6. The apparatus for identifying all of the known and unknown pathogenic or non-pathogenic organisms in a sample of claim 1 wherein said lysis device comprises a wire-based ultrasound-generator inside the microchannel.

7. The apparatus for identifying all of the known and unknown pathogenic or non-pathogenic organisms in a sample of claim 1 wherein said lysis device comprises a resistive, conductive, or radiative heater that causes lysis through hyperthermia of the target organism.

8. The apparatus for identifying all of the known and unknown pathogenic or non-pathogenic organisms in a sample of claim 1 wherein said amplifier comprises a thermocycler.

9. The apparatus for identifying all of the known and unknown pathogenic or non-pathogenic organisms in a sample of claim 1 wherein said parallel analyzer comprises an electrophoresis system that includes capillary electrophoresis lanes.

10. The apparatus for identifying all of the known and unknown pathogenic or non-pathogenic organisms in a sample of claim 1 wherein said parallel analyzer comprises a capillary electrophoresis system.

11. The apparatus for identifying all of the known and unknown pathogenic or non-pathogenic organisms in a sample of claim 1 wherein said parallel analyzer comprises a genomic analyzer.

12. The apparatus for identifying all of the known and unknown pathogenic or non-pathogenic organisms in a sample of claim 1 wherein said parallel analyzer comprises a direct sequencer.

13. An apparatus for identifying all of the known and unknown pathogenic or non-pathogenic organisms in a sample wherein the organisms include nucleic acids, comprising:

a chip,

a microfluidic flow channel in said chip,

an emulsifier source directing an emulsifier into said microfluidic flow channel,

a droplet generator in said microfluidic flow channel that directs the sample into said emulsifier and creates discrete droplets from the sample wherein said droplets constitute sub-nanoliter volume reactors containing the organisms;

a lysis device in said microfluidic flow channel for performing lysis of the organisms to release the nucleic acids;

an amplifier in said microfluidic flow channel for amplifying the nucleic acids;

a fractionater in said microfluidic flow channel that releases the nucleic acids from said droplets; and

a parallel analyzer connected to said microfluidic flow channel for identifying all of the known and unknown pathogenic or non-pathogenic organisms in the sample.

14. The apparatus for identifying all of the known and unknown pathogenic or non-pathogenic organisms in a sample of claim 13 wherein said droplet generator comprises a mechanical orifice and the sample is forced through said mechanical orifice into said emulsifier in said microfluidic flow channel creating said discrete droplets and wherein said discrete droplets have emulsified shells.

15. The apparatus for identifying all of the known and unknown pathogenic or non-pathogenic organisms in a sample of claim 13 wherein said lysis device comprises an optical window.

16. The apparatus for identifying all of the known and unknown pathogenic or non-pathogenic organisms in a sample of claim 13 wherein said lysis device comprises a laser for directing electromagnetic radiation through an optical window.

17. The apparatus for identifying all of the known and unknown pathogenic or non-pathogenic organisms in a sample of claim 13 wherein said lysis device comprises an ultrasound-generating piezoelectric actuator.

18. The apparatus for identifying all of the known and unknown pathogenic or non-pathogenic organisms in a sample of claim 13 wherein said amplifier comprises a thermocycler.

19. The apparatus for identifying all of the known and unknown pathogenic or non-pathogenic organisms in a sample of claim 13 wherein said parallel analyzer comprises an electrophoresis system.

20. The apparatus for identifying all of the known and unknown pathogenic or non-pathogenic organisms in a sample of claim 13 wherein said parallel analyzer comprises a capillary electrophoresis system that includes capillary electrophoresis lanes.

21. The apparatus for identifying all of the known and unknown pathogenic or non-pathogenic organisms in a sample of claim 13 wherein said parallel analyzer comprises a genomic analyzer.

22. The apparatus for identifying all of the known and unknown pathogenic or non-pathogenic organisms in a sample of claim 13 wherein said parallel analyzer comprises a direct sequencer.

23. The apparatus for identifying all of the known and unknown pathogenic or non-pathogenic organisms in a sample of claim 13 including an optical detector connected to said microfluidic flow channel for analysis of the known and unknown pathogenic or non-pathogenic organisms in the sample.

24. The apparatus for identifying all of the known and unknown pathogenic or non-pathogenic organisms in a sample of claim 13 wherein said chip comprises a multiplicity of wafers produced by a wet etch in glass or borosilicate and wherein said wafers are then aligned and bonded together to produce bonded wafers.

25. The apparatus for identifying all of the known and unknown pathogenic or non-pathogenic organisms in a sample of claim 24 wherein said bonded wafers are then cut by a diamond saw to produce said chip.

26. The apparatus for identifying all of the known and unknown pathogenic or non-pathogenic organisms in a sample of claim 24 including a borosilicate or glass cover layer positioned over said microfluidic flow channel wherein said chip comprises a multiplicity of wafers produced by a photolithography process utilizing a front and backside Deep Reactive Ion Etch (DRIE) process where the front side of a Si wafer contains said microfluidic channel and the back side etch creates the fluid vias to connect to said microfluidic channel and said front side of said Si wafer is then sealed by anodic bonding of said borosilicate or glass cover layer to said front side of said Si wafer.

27. The apparatus for identifying all of the known and unknown pathogenic or non-pathogenic organisms in a sample of claim 13 wherein said chip comprises a multiplicity of wafers produced by a soft lithography process where SU-8 photoresist is patterned into a positive-relief on a channel architecture using a standard photolithography process and said patterned structure becomes a mold for the addition of liquid polydimethylsiloxane which is flowed over the SU-8 and cured.

28. The apparatus for identifying all of the known and unknown pathogenic or non-pathogenic organisms in a sample of claim 27 wherein said cured polydimethylsiloxane (PDMS) is then pulled from the mold, a glass coverslip is spin coated with a small layer of polydimethylsiloxane and cured, layers of glass plus PDMS with channels are brought together and cured such that the polydimethylsiloxane forms a complete seal around the channel geometry and fluidic ports are cored out of the polymeric polydimethylsiloxane.