Methods and systems for detecting biological components

• US 10,161,007 B2
• Filed: 08/12/2013
• Issued: 12/25/2018
• Est. Priority Date: 08/13/2012
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Abate, Adam R., Eastbum, Dennis Jay, Sciambi, Adam R.
• Primary Examiner(s): Chunduru, Suryaprabha
• Application Number:
  US14/420,646
• Publication Number:
  US 20150232942A1
• Time in Patent Office: 1,961 Days
• Field of Search: None
• CPC Class Codes: B01F 23/41 Emulsifying
• CPC Class Codes: B01F 25/4335 Mixers with a converging-diverging 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 33/3031 using electro-hydrodynamic [EHD] or electro-kinetic [EKI] phenomena to mix or move the fluids
• CPC Class Codes: B01L 2200/0652 Sorting or classification of particles or molecules
• 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 2300/0883 Serpentine channels
• CPC Class Codes: B01L 2300/1822 using Peltier elements
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 3/502784 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: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• 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 2565/629 : being a microfluidic device
• CPC Class Codes: C12Q 2600/118 : Prognosis of disease development
• CPC Class Codes: C12Q 2600/158 : Expression markers
• CPC Class Codes: C12Q 2600/16 : Primer sets for multiplex assays

Abstract

Methods for the detection of components from biological samples are provided. In certain aspects, the methods may be used to detect and/or quantify specific components in a biological sample, such as tumor cells (e.g., circulating tumor cells). Systems and devices for practicing the subject methods are also provided.

Claims

1. A method of amplifying a target polynucleotide, the method comprising:

(a) contacting on a microfluidic device a biological sample comprising a cell containing a target polynucleotide with lysis reagents in a microdroplet, the lysis reagents comprising an enzyme having protease activity, wherein the microdroplet is encapsulated in an immiscible carrier fluid;

(b) incubating the microdroplet containing the sample and the lysis reagents comprising the enzyme having protease activity for a period of time sufficient to allow the cell to lyse and form a cell lysate and the protease to digest inhibitory proteins;

(c) incubating the microdroplet containing the cell lysate and the enzyme having the protease activity at a temperature sufficient to inactivate the enzyme having protease activity;

(d) adding to the microdroplet containing the cell lysate, amplification reagents to form a microdroplet containing the cell lysate and the amplification reagents in the immiscible carrier fluid; and

(e) amplifying the target polynucleotide within the microdroplet containing the cell lysate and the amplification reagents;

wherein the method does not comprise a step of selectively removing reagents from the microdroplet containing the cell lysate and amplification reagents prior to step (e).

2. The method of claim 1, wherein the enzyme is a protease.

3. The method of claim 1, wherein the enzyme is proteinase K.

4. The method of claim 1, wherein the microdroplet containing the cell lysate and the amplification reagents has a volume of 0.001 to 1000 picoliters.

5. The method of claim 1, wherein the microdroplet containing the cell lysate and the amplification reagents has a diameter of between 0.1 microns and 1000 microns.

6. The method of claim 1, wherein the microdroplet containing the sample contains a single cell.

7. The method of claim 1, wherein the step (d) comprises (i) contacting the microdroplet containing the cell lysate with a continuous stream of fluid comprising the amplification reagents, and (ii) forming the microdroplet containing the cell lysate and the amplification reagents from a portion of the continuous stream of fluid and the microdroplet containing the cell lysate.

8. The method of claim 1, wherein the amplification comprises reverse transcription to produce a reverse transcription product.

9. The method of claim 1, wherein the amplification comprises polymerase chain reaction.

10. The method of claim 1, further comprising detecting an amplification product of the target polynucleotide.

11. The method of claim 10, wherein detecting the amplification product of the target polynucleotide comprises determining a sequence of the amplification product.

12. The method of claim 10, wherein detecting the amplification product of the target polynucleotide comprises forming a double-emulsion comprising the microdroplet containing amplification product and sorting the double-emulsion based on at least one of droplet size and fluorescence.

13. The method of claim 10, further comprising the step of sorting the microdroplet based on results of detecting the amplification product of the target polynucleotide.

14. The method of claim 10, further comprising determining a number of microdroplets containing the target polynucleotide based on results of detecting the amplification product of the target polynucleotide.

15. The method of claim 10, further comprising determining percentage of microdroplets containing the target polynucleotide based on results of detecting the amplification product of the target polynucleotide.

16. The method of claim 10, wherein amplifying the target polynucleotide comprises extension of a primer comprising a capture sequence and detecting the target polynucleotide comprises detecting the capture sequence in the amplification product.

17. The method of claim 16, wherein detecting presence of the capture sequence comprises introducing a fluorescent bead into the microdroplet containing the amplification product, wherein the fluorescent bead comprises a nucleotide sequence complementary to the capture sequence.

18. The method of claim 1, wherein at least one of the steps (d) and (e) are performed under microfluidic control.

19. The method of claim 1, wherein step (d) is performed under microfluidic control.

20. The method of claim 1, wherein the step (d) comprises droplet coalescence.

21. The method of claim 1, wherein the step (d) comprises picoinjection.

22. The method of claim 1, further comprising sorting prior to lysing the sample containing target polynucleotide.

23. The method of claim 1, further comprising sorting between step (a) and step (d).