Mixed-phase fluids for nucleic acid sequencing and other analytical assays

• US 10,710,076 B2
• Filed: 12/02/2019
• Issued: 07/14/2020
• Est. Priority Date: 12/04/2018
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Omniome, Inc.
• Inventors: Oliphant, Arnold, Nemiroski, Alex, Alberni, Julian Sean, Erickstad, Michael John, McGinley, Rebecca, Fleischer, Chad, Villarreal, Eric
• Primary Examiner(s): Bhat, Narayan K
• Application Number:
  US16/700,422
• Publication Number:
  US 20200171498A1
• Time in Patent Office: 225 Days
• CPC Class Codes: B01L 2200/16 Reagents, handling or storing thereof
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0819 Microarrays; Biochips
• CPC Class Codes: B01L 2300/0883 Serpentine channels
• CPC Class Codes: B01L 2400/0403 specific forces
• 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 3/502776 specially adapted for focusing or laminating flows
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• 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 2565/629 being a microfluidic device

Abstract

An analytical system that includes a flow cell, a liquid delivery component, a gas delivery component and a bubble generator component, wherein the liquid delivery component is configured to deliver liquid from one or more reservoirs to the bubble generator component, wherein the gas delivery component is configured to deliver gas from one or more source to the bubble generator component, and wherein the bubble generator component is configured to mix liquids from the liquid delivery component with gas from the gas delivery component to deliver a fluid foam to the inside of the flow cell, wherein the fluid foam includes bubbles of the gas in the liquid.

Claims

1. A method for sequencing a nucleic acid, comprising

(a) providing a sequencing system comprising

(i) a flow cell comprising a nucleic acid immobilized therein,

(ii) a bubble generator component that delivers gas bubbles to a liquid at a predefined rate, wherein the bubble generator component comprises a filter membrane located at a junction through which the gas is delivered to the liquid;

(b) delivering a series of fluids to the inside of the flow cell to perform a cycle of a sequencing process, wherein at least one of the fluids is a fluid foam produced by the bubble generator component comprising gas bubbles in the liquid; and

(c) repeating step (b), thereby determining the sequence for the nucleic acid.

2. The method of claim 1, wherein the nucleic acid is immobilized on a surface inside the flow cell.

3. The method of claim 2, wherein the nucleic acid is present at a site in an array of nucleic acids immobilized on the surface.

4. The sequencing system of claim 3, wherein the sequencing process comprises optically resolving the site from other sites in the array.

5. The method of claim 1, wherein at least one of the fluids that is delivered to the flow cell is fluid foam containing a reversibly terminated nucleotide.

6. The method of claim 5, wherein at least one of the fluids that is delivered to the flow cell is fluid foam containing a deblocking reagent.

7. The method of claim 1, wherein at least one of the fluids that is delivered to the flow cell is fluid foam containing a fluorescently labeled nucleotide.

8. The method of claim 7, wherein the fluorescently labeled nucleotide comprises a reversible terminator moiety.

9. The method of claim 1, wherein at least one of the fluids that is delivered to the flow cell is fluid foam containing a polymerase.

10. The method of claim 9, wherein the polymerase forms a stabilized ternary complex with a fluorescently labeled nucleotide and the nucleic acid in fluid foam.

11. The method of claim 10, wherein the sequencing process comprises optical detection of the stabilized ternary complex.

12. The method of claim 9, wherein the polymerase covalently adds a fluorescently labeled nucleotide to the nucleic acid in fluid foam.

13. The method of claim 12, wherein the sequencing process comprises optical detection of the fluorescently labeled nucleotide added to the nucleic acid.

14. The method of claim 1, wherein the optical detection comprises irradiating the inside of the flow cell and collecting luminescence emission from the inside of the flow cell.

15. The method of claim 1, wherein the bubbles are substantially devoid of oxygen gas.

16. The method of claim 1, wherein the filter membrane comprises a hydrophobic material having a pattern of holes therein.

17. The method of claim 1, wherein at least one of the fluids that is delivered to the flow cell is substantially devoid of bubbles.

18. The method of claim 17, wherein the sequencing process comprises optical detection of the nucleic acid in the fluid that is devoid of bubbles.

19. The method of claim 1, wherein the delivering comprises delivering at least a portion of the fluid foam into the flow cell, out of the flow cell and back into the flow cell.

20. The method of claim 1, wherein the fluid foam comprises a volume fraction of bubbles that is at least 25% of the total volume of the fluid foam.

21. The method of claim 20, wherein the average diameter of the bubbles is at most 95%, of the diameter of the inside of the flow cell.

22. The method of claim 1, wherein the cross-sectional area of the inside of the flow cell is between 10 μ

m² and 100 mm².

23. The method of claim 1, wherein the volume of the inside of the flow cell is between 1 μ

l and 10 ml.

24. The method of claim 1, wherein the bubbles in the fluid foam have an average diameter that is between 500 nm and 500 μ

m.