Channels with cross-sectional thermal gradients

• US 10,052,632 B2
• Filed: 12/28/2015
• Issued: 08/21/2018
• Est. Priority Date: 12/30/2011
• Status: Active Grant

• Current Assignee: Abbott Molecular Incorporated (Abbott Laboratories Incorporated)
• Original Assignee: Abbott Molecular Incorporated (Abbott Laboratories Incorporated)
• Inventors: Marble, Herbert A., Laffler, Thomas
• Primary Examiner(s): Kim, Young J
• Application Number:
  US14/981,158
• Publication Number:
  US 20160318023A1
• Time in Patent Office: 967 Days
• Field of Search: None
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/0433 vibrational forces
• CPC Class Codes: B01L 2400/0445 Natural or forced convection
• CPC Class Codes: B01L 2400/086 using baffles or other fixed flow obstructions
• 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 7/525 with physical movement of samples between temperature zones
• CPC Class Codes: B01L 7/54 using spatial temperature gradients
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2527/15 Gradients
• 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

Abstract

Provided herein are systems, devices, and methods for generating thermal gradients in channels and uses thereof. In particular, provided herein are system, methods, and devices employing first and second thermal layers positioned around a channel in order to create a thermal gradient across a cross-section of the channel having, for example, a nucleic acid denaturation zone, a nucleic acid annealing zone, and a nucleic acid polymerization zone. Such devices find use in, for example, nucleic acid amplification procedures, including digital polymerase chain reaction (dPCR) to temperature cycle droplets for amplification of nucleic acid templates within the droplets.

Claims

1. A method comprising:

a) providing a device comprising;

i) a microchannel comprising a surface configured for transmission a carrier medium comprising droplets,

ii) a first thermal layer positioned along said microchannel configured to heat a portion of said microchannel surface to a nucleic acid denaturation temperature; and

iii) a second thermal layer positioned along said microchannel configured to heat or cool a portion of said microchannel surface to a nucleic acid annealing temperature;

wherein said first and said second thermal layers are positioned along opposing surface walls of said microchannel to create a thermal gradient across a cross-section of said microchannel, and wherein said thermal gradient comprises a nucleic acid denaturation zone, a nucleic acid annealing zone, and a nucleic acid polymerization zone; and

b) flowing said carrier medium through said microchannel such that said droplets repeatedly pass through said thermal gradient causing temperature cycling in said droplets, wherein said droplets each comprise primers, a nucleic acid template, a polymerase, and nucleotides, and wherein said temperature cycling causes amplification of said nucleic acid templates in said droplets.

2. The method of claim 1, further comprising sequencing the amplified templates in at least some of the droplets.

3. The method of claim 2, wherein said sequencing comprises use of reversible dye terminators.

4. The method of claim 1, wherein flowing said carrier medium through said microchannel is performed in a continuous manner, a pulsed manner, a batch manner, or fed-batch manner.

5. The method of claim 1, wherein said device further comprises a plurality of motion components positioned along said microchannel, wherein said plurality of motion components are configured to impart motion to said droplets in said carrier medium when present in said microchannel such that stochastic contact of said droplet with said microchannel surface is increased.

6. The method of claim 5, wherein said motion is imparted via mechanical agitation.

7. The method of claim 6, wherein said mechanical agitation is selected from the group consisting of:

vibration, low-frequency sonication, and acoustic waves.

8. The method of claim 5, wherein said motion is imparted via convection and/or turbulent flow.

9. The method of claim 5, wherein said motion is imparted via an electrical field.

10. The method of claim 5, wherein said droplets comprise paramagnetic microparticles, and wherein said plurality of motion components are configured to induce an oscillating magnetic field along said channel.

11. The method of claim 5, wherein said plurality of motion components are positioned intermittently along said microchannel.

12. The method of claim 1, further comprising monitoring template amplification with an optical array sensor positioned along said microchannel configured to monitor PCR signal output in said droplets.

13. The method of claim 1, wherein said optical array sensor comprises a component selected from the group consisting of:

a light-emitting diode, a photodiode array, an optical fiber bundle, a laser configured for fluorometry, a piezoelectric crystal, an interdigital cantilever, a photovoltaic cell, and a gated linear CCD array.

14. The method of claim 1, wherein said droplets comprise an emulsion or a macromolecular assembly selected from the group consisting of:

unilamellar vesicles, bilamellar vesicles, micelles, liposomes, nanostructures, macromolecular cages, carbon nanotubes, fullerenes or paramagnetic particles.

15. The method of claim 1, wherein said carrier medium is liquid carrier medium or a gas carrier medium.

16. The method of claim 1, wherein said droplets further comprise a detection reagent.

17. The method of claim 1, wherein said device further comprises a nucleic acid purification component.