Thermal reaction device and method for using the same

• US 10,131,934 B2
• Filed: 11/07/2017
• Issued: 11/20/2018
• Est. Priority Date: 04/03/2003
• Status: Active Grant

• Current Assignee: Standard Biotools Incorporated
• Original Assignee: Fluidigm Corporation
• Inventors: Unger, Marc, Manger, Ian D., Lucero, Michael, Yi, Yong, Miyashita-Lin, Emily, Wienecke, Anja, Facer, Geoffrey
• Primary Examiner(s): Kim, Young J
• Application Number:
  US15/805,720
• Publication Number:
  US 20180073051A1
• Time in Patent Office: 378 Days
• Field of Search: None
• CPC Class Codes: B01L 2200/0642 Filling fluids into wells by specific techniques
• 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 2200/142 Preventing evaporation
• CPC Class Codes: B01L 2200/147 Employing temperature sensors
• CPC Class Codes: B01L 2300/06 Auxiliary integrated devices, integrated components
• 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 2300/0819 Microarrays; Biochips
• 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 2300/0874 Three dimensional network
• CPC Class Codes: B01L 2300/123 Flexible; Elastomeric
• CPC Class Codes: B01L 2300/1805 Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
• CPC Class Codes: B01L 2400/0481 squeezing of channels or chambers
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/06 Valves, specific forms thereof
• CPC Class Codes: B01L 2400/0638 membrane valves, flap valves
• CPC Class Codes: B01L 2400/0655 pinch valves
• CPC Class Codes: B01L 3/0248 Prongs, quill pen type dispenser
• CPC Class Codes: B01L 3/5025 : for parallel transport of multiple samples
• CPC Class Codes: B01L 3/5027 : by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: B01L 3/502707 : characterised by the manufacture of the container or its components by shaping or joining plastic parts B29C59/00, B29C65/00; by laminating B32B37/00; manufacture of microstructural devices in general B81C
• CPC Class Codes: B01L 3/502715 : characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/50273 : characterised by the means or forces applied to move the fluids micropumps F04B19/006, of the membrane type F04B43/043
• CPC Class Codes: B01L 3/502738 : characterised by integrated valves microvalves F16K99/0001
• 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 1/68 : involving nucleic acids
• CPC Class Codes: C12Q 1/686 : Polymerase chain reaction [PCR]
• CPC Class Codes: G01N 27/453 : Cells therefor

Abstract

A method for carrying out nucleic acid amplification reactions using a microfluidic device is described. Amplification primers and other amplification reagents are deposited at a plurality of reaction sites in the device, a sample solution containing amplifiable polynucleotides is introduced into the reaction sites, and amplification is carried out.

Claims

1. A method for carrying out nucleic acid amplification reactions to detect the quantity, presence, or absence of a plurality of target polynucleotide sequences in a sample, the method comprising:

(a) providing a microfluidic device comprising

(i) an inlet,

(ii) a first flow channel in fluid communication with the inlet, and

(iii) a plurality of second channels in fluid communication with the first flow channel, wherein each second channel comprises an entrance and a dead end,

wherein amplification primers, and optionally other amplification reagents, are non-covalently deposited at reaction sites at the dead ends of each second channel, and amplification primers deposited in different second channels amplify different target polynucleotide sequences,

(b) introducing a sample solution comprising the sample through the inlet such that the sample solution flows through the first flow channel and into the plurality of second channels, wherein the sample comprises a plurality of polynucleotides, and wherein introduction of the sample solution into the second channels causes the deposited amplification primers to be dissolved into the sample solution, and

(c) isolating the reaction sites from the first flow channel and from each other,

(d) conducting polynucleotide amplification reactions in the reaction sites, and

(e) detecting the quantity, presence or absence of polynucleotide amplification products in the reaction sites to determine the quantity, presence, or absence of said target polynucleotide sequences in the sample, wherein in Step (c) an elastomeric membrane is deflected into the plurality of second channels to block flow from said reaction sites to said first channel, thereby isolating the reaction sites from the first flow channel and from each other.

2. The method of claim 1, wherein the microfluidic device comprises at least 100 reaction sites.

3. The method of claim 2, wherein the microfluidic device comprises 100 to 1,000 reaction sites.

4. The method of claim 1, wherein said target polynucleotide sequences comprise viral or bacterial sequences.

5. The method of claim 1, wherein the target polynucleotide sequences are cDNA sequences.

6. The method of claim 1, wherein the quantity, presence or absence of polynucleotide amplification products is detected using a DNA intercalation dye.

7. The method of claim 1 wherein primers suitable for multiplex amplifications are deposited in at least one of said reaction sites.

8. The method of claim 1, wherein the polynucleotide amplification reactions are polymerase chain reactions (PCR).

9. The method of claim 8, wherein the polynucleotide amplification reactions are real time quantitative polymerase chain reactions.

10. A method for carrying out nucleic acid amplification reactions to detect the presence or absence of a plurality of different target viral or bacterial polynucleotide sequences in a biological sample obtained from a human patient, the method comprising:

(a) providing a microfluidic device comprising

(i) an inlet,

(ii) a first flow channel in fluid communication with the inlet, and

(iii) a plurality of second channels in fluid communication with the first flow channel, wherein each second channel comprises an entrance and a dead end,

wherein amplification primers, and optionally other amplification reagents, are non-covalently deposited at a reaction site at the dead-ends of each second channel, and amplification primers deposited in different second channels amplify different target polynucleotide sequences; and

wherein the different target polynucleotide sequences are viral or bacterial sequences,

(b) introducing a sample solution comprising the sample through the inlet such that the sample solution flows through the first flow channel and into the plurality of second channels, wherein the sample comprises a plurality of polynucleotides, and wherein introduction of the sample solution into the second channels causes the deposited amplification primers to be dissolved into the sample solution,

(c) isolating the reaction sites from the first flow channel and from each other,

(d) conducting polynucleotide amplification reactions in the reaction sites, and

(e) detecting the presence or absence of a polynucleotide amplification products in the reaction sites to determine the presence or absence of a viral or bacterial target polynucleotide sequence in the biological sample obtained from the human patient, wherein in Step (c) an elastomeric membrane is deflected into the plurality of second channels to block flow from said reaction sites to said first channel, thereby isolating the reaction sites from the first flow channel and from each other.