Thermal reaction device and method for using the same

• US 7,749,737 B2
• Filed: 10/30/2007
• Issued: 07/06/2010
• Est. Priority Date: 04/03/2003
• Status: Active Grant

• Current Assignee: Standard Biotools Incorporated
• Original Assignee: Fluidigm Corporation
• Inventors: Unger, Marc, Facer, Geoffrey, Nassef, Hany Ramez, Lucero, Michael, McBride, Lincoln
• Primary Examiner(s): Kim; Young J
• Application Number:
  US11/929,436
• Publication Number:
  US 20090061428A1
• Time in Patent Office: 980 Days
• Field of Search: None
• US Class Current: 435/91.2
• 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

An M times.N matrix microfluidic device for performing a matrix of reactions, the device having a plurality of reaction cells in communication with one of either a sample inlet or a reagent inlet through a via formed within an elastomeric block of the device. Methods provided include a method for forming vias in parallel in an elastomeric layer of an elastomeric block of a microfluidic device, the method comprising using patterned photoresist masks and etching reagents to etch away regions or portions of an elastomeric layer of the elastomeric block.

Claims

1. A method of analyzing a sample fluid comprising at least one nucleic acid molecule comprising a target sequence, and reagents for amplifying said target sequence, said method comprising the steps of:

i) providing a device having a microfluidic fluid channel therein, said fluid channel having a plurality of valves associated therewith which when activated partition the fluid channel into a plurality of fluidically isolated reaction sites, wherein said fluidically isolated reaction sites are not located at junctions formed by the fluid channel and other channels that are in fluid communication with the fluid channel;

ii) introducing the sample fluid into the fluid channel, wherein the sample fluid contains the target sequence in a concentration such that upon closing the valves to partition the fluid channel into a plurality of fluidically isolated reaction sites, thereby partitioning the sample fluid into a plurality of fluid portions, at least one fluid portion contains the target sequence and at least one fluid portion does not contain the target sequence;

iii) closing the valves to partition the sample fluid into the said fluid portions;

iv) amplifying the target sequence in said fluidically isolated reaction site(s) in which it is present; and

v) determining the number of said fluidically isolated reaction site(s) that contain products of said amplification of said target sequence.

2. A method of claim 1 wherein the amplification is achieved by PCR or by an isothermal amplification reaction.

3. A method of claim 1 wherein the analysis of the sample fluid comprises single nucleotide polymorphism (SNP) analysis.

4. A method of claim 1 wherein the analysis of the sample fluid comprises genotyping analysis.

5. A method of claim 1 wherein the sample fluid comprises at least one nucleic acid molecule comprising a first target sequence and at least one nucleic acid molecule comprising a second target sequence, wherein both the first and second target sequences are amplified, and wherein the products of said amplification of the first and second target sequences are distinguishable.

6. A method of claim 1 wherein determining the number of said fluidically isolated reaction site(s) that contain products of said amplification comprises detecting signal from a dual-labeled fluorogenic oligonucleotide probe in said fluidically isolated reaction site(s).

7. A method of claim 1 wherein the nucleic acid molecule comprising the target sequence is from genomic DNA.

8. A method of claim 7 wherein the nucleic acid molecule is DNA released into a subject'"'"'s blood stream.