Microfluidic device with reaction sites configured for blind filling

• US 9,643,178 B2
• Filed: 04/04/2012
• Issued: 05/09/2017
• Est. Priority Date: 11/30/2001
• 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): Shibuya, Mark
• Assistant Examiner(s): MOERSCHELL, RICHARD P
• Application Number:
  US13/439,364
• Publication Number:
  US 20130065773A1
• Time in Patent Office: 1,861 Days
• Field of Search: None
• CPC Class Codes: B01F 33/30 Micromixers
• CPC Class Codes: B01J 2219/00337 Valves
• CPC Class Codes: B01J 2219/00353 Pumps
• CPC Class Codes: B01J 2219/00403 in multiple arrangements
• CPC Class Codes: B01J 2219/00495 Means for heating or cooling the reaction vessels
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/0072 Organic compounds
• CPC Class Codes: B01L 2300/0627 Sensor or part of a sensor is integrated
• CPC Class Codes: B01L 2300/123 Flexible; Elastomeric
• CPC Class Codes: B01L 2400/06 Valves, specific forms thereof
• 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 3/50851 specially adapted for heating or cooling samples laboratory heating apparatus B01L7/00; incubators C12M
• CPC Class Codes: C12M 1/38 Temperature-responsive control
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C40B 60/14 for creating libraries
• CPC Class Codes: F16K 11/20 operated by separate actuating members with one plug turning in another F16K11/12
• CPC Class Codes: G01N 2035/00366 : Several different temperatures used
• CPC Class Codes: G01N 35/00029 : provided with flat sample substrates, e.g. slides G01N35/028 takes precedence
• CPC Class Codes: G01N 35/0098 : involving analyte bound to insoluble magnetic carrier, e.g. using magnetic separation magnetic particles used in immunoassays G01N33/54326; magnetic separation in general B03C
• CPC Class Codes: Y10T 137/2076 : Utilizing diverse fluids
• CPC Class Codes: Y10T 137/2496 : Self-proportioning or correlating systems
• CPC Class Codes: Y10T 137/86292 : System with plural openings, one a gas vent or access opening
• CPC Class Codes: Y10T 137/87249 : Multiple inlet with multiple outlet
• CPC Class Codes: Y10T 436/2575 : Volumetric liquid transfer

Abstract

A variety of elastomeric-based microfluidic devices and methods for using and manufacturing such devices are provided. Certain of the devices have arrays of reaction sites to facilitate high throughput analyzes. Some devices also include reaction sites located at the end of blind channels at which reagents have been previously deposited during manufacture. The reagents become suspended once sample is introduced into the reaction site. The devices can be utilized with a variety of heating devices and thus can be used in a variety of analyzes requiring temperature control, including thermocycling applications such as nucleic acid amplification reactions, genotyping and gene expression analyzes.

Claims

1. A microfluidic device formed in an elastomeric material, comprising:

(a) an inlet that receives a liquid sample into the device;

(b) an input flow channel or an interconnected network of such channels connected to the inlet;

(c) a plurality of blind flow channels each having only a single connection through which to introduce a fluid and otherwise surrounded by the elastomeric material, the single connection being a connection from the input flow channel or network; and

(d) a valve associated with each of a plurality of the blind flow channels in such a manner that when the valve closes, a portion of the blind flow channel is fluidically isolated from the input flow channel, thereby forming an isolated reaction site;

wherein the blind flow channels are formed in an elastomeric material that is sufficiently porous such that when a liquid sample is introduced through the inlet into the input flow channel or network, it flows into the blind flow channels while air passes out through the elastomeric material.

2. The microfluidic device of claim 1, comprising an array of reaction sites having a density of at least 100 sites/cm².

3. The microfluidic device of claim 2, comprising an array of reaction sites having a density of at least 1000 sites/cm².

16. The microfluidic device of claim 1, wherein the elastomeric material is a polydimethylsiloxane elastomer.

17. The microfluidic device of claim 1, wherein a plurality of the blind flow channels comprise a terminal compartment and a branch channel connecting the terminal compartment to the input flow channel or network, wherein the terminal compartment is wider than the branch channel.

18. The microfluidic device of claim 1, wherein the blind flow channels are connected to a plurality of interconnected parallel input flow channels.

19. The microfluidic device of claim 18, wherein a plurality of blind flow channels connected to one of the parallel input flow channels interleave with a plurality of blind flow channels connected to an adjacent parallel input flow channel.

20. The microfluidic device of claim 1, comprising a plurality of sample inlets connected to separate input flow channels or networks thereof, each of which branches into a plurality of blind flow channels.

21. The microfluidic device of claim 1, comprising guard channels that overlay the blind flow channels such that when water is flowed through the guard channels during heating of the device, evaporation of water from the blind flow channels is reduced.

22. The microfluidic device of claim 1, wherein reaction sites in a plurality of the blind flow channels each comprises a reagent composition that contains components that will react with an analyte if present in a sample that is combined with the composition.

23. The microfluidic device of claim 21, wherein different reagent compositions are present in different blind flow channels.

32. A method of analyzing nucleic acid in a sample fluid comprising the steps of:

(i) providing a microfluidic device according to claim 1;

(ii) introducing the sample fluid into the inlet so as to enter a plurality of the blind flow channels;

(iii) closing the valves so as to isolate the reaction sites from each other;

(iv) amplifying the nucleic acid in amplification reactions in at least some of the isolated reaction sites; and

(v) detecting the amplification reactions.

4. A microfluidic device comprising:

(a) an input flow channel or an interconnected network of such channels that receives a liquid sample into the device;

(b) a plurality of blind flow channels each having only a single connection through which to introduce a fluid, the single connection being a connection from the input flow channel or network;

wherein the blind flow channels have at least one wall made from a material that is sufficiently porous such that when a liquid sample is introduced into the input flow channel or network, it flows into the blind flow channels while air passes out through the permeable material; and

(c) a valve controlling flow through each of a plurality of the blind flow channels, wherein each valve is situated so that when the valve closes, a portion of the respective blind flow channel is fluidically isolated from the input flow channel, thereby forming an isolated reaction site;

wherein a plurality of the valves is coordinately controlled with a control line or an interconnected network of control lines that can be actuated by way of a single port or inlet to close all the valves at the same time when sufficient pressure is applied.

5. The microfluidic device of claim 4, that comprises two or more interconnected input flow channels that are substantially parallel to each other.

6. A method of analyzing nucleic acid in a sample fluid comprising the steps of:

(i) providing a microfluidic device according to claim 4;

(ii) introducing the sample fluid into the input flow channel so as to fill the r blind flow channels;

(iii) closing the valves so as to isolate the reaction sites from each other;

(iv) amplifying the nucleic acid in amplification reactions in at least some of the isolated reaction sites;

(v) detecting the amplification reactions.

7. The method of claim 6, wherein the nucleic acid is amplified by a polymerase chain reaction (PCR).

8. The method of claim 6, which is a method of single nucleotide polymorphism analysis.

9. The method of claim 6, which is a method of genotyping.

10. The method of claim 7, wherein the PCR is real-time PCR.

11. The method of claim 6, wherein step (iv) is conducted in an array of reaction sites having a density of at least 100 sites/cm².

12. The method of claim 6, wherein step (iv) is conducted in an array of reaction sites having a density of at least 1000 sites/cm².

13. The method of claim 6, wherein the amplifying is achieved by thermocycling.

14. The microfluidic device of claim 4, wherein the plurality of valves is coordinately controlled by a control line or an interconnected network of such lines that overlays the blind flow channels so that actuation of the line or network via a single port or inlet closes all the valves at the same time.

15. The microfluidic device of claim 4, wherein the permeable material is an elastomeric material.

24. A microfluidic device comprising:

(a) an input flow channel or an interconnected network of such channels that receives a liquid sample into the device;

(b) a plurality of blind flow channels each having only a single connection through which to introduce a fluid, the single connection being a connection from the input flow channel or network;

wherein the blind flow channels are formed in a material that is permeable to air but not liquid, such that when a liquid sample is introduced into the input flow channel or network, it flows into the blind flow channels while air passes out through the permeable material; and

(c) different reagent compositions deposited on one side of each of a plurality of the blind flow channels, the reagent compositions each containing a component that will react with an analyte if the analyte is present in a sample introduced into the respective blind flow channel,

wherein the compositions are incorporated during manufacture of the device by depositing them onto an elastomeric layer, and then surrounding each deposit with additional elastomeric layers to form one of the blind flow channels.

25. The microfluidic device of claim 24, wherein flow through a plurality of the blind flow channels is coordinately controlled by a single control line or an interconnected network of such lines such that the blind flow channels are necessarily closed and isolated at the same time.

26. The microfluidic device of claim 24, wherein the permeable material is an elastomeric material.

27. The microfluidic device of claim 24, wherein the reagent composition in each of a plurality of the blind flow channels is dry and spotted in the channel so as to dissolve when a sample is introduced into the channel.

28. The microfluidic device of claim 24, wherein the reagent composition in each of a plurality of the blind flow channels is covalently linked to a channel wall.

29. The microfluidic device of claim 24, wherein contacting at least some of the reagent compositions with a sample comprising DNA under thermocycling conditions initiates a polymerase chain reaction.

30. The microfluidic device of claim 24, wherein the reagent compositions in each of a plurality of the blind flow channels contain an oligonucleotide primer.

31. The microfluidic device of claim 24, wherein different primers selected to amplify different regions of a target are present in compositions deposited at each of a plurality of the blind flow channels.

33. A method of analyzing nucleic acid in a sample fluid comprising the steps of:

(i) providing a microfluidic device according to claim 24;

(ii) introducing the sample fluid into the input flow channel so as to contact the reagent compositions in a plurality of the blind flow channels;

(iv) thermocycling the device so as to promote amplification reactions where the sample fluid has combined with the reagent compositions; and

(v) detecting the amplification reactions.

34. A method of preparing a device according to claim 24, comprising forming spots of different reagent compositions on the surface of a layer of the device that is exposed during manufacture;

and then forming subsequent layers of the device on top of the exposed layer such that the spots are positioned within separate reaction sites.

35. The method of claim 34, wherein the spots contain different oligonucleotide primers or probes.