Integrated microfluidic device and methods

• US 9,132,398 B2
• Filed: 10/10/2008
• Issued: 09/15/2015
• Est. Priority Date: 10/12/2007
• Status: Active Grant

• Current Assignee: Rheonix, Inc.
• Original Assignee: Rheonix, Inc.
• Inventors: Zhou, Peng, Young, Lincoln C., Roswech, Todd, Spizz, Gwendolyn, Chen, Zongyuan, Thomas, Benjamin W., Lee, Travis
• Primary Examiner(s): HOBBS, MICHAEL L
• Application Number:
  US12/249,872
• Publication Number:
  US 20120115738A1
• Time in Patent Office: 2,531 Days
• Field of Search: 435/6.12, 435/287.2, 506/9
• US Class Current: 1/1
• CPC Class Codes: B01F 25/451 characterised by means for moving the materials to be mixed or the mixture
• CPC Class Codes: B01F 25/4521 the components being pressed through orifices in elements, e.g. flat plates or cylinders, which obstruct the whole diameter of the tube
• CPC Class Codes: B01F 31/651 Mixing by successively aspirating a part of the mixture in a conduit, e.g. a piston, and reinjecting it through the same conduit into the receptacle
• CPC Class Codes: B01F 33/30 Micromixers
• CPC Class Codes: B01L 2200/027 for microfluidic devices
• CPC Class Codes: B01L 2200/0621 Control of the sequence of chambers filled or emptied
• 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 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/1816 using induction heating
• CPC Class Codes: B01L 2300/1822 using Peltier elements
• CPC Class Codes: B01L 2300/1827 using resistive heater
• CPC Class Codes: B01L 2300/1844 using fans
• CPC Class Codes: B01L 2300/185 using a liquid as fluid
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/0638 membrane valves, flap valves
• CPC Class Codes: B01L 2400/084 Passive control of flow resistance
• 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 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

Abstract

A microfluidic device for analyzing a sample of interest is provided. The microfluidic device can comprise a microfluidic device body, wherein the microfluidic device body comprises a sample preparation area, a nucleic acid amplification area, a nucleic acid analysis area, and a network of fluid channels. Each of the sample preparation area, the nucleic acid amplification area and the nucleic acid analysis area are fluidly interconnected to at least one of the other two areas by at least one of the fluid channels. Using the microfluidic device, sample preparation can be combined with amplification of a biologically active molecule, and a suitable biological sample can be provided for analysis and/or detection of a molecule of interest. The small-scale apparatus and methods provided are easier, faster, less expensive, and equally efficacious compared to larger scale equipment for the preparation and analysis of a biological sample.

Claims

1. A microfluidic device for analyzing a sample of interest comprising:

a) a microfluidic device body, wherein the microfluidic device body comprises;

i) a sample preparation area,

ii) a non-actuated, fixed volume nucleic acid amplification area, and

iii) a plurality of fluid channels interconnected in a network,

and wherein each of the sample preparation area and the nucleic acid amplification area are fluidly interconnected to the other area by at least one of the plurality of fluid channels in the network, characterized in that the device body further comprises;

a single, non-elastomeric substrate layer having upper and lower surfaces, and having one or more microfeatures in the upper surface thereof; and

a single, non-elastomeric membrane layer contacting and joined with the upper surface of the substrate and disposed over the one or more microfeatures to form a diaphragm valve, having a relaxed state wherein the non-elastomeric membrane lies substantially against the upper surface of the substrate and an actuated state wherein the membrane is moved away from the upper surface of the substrate,

further wherein the device is a two-layer device consisting of the substrate layer and the membrane layer.

2. The microfluidic device of claim 1, further comprising:

iv)) a nucleic acid analysis area,

and wherein the nucleic acid analysis area is fluidly interconnected to at least one of the other two areas by at least one of the plurality of fluid channels in the network.

3. The microfluidic device of claim 2, comprising a differential pressure source capable of exerting a positive pressure or a negative pressure with respect to ambient pressure on a selected area of the microfluidic device body.

4. The microfluidic device of claim 3, comprising a differential pressure delivery system operably connected to the differential pressure source and to the microfluidic device body.

5. The microfluidic device of claim 3, comprising at least one diaphragm disposed in at least two of the plurality of fluid channels for transforming a pressure from the differential pressure source to a desired open or closed position.

6. The microfluidic device of claim 2, wherein the sample preparation area comprises:

a sample intake reservoir;

a reservoir for a sample preparation reagent; and

sample purification media;

wherein the sample intake reservoir, the reservoir for the sample preparation reagent, and the sample purification media are fluidly interconnected.

7. The microfluidic device of claim 6, comprising a sample purification media reservoir, wherein the sample purification media is disposed in the sample purification media reservoir.

8. The microfluidic device of claim 7, wherein the sample purification media is disposed in the bottom of the sample purification media reservoir.

9. The microfluidic device of claim 6 wherein the sample purification media is disposed in one of the plurality of fluidic channels.

10. The microfluidic device of claim 2, wherein the nucleic acid amplification area comprises:

a nucleic acid amplification reactor;

a nucleic acid amplification reagent reservoir; and

a nucleic acid amplification product reservoir;

wherein the nucleic acid amplification reactor, the nucleic acid amplification reagent reservoir, and the nucleic acid amplification product reservoir are fluidly interconnected.

11. The microfluidic device of claim 2, comprising a nucleic acid amplification products analysis area.

12. The microfluidic device of claim 11, wherein the nucleic acid amplification products analysis area comprises a nucleic acid analysis reservoir.

13. The microfluidic device of claim 6, wherein the sample purification media is a silica membrane.

14. The microfluidic device of claim 2, wherein the sample of interest is a fluid material, a gaseous material, a solid material substantially dissolved in a liquid material, an emulsion material, a slurry material, or a fluid material with particles suspended therein, and further comprising at least one of a biological material and a suspension of cells in a fluid.

15. The microfluidic device of claim 2, wherein the microfluidic device body comprises a plurality of layers of weak solvent-bonded polystyrene.

16. The microfluidic device of claim 2 wherein the sample preparation area comprises a sample mixing diaphragm fluidly connected to the sample intake reservoir.

17. The microfluidic device of claim 2, wherein the microfluidic device body comprises a means for air-drying the sample purification media.

18. The microfluidic device of claim 2, wherein the sample preparation area comprises at least one of a waste reservoir and an elution reagent reservoir.

19. The microfluidic device of claim 6, wherein the sample preparation reagent comprises magnetic beads.

20. The microfluidic device of claim 6, wherein the sample preparation reagent comprises a lysing reagent.

21. The microfluidic device of claim 10, wherein the nucleic acid amplification reactor is a thermal cycling reactor.

22. The microfluidic device of claim 21, wherein the bottom of the thermal cycling reactor is a thin layer of polystyrene.

23. The microfluidic device of claim 21, wherein the bottom of the thermal cycling reactor is heated during thermal cycling by a heater that is not disposed on or in the microfluidic device body.

24. A method for detecting a nucleic acid of interest comprising the steps of:

obtaining a sample suspected of containing the nucleic acid of interest;

providing the microfluidic device of claim 1;

introducing the sample into the sample preparation area;

preparing the sample for nucleic acid amplification;

introducing the prepared sample into the nucleic acid amplification area;

performing a nucleic acid amplification reaction in the nucleic acid amplification area to amplify the nucleic acid of interest; and

detecting the amplified nucleic acid of interest.

25. The microfluidic device of claim 2, wherein the nucleic acid analysis area comprises an area for detecting an interaction between the nucleic acid of interest and a probe for the nucleic acid of interest.

26. The method of claim 24, wherein the nucleic acid amplification is selected from the group consisting of polymerase chain reaction (PCR), reverse-transcriptase (RT-) PCR, Rapid Amplification of cDNA Ends (RACE), rolling circle amplification, nucleic Acid Sequence Based Amplification (NASBA), Transcript Mediated Amplification (TMA), and Ligase Chain Reaction.

27. The method of claim 24, wherein the detecting step comprises detecting an interaction between the amplified nucleic acid of interest and a probe for the nucleic acid of interest.

28. The method of claim 24, wherein the detecting step comprises visualizing color intensity, fluorescence intensity, electrical signal intensity or chemiluminescence intensity.

29. The method of claim 24, wherein the detecting step comprises generating an intensity value corresponding to at least one molecule of interest in the sample.

30. The method of claim 29, wherein the intensity value is selected from the group consisting of color intensity value, fluorescence intensity value and chemiluminescence intensity value, current or voltage.

31. The method of claim 30, wherein generating the color intensity value comprises:

digitizing an image corresponding to the sample to generate a plurality of pixels;

providing a plurality of numerical values for respective ones of the plurality of pixels;

and

producing numerical values to provide the color intensity value.

32. The method of claim 30, further comprising computing a threshold value and comparing the color intensity value to the threshold value to detect the molecule of interest.

33. The method of claim 24, further characterized by the step of:

introducing the amplified nucleic acid of interest into the nucleic acid analysis area.

34. A method for determining presence of or predisposition for a disease or disorder of interest in a subject comprising:

a) obtaining a sample from the subject, wherein the sample is suspected of containing a nucleic acid associated with the disease or disorder of interest; and

b) detecting the nucleic acid associated with the disease or disorder of interest in the sample, wherein the detecting comprises the steps of;

providing the microfluidic device of claim 1,

introducing the sample into the sample preparation area,

preparing the sample for nucleic acid amplification,

introducing the prepared sample into the nucleic acid amplification area,

performing a nucleic acid amplification reaction in the nucleic acid amplification area to amplify the nucleic acid of interest, and

detecting the amplified nucleic acid of interest;

wherein detecting the amplified nucleic acid of interest is associated with presence of or predisposition for the disease or disorder of interest.

35. The method of claim 34 wherein the detecting step comprises determining an amount (or level) of the amplified nucleic acid of interest and wherein the method further comprises comparing the amount (or level) with a preselected amount (or level) of the nucleic acid of interest.

36. The method of claim 35 wherein a difference between the amount (or level) with the preselected amount (or level) is indicative of presence or predisposition for the disease or disorder of interest.