Microfluidic flow cell

• US 8,277,759 B2
• Filed: 03/03/2009
• Issued: 10/02/2012
• Est. Priority Date: 03/04/2008
• Status: Expired due to Fees

• Current Assignee: University of Utah Research Foundation (State of Utah)
• Original Assignee: University of Utah Research Foundation (State of Utah)
• Inventors: Sundberg, Scott O., Wittner, Carl T., Gale, Bruce K.
• Primary Examiner(s): Bullock, In Suk
• Assistant Examiner(s): Kingan, Timothy G
• Application Number:
  US12/920,577
• Publication Number:
  US 20110104688A1
• Time in Patent Office: 1,309 Days
• Field of Search: None
• US Class Current: 422/503
• CPC Class Codes: B01L 2200/0642 Filling fluids into wells by specific techniques
• CPC Class Codes: B01L 2300/0806 Standardised forms, e.g. compact disc [CD] format
• CPC Class Codes: B01L 2300/0887 Laminated structure
• CPC Class Codes: B01L 2400/0409 centrifugal forces
• 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 3/50851 specially adapted for heating or cooling samples laboratory heating apparatus B01L7/00; incubators C12M
• 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 1/6844 Nucleic acid amplification reactions

Abstract

A microfluidic flow cell having a body with a fluid transport channel disposed therein, the fluid transport channel having a proximal end and a distal end defining a fluid flow path, a fluid inlet port disposed at the proximal end of the fluid transport channel at a central portion of the body and an outlet port disposed at the distal end of the fluid transport channel at an outer portion of the body, and a plurality sample wells disposed in the fluid transport channel substantially perpendicular to the fluid flow path in the fluid transport channel. The microfluidic flow cell may have hundreds or thousands of individual, sub-microliter sample wells. The microfluidic flow cell can be filled by applying a flowable liquid to the inlet port and spinning the flow cell to cause fluid to flow into fluid transport channel. The microfluidic flow cells described herein can be used in a variety of applications where small sample size and/or a large number of replicates are desirable.

Claims

1. A microfluidic flow cell, comprising:

a body having a fluid transport channel disposed therein, the fluid transport channel having a proximal end and a distal end defining a fluid flow path;

a fluid inlet port disposed at the proximal end of the fluid transport channel at a central portion of the body and an outlet port disposed at the distal end of the fluid transport channel at an outer portion of the body; and

a plurality of sample wells in direct fluid communication with the fluid transport channel and substantially perpendicular to the fluid flow path in the fluid transport channel such that a volume of a liquid medium flowing through the fluid transport channel will flow into at least a first sample well with an excess of the liquid medium flowing into at least a second sample well.

2. The microfluidic flow cell as recited in claim 1, each of the sample wells having a volume between about 1 nl and about 100 nl.

3. The microfluidic flow cell as recited in claim 1, each of the sample wells having a volume between about 10 nl and about 80 nl.

4. The microfluidic flow cell as recited in claim 1, each of the sample wells having a volume between about 20 nl and about 50 nl.

5. The microfluidic flow cell as recited in claim 1, the body further comprising at least first and second outer layers and an inner layer disposed between the first and second outer layers;

the fluid transport channel being formed in the inner layer; and

the fluid inlet and the fluid outlet ports being disposed in the first or second outer layers.

6. The microfluidic flow cell as recited in claim 5, the fluid inlet port being disposed in the first outer layer and the fluid outlet port being disposed in the second outer layer.

7. The microfluidic flow cell as recited in claim 1, the body further comprising at least a first layer and a second layer;

the fluid transport channel being formed in the second layer;

the first layer being disposed over the second layer and over the fluid transport channel; and

the fluid inlet and the fluid outlet ports being disposed in the first or second layers.

8. The microfluidic flow cell as recited in claim 7, the fluid inlet port being disposed in the first layer and the fluid outlet port being disposed in the second layer.

9. The microfluidic flow cell as recited in claim 1, the body being a rotatable disc such that a fluid applied at the inlet port will flow through the fluid path toward the outlet port when the disc is rotated.

17. A method for filling a microfluidic flow cell, comprising:

providing a microfluidic flow cell as recited in one of claim 1 or claim 10;

applying a volume of a flowable fluid to the fluid inlet port; and

flowing the flowable fluid through the fluid transport channel toward the fluid outlet port such that the flowable fluid will flow into at least a first sample well with an excess of the flowable fluid flowing into at least a second sample well.

18. The method as recited in claim 17, the flowing further comprising spinning the flow cell about its central axis in a centrifuge so as to cause the flowable fluid to flow from the fluid inlet port, through the fluid transport channel, and toward the fluid outlet port.

19. The method as recited in claim 18, further comprising spinning the flow cell at a rate between about 500 rpm and about 10,000 rpm.

20. The method as recited in claim 18, further comprising spinning the flow cell at a rate between about 2000 rpm and about 8000 rpm.

21. The method as recited in claim 18, further comprising spinning the flow cell at a rate between about 3000 rpm and about 5000 rpm.

22. The method as recited in claim 17, wherein the volume of flowable fluid that is applied to the inlet port is sufficient to fill each of the plurality of samples well that are disposed in the fluid transport channel, and wherein excess flowable fluid flows out of the fluid transport channel through the fluid outlet port.

23. The method as recited in claim 22, wherein the filled sample wells are not in fluid communication with adjacent filled sample wells.

33. A method for DNA quantification, comprising:

providing a microfluidic flow cell as recited in one of claim 1 or claim 10;

loading the plurality of sample wells in the microfluidic flow cell with a reaction mixture, the reaction mixture including a nucleic acid template, a plurality of primer strands for amplifying a sequence of interest, and means for amplifying the nucleic acid template;

amplifying the nucleic acid template to generate an amplified product therefrom;

detecting the presence or absence of the amplified product in a plurality of wells; and

quantifying the amount of the nucleic acid template amplified.

34. The method as recited in claim 33, wherein means for amplifying the nucleic acid template include at least one of polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription mediated amplification (TMA), rolling circle amplification (RCA), strand displacement amplification (SDA), or loop-mediated amplification (LAMP).

35. The method as recited in claim 33, wherein the plurality of primer strands includes at least one of a forward primer or a reverse primer for amplifying the sequence of interest.

36. The method as recited in claim 33, further comprising sealing the fluid transport channel so as to prevent evaporation of the fluid in the sample wells.

37. The method as recited in claim 33, each of the sample wells in the flow cell having a volume between about 1 nl and about 100 nl.

38. The method as recited in claim 33, wherein the nucleic acid template is provided at a concentration such that the plurality of sample wells contain an average of about 0.1 copies of the nucleic acid template per sample well to about 1 copy of the nucleic acid template per sample well.

39. The method as recited in claim 33, wherein the nucleic acid template is provided at a concentration such that the plurality of sample wells contain an average of about 0.2 copies of the nucleic acid template per sample well to about 0.8 copies of the nucleic acid template per sample well.

40. The method as recited in claim 33, wherein the nucleic acid template is provided at a concentration such that the plurality of sample wells contain an average of about 0.3 copies of the nucleic acid template per sample well to about 0.5 copies of the nucleic acid template per sample well.

41. The method as recited in claim 33, further comprising:

providing the nucleic acid template from a diploid organism at a concentration such that each of the plurality of sample wells contain no more than one copy of the organism'"'"'s two copies of the sequence of interest.

42. The method as recited in claim 33, further comprising:

providing a microfluidic flow cell formed from a material having an affinity for the nucleic acid template such that the nucleic acid template is progressively diluted as it flows down the fluid transport channel; and

plotting a frequency of sample wells having amplified product versus sample wells having no amplified product as a function of distance along the fluid transport channel so as to find a subset of sample wells having a concentration of the nucleic acid template usable for quantification.

43. The method as recited in claim 33, wherein quantifying the amount of the nucleic acid template amplified provides for at least one of cancer detection, cancer therapy response monitoring, quantification of viral load, prenatal genetic testing, or direct haplotyping.

44. The method as recited in claim 33, wherein quantifying the amount of the nucleic acid template amplified further comprises:

performing melting analysis on the amplified product, the melting analysis including;

raising the temperature of the microfluidic flow cell and the contents therein; and

monitoring the absorbance and/or fluorescence in each of the sample wells while raising the temperature to generate a melting profile for the amplified product,

wherein a change in absorbance and/or fluorescence indicates denaturation of a double stranded nucleic acid.

45. The method as recited in claim 44, further comprising analyzing the results of the melting analysis, wherein a shift in the melting profile of the amplified product indicates the presence of sequence variants in the nucleic acid template.

46. The method as recited in claim 44, the reaction mixture further comprising a plurality of DNA probe strands that are capable of forming a double stranded DNA structure with the sequence of interest and/or a fluorescent dye that fluoresces in the presence of double-stranded DNA.

47. The method as recited in claim 46, further comprising:

providing a light source that is positionable and timeable for providing light at a fluorescence excitation wavelength to at least one sample well;

flashing the light source at the fluorescence excitation wavelength so as to excite fluorescent emission from the fluorescent dye in the at least one sample well; and

detecting the fluorescent emission from the fluorescent dye in the at least one sample well using a positionable detector means,

wherein flashing the light source substantially prevents photobleaching of the fluorescent dye in the sample well.

10. A microfluidic flow cell, comprising:

a disc-shaped body configured for spinning about a central axis, the body further including;

at least one fluid inlet port situated toward a center portion of the disc and at least one fluid outlet port situated toward an outer edge of the disc;

at least one fluid transport channel defining a fluid flow path in the body, the fluid transport channel being disposed in the body such that it is in fluid communication with a fluid inlet port and a fluid outlet port; and

a plurality of sample wells in direct fluid communication with the at least one fluid transport channel such that the sample wells are substantially perpendicular to the fluid flow path, wherein a fluid applied at the inlet port will flow through the body along the fluid path toward the outlet port in response to spinning the disc-shaped body about the central axis.

11. The microfluidic flow cell as recited in claim 10, each of the sample wells having a volume between about 20 nl and about 50 nl.

12. The microfluidic flow cell as recited in claim 10, the body further comprising at least first and second outer layers and an inner layer disposed between the first and second outer layers;

the fluid transport channel being formed in the inner layer;

the fluid inlet port being disposed in the first outer layer; and

the fluid outlet port being disposed in the first outer layer or the second outer layer.

13. The microfluidic flow cell as recited in claim 12, the fluid inlet port being disposed in the first outer layer and the fluid outlet port being disposed in the second outer layer.

14. The microfluidic flow cell as recited in claim 10, the body further comprising at least a first layer and a second layer;

the fluid transport channel being formed in the second layer;

the first layer being disposed over the second layer and over the fluid transport channel; and

the fluid inlet port being disposed in the first layer; and

the fluid outlet port being disposed in the first layer or the second layer.

15. The microfluidic flow cell as recited in claim 14, the fluid inlet port being disposed in the first layer and the fluid outlet port being disposed in the second layer.

16. The microfluidic flow cell as recited in claim 10, the body further comprising:

a plurality of inlet ports situated toward the center of the disc;

a plurality of outlet ports situated toward the outer edge of the disc; and

a plurality of fluid transport channels for transporting a flowable fluid with each channel being connected to one inlet port and one outlet port.

24. A method for manufacturing a microfluidic flow cell, comprising:

providing a fluid transport layer, comprising;

forming a fluid transport channel defining a fluid flow path in the fluid transport layer, the fluid transport channel having a proximal end at a central portion of the fluid transport layer and a distal end at an outer portion of the fluid transport layer;

forming a plurality sample wells in direct fluid communication with the fluid transport channel, the plurality sample wells being substantially perpendicular to the fluid flow path in the fluid transport channel; and

sealing the fluid transport layer with at least one outer layer.

25. The method as recited in claim 24, the forming including at least one of xurography, lithography and etching, laser cutting, stamping, hot embossing, injection molding, and micro machining.

26. The method as recited in claim 24, each of the sample wells having a volume between about 1 nl and about 100 nl.

27. The method as recited in claim 24, the fluid transport layer being formed from a polymeric, thermoplastic material.

28. The method as recited in claim 24, the fluid transport layer being formed from a glass material.

29. The method as recited in claim 24, the fluid transport layer being formed from an inert material.

30. The method as recited in claim 24, the fluid transport layer being formed from a material having an affinity for DNA and/or protein.

31. The method as recited in claim 24, further comprising:

disposing an outer layer over the fluid transport layer, the outer layer including at least inlet and outlet ports that are in fluid communication with the fluid transport channel.

32. The method as recited in claim 24, further comprising:

disposing the fluid transport layer between the first and second outer layers, the first and second outer layers including at least inlet and outlet ports that are in fluid communication with the fluid transport channel.