METHOD OF LOADING SAMPLE INTO A MICROFLUIDIC DEVICE

• US 20080138815A1
• Filed: 08/13/2007
• Published: 06/12/2008
• Est. Priority Date: 04/17/1997
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated), United States Department Of Health And Human Services
• Original Assignee: Cytonix, The United States of America As Represented By The Secretary of Agriculture
• Inventors: SILVER, Jonathan E., BROWN, James F.
• Granted Patent: US 8,551,698 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00621 by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00644 the porous medium being present in discrete locations, e.g. gel pads
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00677 Ex-situ synthesis followed by deposition on the substrate
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01L 2200/06 Fluid handling related problems
• 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 2200/16 Reagents, handling or storing thereof
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0819 Microarrays; Biochips
• CPC Class Codes: B01L 2400/0409 centrifugal forces
• 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 3/5085 for multiple samples, e.g. microtitration plates
• CPC Class Codes: B01L 3/50851 specially adapted for heating or cooling samples laboratory heating apparatus B01L7/00; incubators C12M
• CPC Class Codes: B01L 3/5088 confining liquids at a location by surface tension, e.g. virtual wells on plates, wires B01L3/50857 takes precedence
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6844 : Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/686 : Polymerase chain reaction [PCR]
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: Y10S 436/805 : Optical property
• CPC Class Codes: Y10S 436/809 : Multifield plates or multicontainer arrays
• CPC Class Codes: Y10T 436/2575 : Volumetric liquid transfer

Abstract

A method comprising loading a sample into a microfluidic device which comprises plural sample chambers, subdividing the sample into a plurality of sample portions, such that respective sample portions are positioned in each of a plurality of the sample chambers, and subjecting the sample portions loaded into the respective sample chambers to at least a first amplification step. Each of the sample chambers has a respective volume such that if a sample portion positioned in the sample chamber comprises at least one molecule of a target nucleic acid, the target nucleic acid would attain a detectable concentration in the sample chamber after a single round of amplification.

Claims

1. A method comprising:

loading a sample into a microfluidic device, said microfluidic device comprising plural sample chambers, said sample comprising at least a portion of a first fluid;

subdividing the sample into a plurality of sample portions, such that respective sample portions are positioned in each of a plurality of said sample chambers, each of said plurality of sample chambers having a respective volume such that if a sample portion positioned in said sample chamber comprises at least one molecule of a first target nucleic acid, said first target nucleic acid would attain a detectable concentration in said sample chamber after a single round of amplification; and

subjecting the sample portions loaded into said respective sample chambers to at least a first amplification step.

2. A method as recited in claim 1, wherein if a first sample portion positioned in said sample chamber comprises at least one molecule of said first target nucleic acid, said first sample portion would attain a detectable concentration of said first target nucleic acid within a portion of said first sample chamber after a single amplification step.

3. A method as recited in claim 1, wherein said loading comprises applying centrifugal force.

4. A method as recited in claim 1, wherein said microfluidic device comprises at least one flow-through surface defining at least one flow-through channel, at least a portion of said at least one flow-through surface being hydrophobic, at least a portion of at least one of said sample chambers being hydrophilic.

5. A method as recited in claim 1, wherein said microfluidic device comprises at least one flow-through surface defining at least one flow-through channel, at least a portion of said at least one flow-through surface having a first affinity to said sample portions and a second affinity to a displacing fluid, at least one of said sample chambers having a third affinity to said sample portions and a fourth affinity to said displacing fluid, said second affinity being greater than said first affinity, said third affinity being greater than said fourth affinity.

6. A method as recited in claim 1, wherein said microfluidic device comprises at least one flow-through channel and said method further comprises supplying a displacing fluid into said flow-through channel, whereby said displacing fluid displaces sample fluid from said flow-through channel.

7. A method as recited in claim 1, wherein:

said loading comprises applying centrifugal force,

said microfluidic device comprises at least one flow-through surface defining at least one flow-through channel,

at least a portion of said at least one flow-through surface is hydrophobic,

at least a portion of at least one of said sample chambers is hydrophilic,

said method further comprises supplying a displacing fluid into said flow-through channel, whereby said displacing fluid displaces first fluid from said flow-through channel

at least one of said sample chambers is in communication with said flow-through channel,

at least a portion of said at least one flow-through surface has a first affinity to said sample portions and a second affinity to said displacing fluid, said second affinity being greater than said first affinity, and

at least one of said sample chambers has a third affinity to said sample portions and a fourth affinity to said displacing fluid, said third affinity being greater than said fourth affinity.

8. A method as recited in claim 1, wherein said method further comprises detecting, for at least one of said individual sample chambers, whether there is amplified product in said individual sample chamber.

9. A method as recited in claim 8, wherein said detecting whether there is amplified product is achieved out by carrying out a procedure which generates signals having magnitude which is higher where said detectable concentration is present than where said detectable concentration is not present.

10. A method as recited in claim 9, wherein said procedure comprises detecting fluorescence from fluor-labeled materials.

11. A method as recited in claim 9, wherein said procedure comprises detecting at least one chemical property which changes upon hybridization.

12. A method as recited in claim 9, wherein said procedure comprises evaluating at least one property selected from among the group consisting of agglutination, turbidity, phosphorescence, light scattering, light absorbance, fluorescence energy transfer, fluorescence quenching, fluorescence dequenching, time-delayed fluorescence, chemiluminescence and calorimetric evaluation.

13. A method as recited in claim 8, wherein said each said sample chamber comprises a microcapillary device, and wherein peaks are generated by detecting regions within said microcapillary devices in which said detectable concentration is present.

14. A method as recited in claim 8, wherein said method further comprises determining a total number of said sample chambers in which there is amplified product.

15. A method as recited in claim 1, wherein each of said sample chambers contains at least one amplification targeting reagent.

16. A method as recited in claim 1, wherein at least said a first of said sample chambers contains at least a first amplification targeting reagent, and at least a second of said sample chambers contains at least a second amplification targeting reagent, said first amplification targeting reagent differing from said second amplification targeting reagent.

17. A method as recited in claim 1, wherein at least a first of said sample chambers further comprises constituents for enabling amplification of said first target nucleic acid.

18. A method as recited in claim 1, wherein at least one of said sample chambers has a volume of about 1 picoliter or less.

19. A method as recited in claim 1, wherein at least one of said sample chambers has a volume in the range of from about 1 picoliter to about 1 microliter.

20. A method as recited in claim 1, wherein at least one of said sample chambers has a volume of about 10 picoliters or less.

21. A method as recited in claim 1, wherein at least one of said sample chambers has a volume of about 100 picoliters.

22. A method as recited in claim 1, wherein at least one of said sample chambers has a volume of about 1 nanoliter.

23. A method as recited in claim 1, wherein at least one of said sample chambers has a volume of 10 nanoliters or less.

24. A method as recited in claim 1, wherein at least one of said sample chambers has a volume of about 10 nanoliters.

25. A method as recited in claim 1, wherein at least one of said sample chambers has at least one dimension of 2 mm or less.

26. A method as recited in claim 1, wherein at least one of said sample chambers has at least one dimension of 1 mm or less.

27. A method as recited in claim 1, wherein at least one of said sample chambers has at least one dimension of 100 microns or less.

28. A method as recited in claim 1, wherein at least one of said sample chambers has at least one dimension of 20 microns or less.

29. A method as recited in claim 1, wherein at least one of said sample chambers has at least one dimension of a few microns or less.

30. A method as recited in claim 1, wherein at least one of said sample portions has a volume of about 1 picoliter or less.

31. A method as recited in claim 1, wherein at least one of said sample portions has a volume in the range of from about 1 picoliter to about 1 microliter.

32. A method as recited in claim 1, wherein at least one of said sample portions has a volume of about 10 picoliters or less.

33. A method as recited in claim 1, wherein at least one of said sample portions has a volume which is nanoliter-sized.

34. A method as recited in claim 1, wherein at least one of said sample portions has a volume of about 1 nanoliter or less.

35. A method as recited in claim 1, wherein at least one of said sample portions has a volume of about 10 nanoliters or less.

36. A method as recited in claim 1, wherein at least one of said sample portions has a volume of about 100 nanoliters or less.

37. A method as recited in claim 1, wherein at least one of said sample portions has a volume of about 1 microliter or less.

38. A method as recited in claim 1, wherein at least one of said sample portions is confined in at least one dimension by opposing barriers separated by about 2 mm or less.

39. A method as recited in claim 1, wherein at least one of said sample portions is confined in at least one dimension by opposing barriers separated by about 1 mm or less.

40. A method as recited in claim 1, wherein at least one of said sample portions is confined in at least one dimension by opposing barriers separated by about 500 microns or less.

41. A method as recited in claim 1, wherein at least one of said sample portions is confined in at least one dimension by opposing barriers separated by about 100 microns or less.

42. A method as recited in claim 1, wherein at least one of said sample portions is confined in at least one dimension by opposing barriers separated by about 20 microns or less.

43. A method as recited in claim 1, wherein at least one of said sample portions is confined in at least one dimension by opposing barriers separated by a few microns or less.

44. A method as recited in claim 1, wherein said microfluidic device comprises an assembly, said assembly comprising:

said plurality of sample chambers, each of said sample chambers being confined in at least one dimension by opposing barriers separated by about 500 microns or less;

means for sealing said plurality of sample chambers to prevent evaporation and contamination of sample confined within said plurality of sample chambers; and

means for restraining amplification product.

45. A method as recited in claim 44, wherein said opposing barriers are separated by 100 microns or less.

46. A method as recited in claim 1, wherein said microfluidic device comprises an assembly, said assembly comprising:

said plurality of sample chambers, each of said sample chambers being confined in at least one dimension by opposing barriers separated by about 500 microns or less;

means for sealing said plurality of sample chambers to prevent evaporation and contamination of sample confined within said plurality of sample chambers; and

means for restraining amplification product, said means for restraining amplification product comprising a patterned layer disposed on at least one of said opposing barriers and at least partially defining each of said plurality of sample chambers.

47. A method as recited in claim 46, wherein said opposing barriers are separated by 100 microns or less.

48. A method as recited in claim 1, wherein said microfluidic device comprises an assembly, said assembly comprising:

a substrate and a cover, said substrate comprising a first surface, said cover comprising a second surface, said first surface facing and being spaced from said second surface, at least one of said first surface and said second surface comprising a first material;

a flow-through channel, said flow-through channel being positioned between said substrate and said cover,

at least a first of said sample chambers being bounded on at least one side thereof by said first material and being in communication with said flow-through channel, said first sample chamber having a first affinity to said sample portions and a second affinity to a displacing fluid, said flow-through channel having a third affinity to said sample portions and a fourth affinity to said displacing fluid, wherein said first, second, third and fourth affinities are such that if a first of said sample portions is loaded into a first of said sample chambers and then said a fluid displaces sample from said flow-through channel, said first sample chamber would retain said first sample portion while said displacing fluid displaces sample from said flow-through channel.

49. A method as recited in claim 1, wherein each of said sample chambers is confined in at least one dimension by opposing barriers separated by a dimension of about 100 microns or less.

50. A method as recited in claim 1, wherein said microfluidic device further comprises:

means for facilitating loading said sample portions into said sample chambers;

means for displacing sample away from open ends of said sample chambers to isolate said sample portions within said sample chambers; and

means for sealing said open ends of said sample chambers immediately after displacing sample away from said open ends.