Method for separating analyte from a sample

• US 20020042125A1
• Filed: 11/07/2001
• Published: 04/11/2002
• Est. Priority Date: 08/13/1997
• Status: Active Grant

• Current Assignee: Cepheid (Danaher Corporation)
• Original Assignee: Cepheid (Danaher Corporation)
• Inventors: Chang, Ronald, Petersen, Kurt E., Northrup, M. Allen, McMillan, William A., Pourahmadi, Farzad, Christel, Lee A., Ching, Jesus, Kovacs, Gregory T.A.
• Granted Patent: US 6,893,879 B2
• Time in Patent Office: Days
• US Class Current: 435/287.2
• CPC Class Codes: B01J 2219/00432 Photolithographic masks
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01L 2200/0621 Control of the sequence of chambers filled or emptied
• CPC Class Codes: B01L 2200/0631 Purification arrangements, e.g. solid phase extraction [SPE]
• CPC Class Codes: B01L 2200/0636 Focussing flows, e.g. to laminate flows
• 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 2300/0883 Serpentine channels
• CPC Class Codes: B01L 2300/1827 using resistive heater
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 2400/046 Chemical or electrochemical formation of bubbles
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/0622 distribution valves, valves having multiple inlets and/or outlets, e.g. metering valves, multi-way valves
• CPC Class Codes: B01L 2400/0644 rotary valves
• CPC Class Codes: B01L 2400/086 using baffles or other fixed flow obstructions
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/502753 characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation separation in general B01D; microapparatus for analysis using electrophoresis G01N27/44791; sample preparation G01N1/28
• 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: C12M 47/02 Separating microorganisms from the culture medium; Concentration of biomass separating microorganisms from their culture media C12N1/02
• CPC Class Codes: C12M 47/06 Hydrolysis; Cell lysis; Extraction of intracellular or cell wall material lysis of microorganisms C12N1/06; extracting or separating nucleic acids from biological samples C12N15/1003
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: G01F 1/708 : Measuring the time taken to traverse a fixed distance
• CPC Class Codes: G01N 30/6091 : Cartridges
• CPC Class Codes: G01N 30/6095 : Micromachined or nanomachined, e.g. micro- or nanosize
• CPC Class Codes: Y10T 436/25375 : Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
• CPC Class Codes: Y10T 436/255 : including use of a solid sorbent, semipermeable membrane, or liquid extraction

Abstract

An analyte is separated from a fluid sample by introducing the sample into a cartridge having a sample port and a first flow path extending from the sample port. The first flow path includes an extraction chamber containing a solid support for capturing the analyte from the sample. The cartridge has a second flow path for eluting the captured analyte from the extraction chamber, the second flow diverging from the first flow path after passing through the extraction chamber. The sample is forced to flow through the extraction chamber and into a waste chamber, thereby capturing the analyte with the solid support as the sample flows through the extraction chamber. The captured analyte is then eluted from the extraction chamber by forcing an elution fluid to flow through the extraction chamber and along the second flow path.

Claims

1. A method for separating an analyte from a fluid sample and for concentrating the analyte into a volume of elution fluid, the method comprising the steps of:

a) introducing the sample into a cartridge having;

i) a sample port;

ii) a first flow path extending from the sample port;

iii) an extraction chamber in the first flow path, wherein the extraction chamber contains at least one solid support for capturing the analyte from the sample; and

iv) a second flow path for eluting the analyte from the extraction chamber, wherein the second flow path passes through the extraction chamber and diverges from the first flow path after passing through the extraction chamber;

b) forcing the sample to flow through the first flow path, thereby capturing the analyte with the solid support as the sample flows through the extraction chamber, wherein the ratio of the volume of sample forced to flow through the extraction chamber to the volume capacity of the extraction chamber is at least 2;

1, and wherein the volume of sample forced to flow through the extraction chamber is at least 0.1 ml; and

c) forcing an elution fluid to flow through the second flow path, thereby releasing the captured analyte from the solid support into the elution fluid.

2. The method of claim 1, wherein the solid support is selected from the group consisting of filters, beads, fibers, membranes, glass wool, filter paper, polymers and gels.

3. The method of claim 1, further comprising the step of heating the solid support while forcing the elution fluid to flow through the extraction chamber.

4. The method of claim 3, wherein the solid support is heated to a temperature in the range of 60 to 95°

C.

5. The method of claim 1, further comprising the step of forcing a gas to flow through the extraction chamber after the step of forcing the sample to flow through the extraction chamber and prior to the step of forcing the elution fluid to flow through the extraction chamber.

6. The method of claim 1, wherein the ratio of the volume of sample forced to flow through the extraction chamber to the volume capacity of the extraction chamber is at least 10:

1.

7. The method of claim 1, wherein the volume of sample forced to flow through the extraction chamber is at least 1 ml.

8. The method of claim 1, wherein the cartridge further comprises a lysing region in the first flow path upstream from the extraction chamber, and the method further comprises the steps of lysing sample components in the lysing region prior to forcing the sample to flow through the extraction chamber.

9. The method of claim 8, wherein the lysing region comprises a lysing chamber containing a solid phase for capturing the sample components, and wherein the sample components are lysed by:

i) capturing the sample components with the solid phase; and

ii) transferring ultrasonic energy to the sample components using an ultrasonic transducer coupled to a wall of the lysing chamber.

10. The method of claim 9, wherein the solid phase comprises a membrane or filter for capturing the components by size exclusion.

11. The method of claim 9, wherein the solid phase comprises beads for capturing the components by affinity retention or chemical selection.

12. The method of claim 9, wherein the transducer comprises an ultrasonic horn.

13. The method of claim 10, further comprising the step of agitating beads in the lysing chamber to rupture the sample components.

14. The method of claim 1, wherein the second flow path leads to a reagent chamber, and the method further comprises the step of mixing the eluted analyte with a reagent in the reagent chamber.

15. The method of claim 1, wherein the second flow path leads to a reaction chamber, the analyte comprises nucleic acid, and the method further comprises the steps of amplifying and detecting the nucleic acid in the reaction chamber.

16. The method of claim 1, wherein the cartridge further includes:

a lysing region in the first flow path upstream of the extraction chamber;

a waste chamber in fluid communication with the extraction chamber via the first flow path; and

a reaction chamber in fluid communication with the extraction chamber via the second flow path;

and wherein the method further comprises the steps of;

lysing sample components in the lysing region prior to forcing the sample to flow through the extraction chamber;

forcing the sample to flow into the waste chamber after the sample flows through the extraction chamber;

forcing the eluted analyte to flow into the reaction chamber; and

amplififying or detecting the analyte in the reaction chamber.

17. The method of claim 16, wherein the cartridge further includes a reagent chamber in fluid communication with the extraction chamber and the reaction chamber, and wherein the method further comprises the step of mixing the eluted analyte with a reagent in the reagent chamber prior to amplifying or detecting the analyte in the reaction chamber.

19. The method of claim 18, further comprising the step of heating the solid support while forcing the elution fluid to flow through the extraction chamber.

20. The method of claim 19, wherein the solid support is heated to a temperature in the range of 60 to 95°

C.

21. The method of claim 18, further comprising the step of forcing a gas to flow through the extraction chamber after the step of forcing the sample to flow through the extraction chamber and prior to the step of forcing the elution fluid to flow through the extraction chamber.

22. The method of claim 18, wherein the volume of sample forced to flow through the extraction chamber is at least 0.1 ml.

23. The method of claim 22, wherein the ratio of the volume of sample forced to flow through the extraction chamber to the volume capacity of the extraction chamber is at least 2:

1.

24. The method of claim 18, wherein the ratio of the volume of sample forced to flow through the extraction chamber to the volume capacity of the extraction chamber is at least 10:

1.

25. The method of claim 18, wherein the volume of sample forced to flow through the extraction chamber is at least 1 ml.

26. The method of claim 18, wherein the cartridge further comprises a lysing region in the first flow path upstream from the extraction chamber, and the method further comprises the steps of lysing sample components in the lysing region prior to forcing the sample to flow through the extraction chamber.

27. The method of claim 26, wherein the lysing region comprises a lysing chamber containing a solid phase for capturing the sample components, and wherein the sample components are lysed by:

i) capturing the sample components with the solid phase; and

ii) transferring ultrasonic energy to the sample components using an ultrasonic transducer coupled to a wall of the lysing chamber.

28. The method of claim 27, wherein the solid phase comprises a membrane or filter for capturing the components by size exclusion.

29. The method of claim 27, wherein the solid phase comprises beads for capturing the components by affinity retention or chemical selection.

30. The method of claim 27, wherein the transducer comprises an ultrasonic horn.

31. The method of claim 28, further comprising the step of agitating beads in the lysing chamber to rupture the sample components.

32. The method of claim 18, wherein the second flow path leads to a reagent chamber, and the method further comprises the step of mixing the eluted analyte with a reagent in the reagent chamber.

33. The method of claim 18, wherein the second flow path leads to a reaction chamber, the analyte comprises nucleic acid, and the method further comprises the steps of amplifying and detecting the nucleic acid in the reaction chamber.

34. The method of claim 18, wherein the cartridge further includes:

a lysing region in the first flow path upstream of the extraction chamber;

a waste chamber in fluid communication with the extraction chamber via the first flow path; and

a reaction chamber in fluid communication with the extraction chamber via the second flow path;

and wherein the method further comprises the steps of;

lysing sample components in the lysing region prior to forcing the sample to flow through the extraction chamber;

forcing the sample to flow into the waste chamber after the sample flows through the extraction chamber;

forcing the eluted analyte to flow into the reaction chamber; and

amplififying or detecting the analyte in the reaction chamber.

35. The method of claim 34, wherein the cartridge further includes a reagent chamber in fluid communication with the extraction chamber and the reaction chamber, and wherein the method further comprises the step of mixing the eluted analyte with a reagent in the reagent chamber prior to amplifying or detecting the analyte in the reaction chamber.

37. The method of claim 36, wherein the flow-through component comprises an extraction chamber containing at least one solid support, and wherein the analyte is captured by binding the analyte to the solid support.

38. The method of claim 37, further comprising the step of heating the extraction chamber while forcing the elution fluid to flow through the extraction chamber.

39. The method of claim 38, wherein the extraction chamber is heated to a temperature in the range of 60 to 95°

C.

40. The method of claim 36, wherein the flow-through component comprises a microfluidic chip having an extraction chamber containing an array of microstructures, the analyte is captured by binding the analyte to the microstructures, and each of the microstructures has an aspect ration of at least 2:

1.

41. The method of claim 40, further comprising the step of heating the microstructures while forcing the elution fluid to flow through the extraction chamber.

42. The method of claim 41, wherein the microstructures are heated to a temperature in the range of 60 to 95°

C.

43. The method of claim 36, wherein the volume of sample forced to flow through the flow-through components is at least 0.1 ml.

44. The method of claim 43, wherein the flow-through component comprises an extraction chamber through which the sample is forced to flow, and wherein the ratio of the volume of sample forced to flow through the extraction chamber to the volume capacity of the extraction chamber is at least 2:

1.

45. The method of claim 44, wherein the ratio of the volume of sample forced to flow through the extraction chamber to the volume capacity of the extraction chamber is at least 10:

1.

46. The method of claim 36, wherein the volume of sample forced to flow through the flow-through components is at least 1 ml.

47. The method of claim 36, wherein the lysing region comprises a lysing chamber containing a solid phase for capturing the sample components, and wherein the sample components are lysed by:

i) capturing the sample components with the solid phase; and

ii) transferring ultrasonic energy to the sample components using an ultrasonic transducer directly coupled to a wall of the lysing chamber.

48. The method of claim 47, wherein the solid phase comprises a membrane or filter for capturing the components by size exclusion.

49. The method of claim 47, wherein the solid phase comprises beads for capturing the components by affinity retention or chemical selection.

50. The method of claim 47, wherein the transducer comprises an ultrasonic horn.

51. The method of claim 48, further comprising the step of agitating beads in the lysing chamber to rupture the sample components.

52. The method of claim 36, wherein the second flow path leads to a reagent chamber, and the method further comprises the step of mixing the eluted analyte with a reagent in the reagent chamber.

53. The method of claim 36, wherein the second flow path leads to a reaction chamber, the analyte comprises nucleic acid, and the method further comprises the steps of amplifying or detecting the nucleic acid in the reaction chamber.

18. A method for separating an analyte from a fluid sample, the method comprising the steps of:

a) introducing the sample into a cartridge having;

i) a sample port;

ii) a first flow path extending from the sample port, wherein the first flow path includes an extraction chamber and a waste chamber downstream of the extraction chamber, the extraction chamber contains at least one solid support for capturing the analyte from the sample, the solid support is selected from the group consisting of filters, beads, fibers, membranes, glass wool, filter paper, polymers and gels, and the cartridge includes means for holding the solid support in the extraction chamber; and

iii) a second flow path for eluting the analyte from the extraction chamber, wherein the second flow path passes through the extraction chamber and diverges from the first flow path after passing through the extraction chamber;

b) forcing the sample to flow through the extraction chamber and into the waste chamber, thereby capturing the analyte with the solid support as the sample flows through the extraction chamber; and

c) eluting the analyte from the extraction chamber by forcing an elution fluid to flow through the extraction chamber while retaining the solid support in the extraction chamber, thereby releasing the analyte from the solid support into the elution fluid.

36. A method for separating an analyte from a fluid sample, the method comprising the steps of:

a) introducing the sample into a cartridge having;

i) a sample port;

ii) a first flow path extending from the sample port, wherein the first flow path includes a lysing region, at least one flow-through component downstream of the lysing region for capturing the analyte, and a waste chamber downstream of the flow-through component for receiving waste fluid;

iii) a second flow path for eluting the analyte from the flow-through component, wherein the second flow path passes through the flow-through component and diverges from the first flow path after passing through the flow-through component;

b) lysing sample components in the lysing region to release the analyte therefrom;

c) forcing the lysed sample to flow through the flow-through component and into the waste chamber, thereby capturing the analyte with the flow-through component; and

d) eluting the analyte from the flow-through component by forcing an elution fluid to flow along the second flow path.