Assays Based On Light Emission From Analyte Complexes Within A Cassette

• US 20080026373A1
• Filed: 07/26/2006
• Published: 01/31/2008
• Est. Priority Date: 07/26/2006
• Status: Abandoned Application

• Current Assignee: Avantra Biosciences Corporation
• Original Assignee: Avantra Biosciences Corporation
• Inventors: Rodionova, Natalia A.
• Application Number:
  US11/460,238
• Publication Number:
  US 20080026373A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01L 2200/0678 Facilitating or initiating evaporation
• 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 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2400/0611 duck bill valves
• CPC Class Codes: B01L 2400/0688 surface tension valves, capillary stop, capillary break
• 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 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/502723 characterised by venting arrangements
• CPC Class Codes: B01L 3/502738 characterised by integrated valves microvalves F16K99/0001
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing
• CPC Class Codes: G01N 33/54373 involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings

Abstract

Assays based on probes attached to surfaces enclosed within cassettes and cassettes and reading stations for the assays. After liquids flow over the probe or probe array to form an array of photo-emissive analyte complexes, and prior to reading, (a) liquid is removed by flow, and (b) a drying gas stream, is forced into the cassette and over the complexes for a drying interval to remove liquid residue. Heating assists the drying. Light from the analyte complexes is then read through a window of the cassette. The interval of drying may be of the order of about one minute. During a preceding wash phase, gas flow bursts through the gas inlet channel purge liquid contaminant. The probes e.g. may be oligonucleotides, peptides, polypeptides, proteins, antibodies, or small molecules (steroids, expression regulators, e.g. siRNA, or other ligands). A cassette has a passage leading from a bubble removal system to the probe-bearing surface and the desiccating gas stream is introduced to that passage. For wide arrays the common passage connects through a widening transition to a wide reaction chamber. A reader station includes an air pump and liquid pumping devices such as linear actuators to deflect liquid-pumping diaphragms of the cassette.

Claims

1. The method of conducting an assay by employing a cassette which encloses a surface to which at least one probe is attached, the surface associated with a liquid passage that enables liquid flow over the probe,

the assay being of the type in which one or more liquids flowing over the probe produce at the probe a bound analyte complex that has a constituent that is capable of emitting light for detection by an external detector after the light passes from the analyte complex through a window of the cassette,

wherein, after formation of the analyte complex within the cassette, the method of conducting the assay includes:

(a) by liquid flow, removing liquid resident at the analyte complex on the enclosed surface and

(b) forcing a stream of drying gas to flow into the cassette, over the enclosed surface and out of the cassette during a drying interval under conditions that substantially volatilize and remove residue of the liquid associated with the analyte complex, and performing the detection on the dried complex enclosed within the cassette.

2. The method of claim 1 conducted in a manner to form and dry an array of said complexes at a corresponding array of surface-bound probes enclosed within the cassette, and performing the detection on the array of complexes within the cassette.

3. The method of claim 2 in which at least some of the analyte complexes in the array are on probes distributed, transversely to the direction of liquid flow, upon a width-wise extended surface, the passage is correspondingly wide, constructed to produce a uniform liquid flow over the array, and the drying gas is introduced through a relatively narrow channel and spreads to flow through the liquid passage over the array.

4. The method of claim 3 in which the extended surface is a planar surface carrying a two-dimensional array of probes and corresponding analyte complexes exposed to the liquid and drying gas flows.

5. The method of claim 2 in which the probes of the array are arranged in a sequence in a line in a flow channel.

6. The method of claim 1 in which an analyte complex comprises a biological receptor probe and biological ligands carried to the probe by liquid flow.

7. The method of claim 1 in which a probe comprises oligonucleotide, peptide, polypeptide, protein or antibody, and the respective analyte comprises biomolecules that bind to the respective probe.

8. The method of claim 1 in which a complex includes a light-emissive tag associated with an analyte complex.

9. The method of claim 8 wherein an analyte complex includes a fluorescent tag.

10. The method of claim 9 in which the tag is a photo-excitable fluorescent compound that is stimulated to fluoresce by light from an external light source passing through a light-transmissive portion of the cassette.

11. The method of claim 10 in which the stimulating light and the light for detection pass through the same light-transmissive window of the cassette.

12. The method of claim 9 in which the tag is subject to electro-stimulation, an electrical pathway is provided in the cassette to the surface to which the complex is attached and the complex is stimulated by electro-stimulation via that pathway.

13. The method of claim 1 in which steps (a) and (b) are performed under automated control.

14. The method of claim 1 in which step (b) includes forcing the drying gas to flow over the enclosed surface for an interval of about one half minute or more.

15. The method of claim 5 in which step (b) includes forcing the drying gas to flow along the flow channel over complexes on the sequence of probes.

16. The method of claim 1 in which heat is delivered to the complex to promote volatilization of the liquid residue.

17. The method of claim 16 in which heat is delivered to the complex at least in part by heating the drying gas before it flows into the cassette to a temperature above ambient but below degradation temperature of the analyte complex or any associated tag.

18. The method of claim 16 in which heat is delivered to the complex at least in part by heating the surface to which the complex is bound to a temperature above ambient but below degradation temperature of the analyte complex or any associated tag.

19. The method of claim 18 comprising employing an external heater to heat the surface by thermal conduction from the exterior through a body portion of the cassette.

20. The method of claim 1 in which the surface with bound complex is a microscope slide or a microscope slide segment incorporated within the cassette.

21. The method of claim 1 in which drying gas flow into or out of the cassette flows through a device that prevents escape of liquid from the cassette.

22. The method of claim 1 in which during a liquid wash phase prior to drying, gas is caused to flow through a gas inlet channel to purge the channel of liquid contaminant.

23. The method of claim 22 in which bursts of such gas flow are introduced at spaced intervals during the wash phase.

24. An assay cassette which encloses a surface to which at least one probe is attached, the surface associated with a liquid passage that enables liquid flow over the probe, the cassette of the type enabling one or more liquids to flow over the probe to produce at the probe an analyte complex that has a constituent that is capable of emitting light that can pass through a window of the cassette for detection by an external detector, wherein, for use after formation of the analyte complex within the cassette, the cassette is constructed and arranged:

(a) to enable, by liquid flow, removal of liquid resident at the analyte complex on the enclosed surface and

(b) to enable a stream of drying gas to be forced to flow into the cassette, over the enclosed surface and out of the cassette during a drying interval under conditions that substantially volatilize and remove residue of the liquid associated with the analyte complex, to provide a dried complex within the cassette.

25. The cassette of claim 24 having an array of surface-attached probes enclosed within the cassette, the cassette constructed to enable formation and drying of an array of analyte complexes corresponding to the array of surface-attached probes and constructed to enable detection of the array of complexes within the cassette.

26. The cassette of claim 25 in which at least some of the probes are distributed transversely to the direction of liquid flow upon a width-wise extended surface, the liquid passage is correspondingly wide, constructed to produce a uniform liquid flow over the array, and the cassette is constructed to introduce the drying gas through a relatively narrow channel and to spread to flow through the liquid passage and over the array.

27. The cassette of claim 26 in which the extended surface is a planar surface carrying a two dimensional array of probes exposed to the liquid and drying gas flows.

28. The cassette of claim 25 in which the probes of the array are arranged in a sequence in a line in a flow channel.

29. The cassette of claim 24 in which a probe comprises a biological receptor probe for biological ligands carried to the probe by liquid flow.

30. The cassette of claim 29 in which a probes comprises oligonucleotide, peptide, polypeptide, protein or antibody.

31. The cassette of claim 24 containing a light-emissive tag material available to be associated with an analyte complex being formed in the cassette.

32. The cassette of claim 31 wherein the tag material is a fluorescent tag material.

33. The cassette of claim 32 in which the tag material is a photo-excitable fluorescent compound, and the cassette includes a light-transmissive portion for transmitting stimulating light from an external light source to an analyte complex that includes the tag material.

34. The cassette of claim 33 constructed to enable the stimulating light and the light for detection to pass through the same light-transmissive window of the cassette.

35. The cassette of claim 32 in which the tag material is subject to electro-stimulation, and an electrical pathway is provided in the cassette to the surface to which the probe is attached to enable a complex at the probe to be stimulated by electro-stimulation via that pathway.

36. The cassette of claim 24 constructed to enable (a) and (b) to be performed under automated control.

37. The cassette of claim 24 combined with a device adapted to force the drying gas stream to flow over the enclosed surface for an interval of about one half minute or more.

38. The cassette of claim 28 combined with a device adapted to force the drying gas stream to flow in the flow channel over a sequence of probes.

39. The cassette of claim 24 constructed to enable heat to be delivered to the complex to promote volatilization of the liquid residue.

40. The cassette of claim 39 combined with a device that heats drying gas to a temperature above ambient but below degradation temperature of the analyte complex or any associated tag.

41. The cassette of claim 39 constructed to enable heat to be delivered to the complex at last in part by heating the surface to which the complex is bound to a temperature above ambient but below degradation temperature of the analyte complex or any associated tag.

42. The cassette of claim 39 adapted for use with an external heater that heats by thermal conduction from the exterior through a body portion of the cassette.

43. The cassette of claim 24 in which the surface to which the probe is bound is a microscope slide or microscope slide segment incorporated within the cassette.

44. The cassette of claim 24 in which drying gas flow into or out of the cassette is arranged to flow through a device that prevents escape of liquid from the cassette.

45. The cassette of claim 24 combined with a control system constructed to produce gas flow during a liquid wash phase for removal of liquid contaminant.

46. The cassette of claim 45 constructed to produce bursts of the gas flow at intervals during the wash phase.

47. The assay cassette of claim 24 having a common passage for introducing liquid and drying gas flows over the surface to which a probe is attached, the cassette having a bubble removal system to which at least some of the liquids are exposed before reaching the common passage, there being multiple connections to the common passage substantially upstream of the surface with attached probe but downstream of the bubble removal system, the connections including an inlet for liquid flow from the bubble removal system and another inlet arranged to receive the drying gas stream.

48. The assay cassette of claim 47 in which there is a widening transition section between the common passage and the surface, the surface bearing an array of probes.

49. The assay cassette of claim 47 in which the bubble removal system comprises a buoyancy chamber through which liquids flow.

50. The assay cassette of claim 49 in which all of the liquids of the assay are forced to flow through a buoyancy chamber.

51. The assay cassette of claim 47 having storage volumes on board the cassette for all liquids employed in the assay.

52. The assay cassette of claim 24 combined with an air pump for producing the stream of drying air and a liquid pumping system for the liquids of the assay.

53. The assay cassette of claim 52 in which the liquid pumping system is a liquid diaphragm pumping system.

54. The cassette of claim 24 in which, the surface enclosed within the cassette supports immobilized binding domains of protein.

55. The cassette of claim 24 in which, the surface enclosed within the cassette supports immobilized binding domains of genomic nature, (oligonucleotides, SNPs, segments of genes, or other genetic material).

56. The cassette of claim 24 in which, the surface enclosed within the cassette supports immobilized binding domains of cells or cell lysate.

57. The cassette of claim 24 in which, the surface enclosed within the cassette supports immobilized binding peptides.

58. The cassette of claim 24 in which the surface enclosed within the cassette supports immobilized binding ligands comprising small organic molecules having molecular weight between about 500 to about 6000 Daltons.

59. The cassette of claim 24 in which the surface enclosed within the cassette supports immobilized binding ligands comprising small organic molecules comprising steroids, peptides or expression regulators comprising RNA primers or siRNAs.

60. An external reader station for an assay cassette which includes a system for causing liquid flows that cause formation of a light-emitting complex on a surface enclosed by the cassette and a detector for detecting light emitted from the complex that passes through a window of the cassette, the reader station including a source of pressurized drying gas and a control constructed to automatically produce a sustained stream of drying gas into and through the cassette, to dry the complex before light detection.

61. The external reader station of claim 60 constructed to automatically introduce one or more bursts of gas flow during a controlled wash phase for the cassette.

62. The external reader station of claim 60 including at least one heater for providing heat to the complex to promote drying.

63. The external reader station of claim 62 in which a heater is arranged to heat the drying gas before it enters the cassette.

64. The external reader station of claim 62 in which a heater is arranged to heat a surface of the cassette that is in heat transfers relation to the complex or to drying gas flowing to the complex.

65. The external reader station of claim 64 constructed, for drying, to heat a surface of the cassette to temperature above that employed during formation of the complex.

66. The external reader station of claim 60 including an air pump for producing the stream of drying air and a liquid pumping system for the liquids of the assay.

67. The external reader station of claim 66 in which the liquid pumping system comprises a linear actuator constructed and arranged to deflect a liquid pumping diaphragm of the cassette.