Assays based on liquid flow over arrays

• US 20060275852A1
• Filed: 10/27/2005
• Published: 12/07/2006
• Est. Priority Date: 06/06/2005
• Status: Abandoned Application

• Current Assignee: Avantra Biosciences Corporation
• Original Assignee: Avantra Biosciences Corporation
• Inventors: Tyburczy, Nathan, Rodionova, Natalia, Deweerd, Herman, Montagu, Jean I., Dowd, Roger, Maimonis, Peter
• Application Number:
  US11/262,115
• Publication Number:
  US 20060275852A1
• Time in Patent Office: Days
• US Class Current: 435/7.930
• CPC Class Codes: B01L 2200/027 for microfluidic devices
• CPC Class Codes: B01L 2200/0684 Venting, avoiding backpressure, avoid gas bubbles
• CPC Class Codes: B01L 2200/16 Reagents, handling or storing thereof
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0672 Integrated piercing tool
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2300/0877 Flow chambers
• CPC Class Codes: B01L 2300/1827 using resistive heater
• CPC Class Codes: B01L 2400/0481 squeezing of channels or chambers
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/0655 pinch valves
• CPC Class Codes: B01L 2400/0683 mechanically breaking a wall or membrane within a channel or chamber
• CPC Class Codes: B01L 2400/0688 surface tension valves, capillary stop, capillary break
• 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 3/50273 characterised by the means or forces applied to move the fluids micropumps F04B19/006, of the membrane type F04B43/043
• CPC Class Codes: B01L 3/502738 characterised by integrated valves microvalves F16K99/0001
• CPC Class Codes: B01L 3/502746 characterised by the means for controlling flow resistance, e.g. flow controllers, baffles B01L3/502738 takes precedence
• CPC Class Codes: B01L 7/00 : Heating or cooling apparatus autoclaves B01J3/04; Heat insulating devices
• CPC Class Codes: B01L 9/527 : for microfluidic devices, e.g. used for lab-on-a-chip
• CPC Class Codes: G01N 33/54366 : Apparatus specially adapted for solid-phase testing
• CPC Class Codes: Y10T 436/117497 : with a continuously flowing sample or carrier stream

Abstract

Cassette (50) performs assays, e.g. multiplexed protein biomarker assays. Wide, bubble-free, slow flows are produced from liquids stored on cassette (50), flowing over wide array (20) of ligand receptors on a capture surface. Flows of Reynolds Number less than about 1, preferably 1×10⁻¹ to 5×10⁻³, are heated in region (34) preceding and including bubble removal system (128). Analyte is introduced through compressed septum (32). External actuations of displacement pumps (30, 37) and valves (137 A, B, and C) produce flows in response to flow-front optical sensors (150, 152). Elastic sheet provides pump and valve diaphragms and resilient expansion of mixing volume (131). Break-away cover portions are pistons. Heating is by conduction through cassette from external contact heater. Planar cassette body, when tilted from horizontal, enables upward flow from pumped storage (134, 135) to reaction (133) to waste (139), with buoyancy bubble removal before reaction. Reading of fluorescence is by external reader, employing calibration, control and reference features on capture surface. Extensive set of calibration features of differing intensities enables self-calibration.

Claims

1. An assay cassette comprising:

a capture surface that carries an array of spaced-apart regions of ligand receptors, and

a liquid passage system constructed to direct over the array a slow flow of assay-supporting liquids having Reynolds numbers less than about 1, the liquids including a ligand-containing liquid,

the liquid passage system including a gas bubble removal system to which the liquids are exposed, the gas bubble removal system constructed and arranged to remove gas micro-bubbles from the liquids prior to exposure of the liquids to the array.

2. The assay cassette of claim 1 further comprising heat transfer surfaces to which flows of the liquids are exposed for heating the liquids prior to exposure of the liquids to the gas bubble removal system, whereby gas micro-bubbles produced in the liquid by heat delivered via the heat transfer surfaces of the cassette can be removed before the liquids are exposed to the array.

3. The assay cassette of claim 2 configured to receive heat from a heater of an external device, the cassette constructed and arranged to enable heat to flow through substance of the cassette to the heat transfer surfaces, thence to the liquid.

4. The assay cassette of claim 1 further comprising a site for storage of an agent useful in the assay, the cassette constructed to enable combining a liquid with the agent to produce an assay-supporting liquid, and, respectively, to enable flow of the assay-supporting liquid through the liquid passage system, or through the liquid passage system with exposure to a heat transfer surface, the cassette constructed to enable exposure of the assay-supporting liquid to the gas bubble removal system prior to reaching the array, whereby gas micro-bubbles previously produced in the liquid can be removed before the assay-supporting liquid is directed over the array.

5. The assay cassette of claim 4 in which the agent to be combined with the liquid is a ligand or a substance comprising a detectable tag.

6. The assay cassette of claim 4 in which the agent, prior to combining, is stored in dry state in a chamber of the cassette, with the agent exposed to air, the cassette constructed to enable introduction of the liquid to the chamber in combining action, and the gas bubble removal system is effective to remove micro-bubbles of the air produced by the combining action.

7. The assay cassette of claim 6 in which the chamber of the cassette has an elastically distensible wall portion adapted to elastically expand in response to liquid displaced into the chamber, and to elastically contract when liquid flows out of the chamber.

8. The assay cassette of claim 1 further comprising at least one liquid storage chamber, the liquid storage chamber associated with a displacement pump constructed to displace liquid in continuous flow from the storage chamber, respectively, to flow through the passage system or through the passage system with exposure to a heat transfer surface, the cassette constructed to enable exposure of the liquid to the gas bubble removal system prior to reaching the array, whereby gas micro-bubbles previously produced in the liquid can be removed before the liquid is directed in continuous flow over the array.

9. The assay cassette of claim 8 in which the displacement pump comprises an elastic diaphragm forming a wall portion of the liquid storage chamber and operable by an external, continuously movable actuator to produce the continuous flow.

10. The assay cassette of claim 9 in which an outlet to the passage system is located at the top of the liquid storage chamber exposed to air present in the chamber, the cassette and diaphragm constructed and arranged to expel air from the chamber followed by forcing liquid through the passage system in continuous flow via the gas bubble removal system and over the array.

11. The assay cassette of claim 8 in which the liquid storage chamber is constructed to contain a sealed pouch of liquid for the assay, the chamber associated with a device for puncturing the pouch to release the liquid.

12. The assay cassette of claim 8 in which the liquid storage chamber is constructed to receive and store a liquid introduced to the cassette from the exterior, whereby gas micro-bubbles produced in the liquid by the step of introduction of the liquid to the cassette can be removed by the gas bubble removal system before the liquid flows over the array.

13. The assay cassette of claim 12 in which the liquid storage chamber is constructed to receive the liquid from the exterior via a needle or pipette projected through a septum, the septum comprising an elastomeric mass that has a pierced passage, the elastomeric mass mounted under substantial compression relative to the pierced passage, the compression effective to maintain the pierced passage closed but enabling insertion and removal of a plastic liquid-supply needle or pipette through the pierced passage.

14. The assay cassette of claim 8 further comprising at least one waste chamber to which the liquid flows after being pumped in continuous flow by the displacement pump, exposed to the gas bubble removal system and directed over the array, whereby the liquid is contained within the cassette throughout the assay.

15. The assay cassette of claim 14 constructed and arranged so that during performance of the assay the capture surface and the liquid flow over it have an upward extent, and the waste chamber is positioned in the cassette to receive gravity flow of liquid that has passed over the capture surface.

16. The assay cassette of claim 15 having a generally planar extent and constructed to be disposed at a substantial angle to the horizontal during performance of the assay to dispose the capture surface to extend upwardly in the direction of the liquid flow to an upper end, and to locate a waste outlet for gravity flow from the upper end of the capture surface to the waste chamber.

17. The assay cassette of claim 1 in which the liquid passage system includes at least one actuatable valve through which liquid flows prior to being exposed to the gas bubble removal system, whereby gas micro-bubbles produced in the liquid by passage through the valve can be removed before the liquid is directed over the array.

18. The assay cassette of claim 17 in which the valve comprises a valve seat across which liquid flows, the valve seat defining inlet and outlet passages, and an elastic diaphragm extends over the valve seat and is displaceable to engage the valve seat to interrupt the flow.

19. The assay cassette of claim 18 in which the valve is a stop valve followed by a surface-tension burst valve, the burst valve being capable of blocking migrating liquid that may leak past the stop valve when the valve is closed, but, at flow pressure, capable of transmitting liquid to flow over the array.

20. The assay cassette of claim 1 in which the flow passage system is constructed and arranged to enable more than one slow flow of liquid of Reynolds number less than about 1 over the array after exposure of the liquids to the gas bubble removal system.

21. The assay cassette of claim 20 constructed to perform a sandwich assay in which liquid flows of the assay are exposed to the bubble removal system, the cassette comprising storage sites for liquid sample and all substances employed in the sandwich assay, the cassette having at least one waste chamber, the cassette being constructed and arranged to contain all liquids throughout the performance of the sandwich assay.

22. The assay cassette of claim 21 in which the capture surface is of extended width and carries a two-dimensional array of ligand-receptor regions comprised of spots of characteristic dimension between about 50 μ

m and 500 μ

m, and the liquid passage system includes a transition section preceding the capture surface that spreads the continuous, slow liquid flow to a width corresponding to the width of the capture surface.

23. The assay cassette of claim 22 in which the capture surface exposed to the slow liquid flow has dimensions of at least about 0.5 cm in the direction of the flow and in the direction transverse to the direction of flow.

24. The assay device of claim 22 in which the cross-section area of flow preceding the flow transition section is about 0.25 mm² or less and the cross-section area of the slow flow over the capture surface is at least 0.75 mm².

25. The assay cassette of claim 22 in which the capture surface carries at least 3 replicate regions of each of a multiplicity of ligand receptors arrayed transversely to the direction of flow of the liquid over the capture surface.

26. The assay cassette of claim 25 in which the array on the capture surface includes, in regions in proximity to the regions of a given ligand receptor, reference regions of known quantity of the ligand to which the receptor is specific.

27. The assay cassette of claim 1 in which the bubble removal system comprises at least one buoyancy chamber to which the liquid is exposed.

28. The assay cassette of claim 27 having a generally planar extent and constructed to be disposed at a substantial angle to the horizontal during performance of the assay to dispose the buoyancy chamber above a discharge outlet through which the liquid is directed to the capture surface.

29. The assay cassette of claim 28 in which the angle to the horizontal locates the capture surface above a flow transition passage that receives liquid from the buoyancy chamber and spreads the liquid flow to a width corresponding with the width of the capture surface, the transition passage constructed to direct the liquid in continuous upward flow over the capture surface.

30. The assay cassette of claim 29 in which the angle to the horizontal locates a waste outlet in position to receive the liquid following flow over the capture surface for gravity flow to a waste chamber in the cassette.

31. The assay cassette of claim 30 further comprising a liquid storage chamber having an outlet passage, the angle to the horizontal locating the storage chamber below the capture surface, the storage chamber associated with an externally actuatable displacement pump that is effective to force liquid from the storage chamber in continuous flow through the buoyancy chamber and upwardly over the capture surface to the waste outlet.

32. The assay cassette of claim 27 in which, in operating orientation, the buoyancy chamber has a top and bottom, a liquid inlet and a liquid outlet, both the liquid inlet and outlet being located near the bottom, in flow-aligned relationship.

33. The assay cassette of claim 27 constructed to enable initial filling of the buoyancy chamber by a liquid stored in the cassette, and to have a plurality of liquids of the cassette flow through the so-filled chamber in sequence, in laminar flow between the inlet and the outlet of the buoyancy chamber.

34. The assay cassette of claim 27 constructed to enable exposure of each increment of liquid flow to the buoyancy chamber for a period between about 1 and 5 seconds.

35. The assay cassette of claim 27 in which the buoyancy chamber comprises a depression molded in a face of a plastic body and channels molded in the face of the plastic body for liquid leading to and from the depression, and an adhesive sheet overlies the molded depression and the channels and is adhered to face portions of the molded body bounding the depression and the channels.

36. The assay cassette of claim 1 in which the bubble removal system, for a given flow, includes, in succession, at least two bubble capture zones to which the flow is exposed.

37. The assay cassette of claim 36 in which the bubble capture zones are constructed and arranged to enable bubbles to rise into the bubble capture zones by buoyancy effects.

38. The assay cassette of claim 37 comprising a buoyancy chamber constructed for liquid flow from an inlet, along a path exposed to enable bubbles to rise by buoyancy effects for capture, to an outlet, there being at least one divider wall spaced along and above the liquid path to define upstream and downstream bubble capture zones exposed to the path such that a large bubble in liquid flow at the inlet will tend to be trapped in the upstream capture zone, leaving the downstream capture zone free to receive liquid flow.

39. The assay cassette of claim 38 in which the divider wall terminates in an upward region above which liquid entering the downstream zone can fill the upstream zone above any bubble lodged in a lower portion of the upstream zone.

40. The assay cassette of claim 38 in which the buoyancy chamber comprises a depression molded in a plastic body, there being a molded upstanding rib defining the divider wall, and an adhesive sheet overlies the molded depression and is adhered to face portions of the molded body bounding the depression, and adhered to an outer edge of the molded rib.

41. The assay cassette of claim 3 having a molded body, the body defining at least one molded receptacle of depth suitable to hold an assay liquid and a molded face wall, the molded receptacle being open at a face-side plane and the molded face wall having an outer face generally aligned with the face-side plane, the outer face of the face wall having at least one molded channel forming a liquid passage for directing liquid from the receptacles to an assay region of the cassette, the face wall having a thickness substantially less than the depth of the receptacle and having a back surface at a heater cavity that is open from the backside of the body, the heater cavity constructed and arranged to removably receive a cooperatively constructed external heater to engage portions of the back surface of the face wall in surface-to-surface heat-transfer contact to heat liquid flowing in the molded channel by heat conduction through the thickness of the molded face wall.

42. The assay cassette of claim 41 having a chip-receiving opening defined in the face wall to receive and position an assay chip so that the chip bounds a reaction chamber into which a molded channel of the face wall directs liquid, the heater cavity in the molded body extending below the chip-receiving opening to expose the chip for surface-to-surface heat-transfer contact with the external heater to heat liquid in the reaction chamber by heat conduction through the thickness of the assay chip.

43. The assay cassette of claim 42 constructed to enable a portion of the backside of the chip to be exposed to a temperature sensor to control the energization of the heater.

44. The assay cassette of claim 41 in which a receptacle is an analyte-receiving receptacle arranged to discharge into a molded channel of heat-exchange contour, preferably of serpentine contour, in a portion of the face wall that is arranged to receive heat from the heater.

45. The assay cassette of claim 41 in which one side of at least one liquefying chamber is molded as a depression in a portion of the face wall through which a liquid channel directs liquid, this portion of the face wall having a back surface exposed for engagement by the external heater for surface-to-surface heat transfer contact to heat liquid in the liquefying chamber by heat conduction through the thickness of the molded face wall.

46. The assay cassette of claim 41 in which one side of a bubble removal device is molded as a depression in a portion of the face wall through which a liquid channel directs liquid, this portion of the face wall having a back surface exposed for engagement by the external heater for surface-to-surface heat transfer contact to heat liquid in the bubble removal device by heat conduction through the thickness of the molded face wall, preferably this bubble removal device being a liquid-filled bubble trap.

47. The assay cassette of claim 41 comprising a cover assembly secured over the receptacle and face-wall of the molded body, the cover assembly including an elastic diaphragm portion lying over the receptacle, the diaphragm portion adapted to be deflected to displace liquid from the receptacle through the molded channel, preferably another portion of the face wall is molded in the form of a valve seat and the cover assembly includes a diaphragm portion adapted to be deflected to engage the valve seat to stop flow.

48. The assay cassette of claim 41 in which the molded body is of generally planar extent and bound, at least substantially, by a perimeter wall of substantially constant depth extending between respective parallel planes.

49. The assay cassette of claim 41 in which the back surface of the face wall of the molded body is planar, preferably parallel to face-side and back-side planes of the cassette, and arranged to be engaged by a planar heat-delivering face of a heater, preferably the heater comprising a flexible, sheet-form resistance heater mounted on a resilient planar pad carried on a rigid, planar plate that is mounted in floating manner enabling the corresponding planar surfaces of the molded body and the heater to self-adjust into face-to-face heat-transfer contact.

50. The assay cassette of claim 1 having a molded body, the body including at least one molded receptacle of depth suitable to hold an assay liquid and a molded face wall, the molded receptacle being open at a face-side plane and the molded face wall having an outer face generally aligned with the face-side plane, the outer face of the face wall having at least one molded channel forming a liquid passage for directing liquid from the receptacle to an assay region of the cassette, the front surface of the face wall being planar and adhered to an adhesive side of an adhesive sheet, portions of the adhesive sheet lying over the channel in the face wall, closing the respective side of the channel.

51. The assay cassette of claim 50 in which the adhesive sheet carries adhesive on its oppositely directed sides, the adhesive sheet having at least one window corresponding to a liquid receptacle or valve seat, one adhesive side of the adhesive sheet being adhered to the face wall, the oppositely directed adhesive side adhered to an elastic diaphragm sheet, a portion of the elastic diaphragm sheet lying over the window defining a deflectable pump or valve diaphragm at the respective receptacle or valve seat in the molded body.

52. The assay cassette of claim 51 including a second adhesive sheet carrying adhesive on its oppositely directed sides, one adhesive side of the second adhesive sheet being adhered to the outer side of the diaphragm sheet and the oppositely directed adhesive side adhered to a relatively rigid cover member.

53. The assay cassette of claim 52 in which there is a window in the second adhesive sheet overlying a pump receptacle, and a breakaway portion of the cover overlying the diaphragm at the pump receptacle is constructed to break from the cover to act as pump piston head for deflecting the respective portion of the diaphragm in response to externally applied actuation force, preferably the break away portion of the cover being adhered to a corresponding outer surface portion of the diaphragm sheet.

54. The assay cassette of claim 1 constructed for use with a protocol which produces light-emitting tags associated with complexes of receptor and ligand, the cassette having a window constructed and arranged to enable reading of light emitted from the tags.

55. The assay cassette of claim 54 in which the capture surface comprises a nitrocellulose layer of less than about 1 micron thickness.

56. The assay cassette of claim 1 constructed as a disposable sandwich assay cassette for optical reading,

the cassette operable by external apparatus, and having:

a liquid storage chamber and associated displacement pump for producing a continuous flow of liquid sample containing an analyte ligand,

at least a second liquid storage chamber and an associated displacement pump for producing continuous flows of assay-supporting liquids for completing the assay,

a flow-through reaction chamber in which a capture surface of extended width is situated,

at least one waste chamber for receiving waste liquid from the reaction chamber,

the liquid passage system including a flow transition section which spreads the liquid flow to the width of the capture surface,

the capture surface carrying a two-dimensional array which includes spaced-apart replicate regions of ligand receptors, the capture surface being positioned and arranged for optical reading,

the liquid passage system comprising a flow network for directing flows of the sample and assay-supporting liquids through the reaction chamber, over the capture surface,

a venting arrangement for air displaced by liquid forced through the system,

and heat transfer surfaces arranged to receive heat to bring the liquids to about a desired assaying temperature prior to entering the bubble removal system and to maintain the reaction chamber at assaying temperature,

the liquid displacement pumps of the storage chambers, the flow network including the associated gas bubble removal system and transition section, and the reaction chamber constructed to produce relatively widened flows of Reynolds numbers less than about 1 of a sequence of liquids over the capture surface, thence to the waste chamber.

57. The assay cassette of claim 56 in which the heat transfer surfaces to which the liquids are exposed are in heat-transfer relationship to an exterior surface of the cassette, the exterior surface of the cassette adapted to be placed in heat-receiving relationship with a heater member of the external apparatus.

58. The assay cassette of claim 56 in which an agent for use in the sandwich assay is stored in dry state in a chamber of the cassette, with the agent exposed to air, the cassette having a heat transfer surface arranged to heat the chamber to about the desired assaying temperature and to enable introduction of the liquid to that chamber in combining action, the gas bubble removal system being effective to remove micro-bubbles of air produced by the heating and the combining action in the chamber.

59. The assay cassette of claim 56 in which the capture surface carries at least 3 replicate regions of each of a multiplicity of ligand receptors arrayed transversely to the direction of flow of the liquid through the reaction chamber.

60. The assay cassette of claim 59 in which the array on the capture surface includes, in regions in proximity to the regions of a given ligand receptor, reference regions of known quantity of the ligand to which the receptor is specific.

61. The assay cassette of claim 59 in which the ligand receptor is an antibody or antigen which is specific, respectively, to an antigen or antibody ligand in the sample.

62. The assay cassette of claim 1 in which the bubble removal system, in operating orientation, comprises an upwardly extending buoyancy chamber adapted to contain liquid and having a top, a liquid inlet and a liquid outlet, the outlet located lower than the top of the buoyancy chamber in position adapted to be submerged in liquid of the buoyancy chamber, there being a vent passage from the upper portion of the buoyancy chamber.

63. The assay cassette of claim 62 in which the buoyancy chamber communicates with an air vent associated with a waste chamber of the cassette until the buoyancy chamber receives liquid, a first surface-tension burst valve being associated with a passage leading from the liquid outlet from the buoyancy chamber, the first burst valve being constructed and arranged to be effective to prevent liquid flow beyond the buoyancy chamber until the chamber is filled with liquid, and the vent passage comprises an air-porous but liquid-blocked element located in the top region of the buoyancy chamber permitting air to exhaust from the chamber but blocking passage of liquid.

64. The assay cassette of claim 62 in which the buoyancy chamber communicates with an air vent associated with a waste chamber of the cassette until the buoyancy chamber is initially filled with liquid, a first surface-tension burst valve being associated with a passage leading from the liquid outlet from the buoyancy chamber, and a second surface-tension burst valve communicating with the top of the buoyancy chamber being associated with the vent passage, the first burst valve being constructed and arranged to be effective to prevent liquid flow beyond the buoyancy chamber until the chamber is filled with liquid, and the second burst valve being constructed and arranged to be effective to prevent liquid flow from the top of the buoyancy chamber after the buoyancy chamber is filled with liquid.

65. The assay cassette of claim 27 having a temperature control region constructed to enable all liquids associated with the assay to be brought approximately to an assaying temperature, the gas bubble removal system following the temperature control region to which the liquid is exposed prior to reaching the capture surface, and comprising an upwardly extending buoyancy chamber adapted to contain liquid and having a top, a liquid inlet and a liquid outlet, the outlet located lower than the top of the buoyancy chamber in position adapted to be submerged in liquid of the buoyancy chamber, there being a vent passage from the upper portion of the buoyancy chamber.

66. The assay cassette of claim 27 in which the liquid passage system is constructed to initially fill the buoyancy chamber with liquid from a first storage volume in a manner that a further flow passage to be connected to provide flow of another liquid through the buoyancy chamber can be isolated and remain empty during the filling of the buoyancy chamber, the buoyancy chamber sized to receive and contain air displaced from the empty passage when liquid is forced through the further passage on its way to the buoyancy chamber without exposing the liquid outlet from the buoyancy chamber to air filling the top of the buoyancy chamber.

67. The assay cassette of claim 1 comprising a generally planar molded body of rectangular form of length of about 8 cm. or less and width of about 5 cm. or less, constructed to be oriented with its longitudinal axis disposed at a substantial angle to the horizontal during use;

in such orientation, substantially the lower half of the body defining, in side-by-side manner, a storage chamber for a pouch of buffer liquid, a chamber for a detection ligand stored therein in desiccated form, and a chamber for a fluorescent tag agent stored therein in desiccated form;

a reaction chamber containing the capture surface located adjacent the opposite longitudinal end of the molded body, at least one storage chamber disposed laterally to one side of the reaction chamber, positioned to receive gravity flow of waste from the reaction chamber; and

a temperature control region in the form of a heated region arranged to heat liquids prior to the liquids entering the gas bubble removal system.

68. The assay cassette of claim 1 constructed to be disposed at an angle in assaying position relative to a rest position, in which a venting arrangement comprises an air vent communicating with the waste chamber, the air vent comprised of material that is permeable to air until wetted, the material located not to be wetted by liquid in the waste chamber when the cassette is in assaying position and to be wetted by liquid from the waste chamber when the cassette is placed in rest position after use.

69. The assay cassette of claim 1 constructed to be controllable by external apparatus the liquid passage system comprising a flow network which includes at least one sensing station adapted to receive an optical sensing beam and enable the beam to pass through a flow passage and thence to a detector in the manner that the beam at the detector is altered in detectable manner by arrival of a liquid-air interface in the flow passage at the sensing station, the altered beam useful as a control signal by the external apparatus during conduct of the assay.

70. A method of conducting an assay comprising providing the assay cassette of claim 1 and external apparatus suitable to control the assay, introducing a sample to a sample chamber of the cassette, and, according to a predetermined assay protocol, conducting the assay under control by the external apparatus, including continuously flowing the sample over the capture surface at Reynolds numbers less than about 1 for a selected duration, and reading the capture surface of the cassette.

71. A method of conducting a sandwich assay comprising providing the assay cassette of claim 56 and external apparatus suitable to control the assay, wherein the capture surface carries an array of replicate regions of ligand receptor specific to a ligand of an analyte molecule, introducing a liquid sample that includes the analyte to the sample chamber and, according to a predetermined sandwich assay protocol suitable for optical reading, under control of the external apparatus, at Reynolds numbers less than about 1, causing, sequentially, continuous flow through the reaction chamber of the sample at a predetermined flow rate for a predetermined time, and continuous flows of assay-supporting liquids for appropriate times, and optically reading the capture surface of the cassette.

72. The method of claim 71 wherein the capture surface carries replicate deposits of receptor ligands in the form of an antigen or antibody specific to an analyte molecule, and the sample contains the analyte molecule.

73. A method for determining the concentration of at least one analyte in a liquid sample employing only liquids contained in a cassette, comprising the steps of:

(a) providing a cassette according to claim 1, the cassette including;

(i) a capture surface having, for each analyte, immobilized binding agent having replicate binding sites specific for the analyte, the binding agent being divided into a set of at least 3 spatially separated locations on the capture surface; and

(ii) a liquid developing system capable of providing at least one liquid for developing the complex of analyte and binding agent by attaching thereto a signal-producing tag in manner to quantitatively indicate by strength of signal the amount of analyte bound to each location;

(b) inserting the liquid sample into a storage volume in the cassette;

(c) producing from the stored sample a continuous flow at controlled rate of the liquid sample over the capture surface for a predetermined interval to enable binding of the at least one analyte to the respective locations of binding agent;

(d) developing the complexes of analyte and binding agent at the sites by producing from the liquid developing system stored on the cassette at least one continuous flow at controlled rate of liquid over the capture surface for predetermined duration, sufficient to bind the tag to complexes at the locations in manner to provide quantitative indication of the amount of analyte bound to each of the locations;

(e) with liquid stored on the cassette, washing the capture surface to remove unbound material capable of producing false signal;

(f) measuring signal produced by the tag at locations on the capture surface to obtain a value representing the fraction of binding sites occupied by the analyte at each location; and

(g) performing an operation on the values for the set of locations to determine a value of the concentration of the analyte in the liquid sample.

74. The method of claim 73 in which each increment of the liquid flows is exposed to heating, and to a bubble removal region for a period of between about 1 and 5 seconds before flowing over the capture surface.

75. The method of claim 73 in which each set of locations comprises at least 5 locations and the operation performed on the set of values comprises discarding at least one highest and one lowest value and employing intermediate values to determine a mean value.

76. The method of claim 73 in which the developing system comprises a detection agent capable of binding at each location in manner based on the quantity of analyte bound at the location, and a signal-producing tag capable of binding to the detection agent, the method including producing, in sequence, continuous controlled flows of a liquid containing the detection agent and a liquid containing the tag.

77. The method of claim 76 in which the binding agent is an antigen or antibody and the analyte is, respectively, an antibody or antigen.

78. The method of claim 73 in which the tag is a fluorescent tag and measuring is performed by exciting the tag and measuring the resultant fluorescence.

79. The method of claim 73 in which the at least 3 locations are distributed in a row across the width of a flow path for the continuous flow over the capture surface.

80. The method of claim 79 including at least one row of locations bearing preformed calibration deposits of analyte of predetermined concentration, which are developed, measured, and employed to correlate the signal with concentration and to determine that the system has performed correctly.

81. The method of claim 80 in which there are calibration locations having analyte deposits of differing known concentrations.

82. The method of claim 73 in which the flows over the capture surface are at Reynolds numbers of the order of 1 or lower.

83. The assay cassette of claim 1 enabling self-calibration, the capture surface bearing a set of replicate deposits of a given ligand receptor for the assay and bearing, in association with that set, a set of calibration deposits that comprises a number of groups of replicate deposits of the ligand for which the ligand receptor is specific, the groups being of respectively different known dilutions of the ligand, the known dilutions selected, when developed, to be sufficient to define a calibration curve for assay measurements made at the deposits of the given ligand receptor after their exposure to a sample containing the ligand, the groups of calibration deposits being adapted to be developed by attachment of a readable tag to all ligand on the capture surface at the deposits of ligand.

84. The assay cassette of claim 83 including at least one row of control deposits of given measurable intensity on the capture surface for verification of operation of the measuring system.

85. The assay cassette of claim 83 in which the deposits comprise spots in a spotted array.

86. The assay cassette of claim 83 in which the tag is a fluorescent tag.

87. The assay cassette of claim 83 constructed for exposure of the capture surface to a sheet-form stream of sample and reagent having a direction of flow, the replicate deposits being spots arranged in at least one row oriented transverse to the direction of the flow, and the groups of calibration deposits being spots arranged in rows transverse to the direction of the flow.

88. The assay cassette of claim 87 in which the replicate deposits of ligand receptor are in a single row transverse to the flow, and calibration deposits of each given dilution are arranged in a single respective row transverse to the flow.

89. The assay cassette of claim 88 in which the capture surface is of extended width carrying, in transverse arrangement, deposits of more than one ligand receptor and associated calibration deposits.

90. An assay method comprising providing an assay cassette according to claim 83, exposing the capture surface to a flow of sample containing the ligand followed by exposing the capture surface to conditions by which a readable tag becomes attached to all ligand present, reading the tag by a reader to obtain measurements of each deposit, analyzing the data from the group of calibration deposits to develop a table of calibration values, comparing a value derived from the group of ligand receptors with values from that table and deriving therefrom a value representing the concentration of the ligand in the analyte.

91. The assay method of claim 90 in which the tag is a fluorescent tag, and the reader is a fluorescence reader.

92. The method of claim 90 in which the ligand receptor is an antigen or antibody and the analyte is respectively an antibody or antigen.

97. The method of claim 96 implemented by use of the cassette of claim 1.

93. A cassette for conducting an assay, the cassette comprising a solid surface carrying an array of spots of ligand receptor of diameter between about 50 micron and 500 micron, with spacing between spots at least about equal to the diameter of the spots, the array having a width greater than about 0.5 cm, the solid surface bearing the array constructed and arranged as one side of a flow passage having a width exceeding the width of the array, and a dimension of the gap between the surface bearing the array and an opposed, parallel, flow-confining surface of between about 80 and 300 micron, a pumping and passage system constructed to create a succession of flows through the width of the flow passage at Reynolds number less than about 1 of liquid sample containing ligand of interest and of developing liquid, or a succession of liquids, capable of attaching detectable tags to spots to which ligand of interest has attached, the cassette constructed to enable reading the detectable tags of the array.

94. The cassette of claim 93 in which the gap is between about 100 and 200 micron and the pumping and passage system is constructed to provide the flows at Reynolds number between about 1×

10<sup>−

1</sup> and 5×

10<sup>−

3</sup>.

95. In a method of conducting an assay employing an array of spots on a solid surface, the steps of (a) providing on a solid surface an array of spots of ligand receptor of diameter between about 50 micron and 500 micron, with spacing between spots at least about equal to the diameter of the spots, the array having a width greater than about 0.5 cm;

(b) arranging the solid surface bearing the array as one side of a flow passage having a width exceeding the width of the array, and a dimension of the gap between the surface bearing the array and an opposed, parallel, flow-confining surface of between about 80 and 300 micron;

(c) creating a succession of flows through the width of the flow passage at Reynolds number less than about 1 of liquid sample containing ligand of interest and of developing liquid, or a succession of liquids, capable of attaching detectable tags to spots to which ligand of interest has attached; and

(d) reading the detectable tags of the array.

96. The method of claim 95 in which the gap is between about 100 and 200 micron and the flow has a Reynolds number between about 1×

10<sup>−

1</sup> and 5×

10<sup>−

3</sup>.