Assays Based on Liquid Flow over Arrays

• US 20110207621A1
• Filed: 02/20/2009
• Published: 08/25/2011
• Est. Priority Date: 02/21/2008
• Status: Abandoned Application

• Current Assignee: Avantra Biosciences Corporation
• Original Assignee: Avantra Biosciences Corporation
• Inventors: Rodionova, Natalia A., Tyburczy, Nathan, Montagu, Jean I., Deweerd, Herman
• Application Number:
  US12/918,757
• Publication Number:
  US 20110207621A1
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: B01L 2200/0684 Venting, avoiding backpressure, avoid gas bubbles
• 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 2300/0672 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 2400/0481 squeezing of channels or chambers
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/0683 mechanically breaking a wall or membrane within a channel or chamber
• CPC Class Codes: B01L 3/5023 with a sample being transported to, and subsequently stored in an absorbent for analysis
• CPC Class Codes: B01L 3/502723 characterised by venting arrangements
• CPC Class Codes: B01L 3/502784 specially adapted for droplet or plug flow, e.g. digital microfluidics automatic analysis using a stream of discrete samples in a tube system G01N35/08

Abstract

Flow-through assay reaction chamber (6) of cassette has back and forth liquid mixing in narrow gap (G) over array of capture agent (S), with net flow advance to waste confinement (19), produced by reversible pumps (3 or 12), operable with rolling diaphragm action with at least limited elastic recovery that advance sample or buffer liquids through conditioning paths (4A, 8, 8′, 9, 14, 15, 15′) before reaching the reaction chamber (6). A single pump produces accurate flow control, liquid conditioning, e.g., liquefying dry reagent from internal surfaces of flow-dividing material (14a, 15A, 15A′, e.g. open cell foam or frit), heating (4A), and air bubble removal (8, 8′, 9), as well as replenishment of reagent while accomplishing mixing within the flow-through reaction chamber (6). Lower viscosity buffer liquid is arranged to propel higher viscosity reagent, the flow-dividing storage material preserving reagent concentration. A blister pack (11) acts as a reversible pump (12) in producing accurate forward and backward flows with the net flow advance. Cascaded bubble traps (8, 9) on the cassette render the system tolerant of minor pumping error during cassette priming.

Claims

1. A method of storing desiccated biological molecules or similar reagent on a flow-dividing material filling the transverse cross-section and a substantial length of a storage passage within a cassette, including so selecting and sizing the material in the passage that in the presence of a liquefying agent that can form reagent liquid with the desiccated reagent, a displacing flow of a viscosity different from that of the reagent liquid, due to the flow-dividing effect of the material, produces plug-like flow of the reagent liquid.

2. The method of claim 1, wherein the flow dividing material is a porous material.

3. The method of claim 2, wherein the porous material is an open cell foam or a frit.

4. The method of claim 1, wherein the flow dividing material defines a multiplicity of parallel flow sub-channels.

5. The method of claim 1, wherein the material is preformed into sets of segments sized to fit in respective sections of a passage of the cassette and, for a particular assay that has been selected, a set of the segments receive selected reagents for the respective assay, which are dried and stored, ready to be installed in the respective passages in the cassette when required.

6. A method of producing a flow of a reagent liquid, comprising storing a reagent according to the method of claim 1, and subjecting flow through the flow-dividing material to forward and backward oscillations during its forward progress out of the storage passage.

7. A cassette having a reaction chamber constructed to conduct a reaction related to an assay, the cassette including buffer liquid storage, a buffer liquid displacement pump for displacing liquids at Reynolds number less than 1 through a passage system, the passage system including a buffer delivery passage for buffer liquid displaced by the pump, a reagent storage passage having extended length in the direction of flow relative to the maximum dimension of its transverse cross-section and capable of storing a liquid reagent of viscosity relatively higher than the viscosity of the buffer liquid, and a relatively small flow cross-section reagent delivery passage leading from the reagent storage passage to the reaction chamber, the buffer delivery passage arranged to deliver displaced buffer liquid into the reagent storage passage, wherein a substantial majority of the length of the reagent storage passage is filled with flow-dividing porous material, or is defined by a multiplicity of substantially parallel flow sub-channels, the porous material or sub-channels providing a multiplicity of paths along the reagent storage passage of transverse cross sections that are small relative to the over-all transverse cross section of the reagent storage passage and distributed across its cross-section and along its length to establish, in response to the pump'"'"'s displacement of buffer liquid, plug-like flow of the relatively higher viscosity reagent liquid from the reagent storage passage into the reagent delivery passage.

8. The cassette of claim 7, wherein the cassette further comprises a positive displacement pump arranged to push liquid through the multiplicity of paths defined by the porous material or sub-channels within the reagent storage passage.

9. The cassette of claim 7, wherein the surface of the porous material or the sub-channels is hydrophilic.

10. The cassette of claim 7, wherein the surface of the porous material or the sub-channels is hydrophilic for supporting reagent material dried thereon, and has a releasable property for the reagent when contacted with liquid, a dried layer of reagent material disposed on the hydrophilic surface, exposed to contact with buffer liquid flowing into the reagent passage to enable the reagent material to be liquefied in situ, to create the relatively viscous reagent liquid that is subject to the plug-like flow.

11. The cassette of claim 7, wherein the substantial majority of the length of the reagent storage passage is filled with the porous material and the size of pores of the porous material is between about 5 to 200 micron.

12. The cassette of claim 11 in which the size of the pores is selected from the group of materials comprising material having a nominal pore size of 30 micron, with variation plus or minus 50%, and material having a nominal pore size of 100 micron, with variation plus or minus 20% .

13. The cassette of claim 7, wherein the reagent storage passage is of rectangular transverse cross-section and porous material of sheet-form open cell foam or frit closely fits the cross-section over more than half of the length of the reagent storage passage.

14. The cassette of claim 13, wherein the reagent storage passage containing porous material is a channel of substantially constant transverse cross-section, of length at least about 60 mm and channel width and depth of about 2 mm and 0.6 mm, respectively.

15. The cassette of claim 7, wherein the substantial majority of the length of the reagent storage passage is filled with flow-dividing porous material.

16. The cassette of claim 15 in which the porous material comprises hydrophilic frit formed of polyethylene.

17. The cassette of claim 15 in which the porous material comprises hydrophilic melamine foam.

18. The cassette of claim 15 in which the porous material comprises hydrophilic polyurethane foam.

19. The cassette of claim 15 in which the porous material comprises porous nitrocellulose in treated state that enables release of deposited bio-material when contacted with liquid.

20. The cassette of claim 19 in which the treated state comprises coating on the nitrocellulose of a mediating substance such as a blocker protein.

21. The cassette of claim 15 in which the porous material comprises hydrophilic polystyrene foam in treated state that enables release of deposited bio-material when contacted with liquid.

22. The cassette of claim 7, wherein a substantial majority of the length of the reagent storage passage is defined by a multiplicity of substantially parallel flow sub-channels each having transverse cross-section dimensions less than 1 mm.

23. The cassette of claim 22 in which the transverse cross-section dimensions are less than about 0.5 mm.

24. The cassette of claim 23 in which the transverse cross-section dimensions are in the range of about 0.5 mm and 0.01 mm.

25. The cassette of claim 7, wherein the reagent storage passage is defined by a multiplicity of sub-channels formed by a molded or extruded resin bearing a hydrophilic surface.

26. The cassette of claim 7, wherein the reagent is a detection reagent.

27. The cassette of claim 26, wherein the detection reagent is an antibody or antigen.

28. The cassette of claim 7, wherein the reagent is a label reagent.

29. The cassette of claim 28, wherein the label reagent includes a fluorescent dye.

30. A method of delivering liquid reagent to a reaction chamber via a reagent delivery passage by displacing reagent liquid from a storage passage by a buffer liquid of viscosity that is low relative to the viscosity of the reagent liquid, comprising

(a) providing a cassette according to claim 7, wherein either the reagent has been provided in liquid form to the cassette, or has been stored in the cassette in dried form and subsequently liquefied to provide the reagent liquid;

and

(b) operating a buffer pump to establish plug-like flow of the relatively higher viscosity reagent liquid from the reagent storage passage into the reagent delivery passage.

31. The method of claim 30 in which the reagent has been stored in the cassette in dried form as a dried layer on a hydrophilic surface of a porous flow-dividing material within the reagent storage passage, or on a hydrophilic surface of a multiplicity of parallel flow sub-channels forming the reagent storage passage, initially operating a buffer displacement pump in manner to introduce buffer liquid into the reagent storage passage to liquefy the reagent, the resulting reagent liquid remaining stored in the cassette, and subsequently operating the buffer pump to pump buffer liquid into the porous material or multiplicity of sub-channels to establish plug-like flow of the relatively higher viscosity reagent liquid from the reagent passage into the reagent delivery passage for supply to the reaction chamber.

32. The method of claim 31 in which the dried reagent layer comprises detection or label bio-material.

33. The method of claim 31, including employing backward and forward oscillations of the liquid with net forward advance, to effectively provide flow to the reaction chamber.

34. A cassette having a liquid storage, pumping and passage system and a reaction chamber, the cassette constructed to conduct a reaction related to an assay by flow of liquids with Reynolds number less than 1 through the system and over a capture surface within the reaction chamber, and constructed to exclude air from reaching the reaction chamber until completion of reactions of the assay, the storage, pumping and passage system including:

an analyte chamber constructed to receive an analyte-containing liquid, an analyte displacement pump for displacing analyte-containing liquid through the system and reaction chamber,

a first buoyancy bubble trap arranged to be filled by displaced analyte-containing liquid, and

a passage leading from the first bubble trap to the reaction chamber;

the storage, pumping and passage system also including;

pre-filled buffer liquid storage,

a buffer liquid displacement pump for displacing liquids through the system and the reaction chamber,

a buffer delivery passage for buffer liquid displaced by the buffer liquid displacement pump,

a reagent storage passage containing a dried reagent and capable of storing the reagent in liquid form when it is liquefied,

a reagent delivery passage leading from the reagent storage passage for flow to the reaction chamber, the buffer delivery passage arranged to deliver displaced buffer liquid into the reagent passage and, alternatively, through a wash passage for flow to the reaction chamber, and

a second buoyancy bubble trap arranged to be filled by displaced buffer liquid and arranged for flow from the reagent store passage to flow through it the discharge of the second bubble trap connected to flow through the first bubble trap (9) and thence to the reaction chamber.

35. The cassette of claim 34, wherein the flow from the wash passage also flows through the second bubble trap, thence through the first bubble trap to the reaction chamber.

36. The cassette of claim 34, wherein a passage is associated with a detector for the air-liquid interface of liquid entering the passage, enabling an external pump and associated control unit responsive to the detector to fill that passage to a predetermined point.

37. The cassette of claim 34, wherein a passage is arranged to fill the second bubble trap by operating the buffer displacement pump over a predetermined pumping volume that results in leaving an indeterminate volume of un-displaced air upstream of the first bubble trap, within a range determined by the predetermined range of flow volume error of the buffer displacement pump, the first bubble trap sized to receive and store said un-displaced air.

38. The cassette of claim 34, wherein there are at least two passages connectible to be filled by the buffer pump by respective operations of the pump, leaving air in each of the respective passages each of these passages arranged to enable its flow to pass through the second bubble trap, the second bubble trap sized to hold the maximum volume of air that may remain within each passage connected to it together with air from liquids passing through the bubble trap.

39. The cassette of claim 38, wherein the at least two passages merge into a common passage leading to the second bubble trap without passing through a valve.

40. The cassette of claim 34, wherein the first bubble trap has an air holding volume of about 10 microliters, and the second bubble trap has an air holding volume of about 50 microliters.

41. The cassette of claim 34, wherein the buffer displacement pump comprises a blister pack containing buffer liquid, a surface of the blister pack being deflectable by an actuator (P) external of the cassette to progressively displace liquid from the blister pack.

42. The cassette of claim 34, wherein the analyte displacement pump comprises a rolling elastic diaphragm pump.

43. A method of conducting an assay employing the cassette of any claim 34, the cassette having storage passages for both a detection reagent and a label reagent.

45. The cassette of claim 34, wherein the buffer pump is in the form of a blister pack filled with buffer fluid, the blister pack having a cover and a volume-defining blister body, the body capable of progressive collapse between a driving piston (P) external of the cassette and an anvil surface to produce a positive liquid displacement pumping action to force liquid forward into the passage system.

46. The cassette of claim 45 in which the cover is adhered and sealed about a piercing device disposed on the anvil surface, and capable of being deformed to be pierced by the device for releasing liquid to a channel associated with the piercing device.

47. The cassette of claim 46, wherein the cover is a metal foil comprised of aluminum of thickness of about 0.001 inch.

48. The cassette of claim 45, wherein the body of the blister pack is capable of elastic recovery upon retraction of the piston sufficient to produce a negative liquid pumping action to draw liquid back within the passage system.

49. The cassette of claim 48, in which the body of the blister pack is defined by a draw-formed sheet that comprises a layer of aluminum, the blister pack subject to permanent deformation when compressed to reduce the blister pack volume and displace liquid from the blister pack forward into the passage system of the cassette in a forward pumping action, for a backward pumping action for a limited distance following forward pumping action, residual elastic recovery of the permanently deformed aluminum wall of the blister body to a less deformed position permitted by progressive retraction of the piston serving as the driving force to increase the volume of the blister pack, drawing liquid back into the blister pack.

50. The cassette of claim 49, in which the blister pack has a volume of about 2 ml and the elastic recovery permitted by progressive retraction of the actuator produces an increase in the volume of the previously deformed blister pack by at least 3 microliters.

52. The method of claim 51, in which the blister pack is constructed according to claim 45.

44. A method of conducting an assay with a cassette having the components indicated below and operated substantially according to the following protocol:

1. Insert analyte liquid in analyte chamber 2 via septum 1

2. Close valves 18 &

17 (wash passage 37 and tag reagent chamber 15)

3. Open valve 16 (detection reagent chamber 14)

4. Operate buffer pump 12 (rotating stepper motor, depressing piston of buffer pump) to

5. Impale pouch 11 on pyramid 30 to release buffer liquid

6. Continue operation of buffer pump 12, (depressing piston and compressing pouch 11) to fill detection reagent passage 14 until

7. Opto-sensor 13 triggers

8. Close valve 16

9. Open valve 17

10. Operate buffer pump 12 a predetermined number of stepper motor steps to fill tag reagent chamber 15 and slightly beyond within error tolerance. Stop.

11. Close valve 17

12. Open valve 18

13. Operate buffer pump 12 a predetermined number of stepper motor steps to fill wash passage 37 and bubble trap 8 and slightly beyond within error tolerance. Stop

14. Close valve 18

15. Operate analyte pump 3 to fill bubble trap 9 until Opto-sensor 5 triggers

16. Continue operation of analyte pump 3 to flow analyte liquid through reaction chamber 6 per protocol.

17. Open valve 18 and operate buffer pump 12 to wash reaction chamber 6 with buffer liquid per protocol

18. Close valve 18

19. Open valve 16 and operate buffer pump 12 to flow detection reagent through reaction chamber 6 per protocol

20. Close valve 16

21. Open valve 18 and operate buffer pump 12 to wash reaction chamber 6 with buffer liquid per protocol

22. Close valve 18

23. Open valve 17 and operate buffer pump 12 to flow tag reagent through reaction chamber 6 per protocol

24. Close valve 17

25. Open valve 18 and operate buffer pump 12 to wash reaction chamber 6 per protocol

26. Prepare chip for imaging.

27. Image the biochip through the window of the reaction chamber 6 and send data to computer for analysis

28. THE END.

51. A method of pumping liquid within a cassette employing a deformable metal blister pack including progressively compressing and permanently deforming a body of the blister pack with an actuator (P) to displace liquid forward, and periodically reversing the movement of the actuator and allowing limited elastic recovery of the permanently deformed blister body to maintain contact with the rearward moving actuator, the increase in volume of the deformed blister pack drawing liquid back into the blister pack.

53. A system for conducting an assay employing a cassette having a liquid displacement pump actuated by an external actuator (P) according to a predetermined automatic pumping protocol, the cassette having a liquid passage system and a reaction chamber having inlet and discharge ends associated respectively with inlet and discharge passages, the cassette constructed to conduct a reaction related to an assay by pumped flow of liquids with Reynolds number less than 1 through the passage system and over a capture surface within the reaction chamber, through the discharge passage to a waste receptacle from which there is no return, wherein;

a control system responsive to the pumping protocol drives the pump in a cyclic operation with forward pumping and backward pumping phases in repeating cycles, the forward pumping phase arranged to produce flow through the reaction chamber out the discharge end, through the discharge passage to the waste receptacle and the backward pumping phase arranged to produce backward flow withdrawing liquid from the inlet end of the reaction chamber and the discharge passage, the net flow per cycle according to the predetermined protocol being in the forward direction out of the discharge end for substantial discharge of liquid to the waste receptacle, and replenishing flow of the liquid to which the capture surface is exposed.

54. The system of claim 53, in which the pump comprises a deformable container having a wall that is resilient within at least a limited elastic range, the container arranged to be compressed by motion of an external actuator (P) and, for backward pumping for a limited distance following forward pumping, the recovery of the wall within its elastic range, to a less deformed position as permitted by retraction of the actuator (P), serving to increase the volume of the container to draw liquid backward into the container, resulting in drawing liquid backward through the inlet of the reaction chamber.

55. The system of claim 54, in which the container comprises a blister pack, the body of the blister pack (which may be defined by a formed sheet that comprises a layer of aluminum) subject to permanent deformation by compression of the body by the external actuator (P) to reduce the volume of the blister pack and displace liquid forward from it.

56. The system of claim 54, wherein the container contains a pre-packaged buffer liquid.

57. The system of claim 53, wherein the pump is a rolling diaphragm pump associated with a storage chamber.

58. The system of claim 57 in which, wherein the storage chamber is an analyte chamber, the analyte chamber associated with a septum for insertion of analyte fluid into the chamber as a preliminary step prior to conducting the assay with the cassette.

59. The system of claim 53, wherein an upwardly extending discharge passage at the discharge end of the reaction chamber terminates at a point of gravity fall of discharge into a waste chamber, the discharge passage sized to contain at least a volume equal to the volume of liquid drawn backward through the inlet during the rearward flow phase of a pumping cycle, so that the backward flow occurs without exposing the reaction chamber to air.

60. The system of claim 53, wherein the reaction chamber and total back flow per cycle determined by the pumping protocol are of substantially the same volume.

61. The system of claim 60, wherein the volume is about 4 microliters.

62. The system of claim 53, wherein the reaction chamber is defined by a capture surface and opposed window spaced apart by a flow gap G of between about 50 and 300 micron, the width (W) and length (L) of the capture surface and opposed window being substantially greater than the dimension (G) of the flow gap, the inlet passage and the discharge passage being of substantially different flow cross-section profile from that of the reaction chamber.

63. The system of claim 62, wherein the depth (G) of the gap between the capture surface and opposed window is of the order of 100 micron, their width (W) being about 4 mm and their length (L) about 12 mm.

65. The system of claim 53, wherein the predetermined pumping protocol provides a forward flow to backward flow volume ratio in the range of about 3/1 to 3/2.

66. The system of claim 65 in which the ratio is about 2/1.

67. The system of claim 53, wherein the flows in both directions are at about the same volumetric flow rates, the forward flow phase lasting longer, e.g., about twice as long as the backward flow phase.

68. The system of claim 53, wherein the flows in the two directions are different, e.g., the forward flow phase having about twice the volumetric flow rate of the backward flow phase.

69. The system of claim 53, wherein the cycles of operation include cycles having dwell phases during which the pump does not pump liquid.

70. The system of claim 53, wherein the control system for producing the set of protocol operations comprising the back and forth flows with net flow advance includes a machine readable medium having instructions stored therein which, when executed, cause the system to perform this set of operations in accordance with the pumping protocol.

71. The system of claim 70 including at least one linear pump actuator driven by a stepper motor to perform the operations.

72. The system of claim 71 in which the linear pump actuator is positioned to drive a pump within an assay cassette, the pump preferably operable with a rolling diaphragm action with at least limited elastic recovery.

73. The system of claim 53, wherein the cyclically operating pump propels the liquid through a conditioning region that conditions the liquid prior to the liquid reaching the reaction chamber.

74. The system of claim 73, wherein the conditioning region includes provisions for heat exchange with the pumped liquid.

75. The system of claim 74 adapted for biological assay in which the heat exchange is regulated to heat the liquid to about 37°

C.

76. The system of claim 73, wherein the conditioning region includes a system for removing gas bubbles from the pumped liquid.

77. The system of claim 73 wherein the pumped liquid passes through a region in which a substance is exposed to the pumped liquid.

78. The system of claim 77, wherein the substance to be exposed to the liquid is a dried substance distributed through the body of flow-dividing open cell foam or frit through which the pumped liquid is directed.

79. The system of claim 78, wherein the open cell foam or frit fills a reagent storage passage of length in the flow direction greater than at least 10 times the largest transverse dimension of the storage passage.

80. The system of claim 79, wherein the reagent storage passage is of rectangular cross-section transverse to the direction of flow and porous material of sheet-form open cell foam or frit fills the cross section of the passage over more than half of the length of the reagent storage passage.

81. The system of claim 79 wherein the storage passage has an open plenum volume at each end into which liquid displaced through the porous material enters.

82. A method of conducting an assay employing the cassette or system of claim 53.

83. The method of claim 82, conducted in manner to cause liquid containing analyte to move in forward and backward directions over the capture surface with net forward flow to the waste receptacle.

84. The method of claim 83, in which the capture surface comprises an array of replicate spots (S) of a given capture reagent arranged transversely to the axis of flow over the capture surface.

85. The method of claim 82 wherein, following pumping of liquid containing analyte to flow over the capture surface in the reaction chamber, the pumping is stopped and a buffer pump is actuated to force buffer liquid to displace a reagent liquid in a reagent storage passage to cause reagent liquid to flow through the reaction chamber.

86. The method of claim 85, wherein the buffer pump is actuated to cause liquid containing reagent to move in forward and backward directions in the reagent storage passage to produce mixing while causing net forward flow of liquid through a reagent delivery passage and the reaction chamber to the waste receptacle.

87. The method of claim 85 conducted with a cassette having a reagent storage passage containing flow-dividing porous material that provides a multiplicity of interlaced flow paths along the reagent storage passage, the flow paths being open to one another and of transverse flow cross-sections that are small relative to the over-all transverse cross-section of the reagent storage passage and the flowpaths distributed across the transverse cross-section of the storage passage and along its length.

88. The method of claim 87 in which the porous material comprises open cell foam or frit.

89. The method of claim 87, conducted with a cassette in which a desiccated reagent is distributed through the porous material.

90. The method of claim 88, wherein the presence of the porous material is effective to produce substantially a plug-like flow of reagent liquid from the reagent storage passage into a reagent delivery passage in response to forward pumping of a buffer liquid.

64. A pumping control system for causing flow of liquid at Reynolds number less that 1 through a reaction chamber to progressively expose an assay capture surface to the liquid, wherein the control system is responsive to a predetermined pumping protocol to drive a pump in a cyclic operation with forward and backward pumping phases in repeating cycles, the forward pumping phase arranged to produce flow through the reaction chamber and out a discharge end, through a discharge passage to waste confinement and the backward pumping phase arranged to produce backward flow withdrawing liquid from an inlet end of the reaction chamber and from the discharge passage, the net flow per cycle according to the predetermined protocol being in the forward direction out of the discharge end for discharge of liquid to the waste confinement, and replenishing fresh liquid to the reaction chamber, preferably the pump located on a cassette that encloses the reaction chamber and preferably the waste confinement is a waste receptacle enclosed within the cassette.

91. An assay cassette having flows limited to Reynolds number NR_e less than 1, comprising a flow mixing channel extending in a general direction and connected to supply reagent to a reaction chamber, the channel filled for a substantial length with a three-dimensional mass of open cell foam or frit to cause fluid flowing in the channel to split into a multiplicity of relatively small flows along differing interlaced flow paths, the paths having flow components transverse to the general direction of the channel along with flow components in the direction of the flow channel, the individual flow paths varying in direction relative to one another and being open to interchange with each other effective to produce a substantially chaotic mixing effect upon liquid flowing into and through the open cell foam or frit material, the output of the channel arranged to supply flow of the thus-mixed liquid to the reaction chamber.

92. The cassette of claim 91 wherein, within the reaction chamber, there is a solid capture surface carrying an array of replicate spots (S) of capture reagent for capturing a reagent carried in the flow from the channel.

93. The cassette of claim 91, wherein surface within the foam or frit is hydrophilic and a desiccated biological agent is supported on the surface, exposed to be hydrated by flow of liquid through the foam or frit.

94. The cassette of claim 93 in which the channel is connected to receive flow of a buffer liquid of viscosity substantially less than the viscosity of reagent exiting the foam or frit material.

95. The cassette of claim 92, wherein the size of pores of the open cell foam or frit is between about 5 to 200 micron.

96. The cassette of claim 95, wherein the size of the pores is selected from the group of open cell foam or frit materials having a nominal pore size of 30 micron, with variation plus or minus 50%, and materials having a nominal pore size of 100 micron, with variation plus or minus 20%.

97. The cassette of claim 92, wherein the flow mixing channel has a transverse cross-section and porous material of sheet-form foam or frit closely fits the transverse cross-section over substantially more than half of the length of the flow mixing channel.

98. The cassette of claim 97 in which the channel is of substantially constant transverse cross-section, of length at least about 60 mm and channel width and depth of about 2 mm and 0.6 mm, respectively.

99. A cassette having a flow-through assay reaction chamber constructed for back and forth liquid mixing in a narrow gap (G) over an array of capture agent (S), with net flow advance to waste confinement produced by a reversible pump, preferably operable with rolling diaphragm action with at least limited elastic recovery, that advances sample or buffer liquids through conditioning paths before reaching the reaction chamber, the pump producing accurate flow control, liquid conditioning, e.g. liquefying dry reagent from internal surfaces of flow-dividing material, heating, and air bubble removal, as well as replenishment of reagent while accomplishing mixing within the flow-through reaction chamber;

in the case of the pumping of buffer liquid;

preferably lower viscosity buffer liquid is arranged to propel higher viscosity reagent liquid, the flow-dividing storage material preserving the concentration of the reagent;

a blister pack on the cassette containing buffer liquid acts as the reversible pump in producing accurate forward and backward flows with the net flow advance; and

cascaded bubble traps on the cassette render the system tolerant of minor pumping error during cassette priming.

100. A cassette having a liquid storage, pumping and passage system and a reaction chamber, the cassette constructed to conduct a reaction related to an assay by flow of liquids with Reynolds number less than 1 through the system and over a capture surface within the reaction chamber, the cassette constructed to be stored with air-filled passages prior to use, but, after initial entry of analyte-containing liquid into the reaction chamber, constructed to exclude air from reaching the reaction chamber until completion of reactions of the assay, the storage, pumping and passage system including:

an analyte chamber constructed to receive an analyte-containing liquid, an analyte displacement pump for displacing analyte-containing liquid through the system and reaction chamber,

a first buoyancy bubble trap arranged to be filled by displaced analyte-containing liquid, and

a passage leading from the first bubble trap to the reaction chamber;

the storage, pumping and passage system also including;

pre-filled buffer liquid storage,

a buffer liquid displacement pump for displacing liquids through the system and the reaction chamber, the buffer liquid displacement pump having a predetermined range of flow volume error,

a buffer delivery passage for buffer liquid displaced by the buffer liquid displacement pump,

a reagent storage passage containing a dried reagent and capable of storing the reagent in liquid form when it is liquified,

a reagent delivery passage leading from the reagent storage passage for flow to the reaction chamber, the buffer delivery passage arranged to deliver displaced buffer liquid into the reagent passage and, alternatively, through a wash passage for flow to the reaction chamber, and

a second buoyancy bubble trap arranged to be filled by displaced buffer liquid;

the reagent storage passage adapted to be filled by the buffer pump by activation for a predetermined pumping volume that results in leaving an indeterminate volume of un-displaced air in the buffer delivery passage of volume within a range determined by the predetermined range of flow volume error of the buffer displacement pump,

the second buoyancy bubble trap sized to hold the maximum volume of air that can remain in the reagent storage passage due to the buffer pump operating for the predetermined pumping volume at the lowest flow volume within its predetermined range of flow volume error together with air released by liquid flowing through the second bubble trap,

the discharge of the second bubble trap connected to flow through the first bubble trap and thence to the reaction chamber, the first bubble trap sized to hold residual air residing between the first and second bubble traps together with air released from the flow of liquids through it.

101. A cassette, system or method in which a reagent storage channel, defined by surfaces, through which liquid flows is filled over a predetermined length with flow-dividing material, the material defining surfaces throughout the material on which dried reagent is deposited, the surfaces throughout this material in aggregate having surface area at least 10 fold greater than the aggregate surface area of the surfaces defining the portion of the channel that is filled by the material.

102. A cassette, system or method in which flow-dividing storage material has internal surfaces carrying a deposit of dried reagent.

103. A cassette, system or method in which the flow-dividing material has length in the direction of flow at least 10 times the width of the material and a width that is at least twice the thickness of the material.

104. The cassette, system or method of claim 103 in which the material is of sheet form of thickness less than 1 mm.

105. A method of priming a cassette passage of known volume with liquid comprising providing in the cassette a pump in the form of a blister pack capable of rolling diaphragm action and containing buffer liquid, and with a linear actuator, displacing the back of the blister pack a predetermined distance inward to displace buffer liquid to fill the known volume.

106. The method of claim 105 in which the linear actuator is driven by a stepper motor and the predetermined distance is controlled by advancing the stepper motor a predetermined number of steps.

107. A method of conducting a flow assay by advancing liquid through a narrow flow gap (G) reaction chamber including the step of providing a storage channel containing open cell foam or frit on the internal surfaces of which reside a predetermined layer of dried reagent, introducing liquid to the storage chamber to liquefy the reagent to a known reagent concentration and advancing the liquid of known reagent concentration through the flow gap (G).

108. The method of claim 107, wherein the reagent is advanced by directing a displacing flow of lower viscosity buffer liquid into the open cell foam or frit.

109. The method of claim 107, wherein advancing of the flow is periodic.

110. The method of claim 109 in which the flow is caused to move rearwardly periodically in manner preserving net forward advance of the flow through the gap.