High-throughput screening assay systems in microscale fluidic devices

• US 6,429,025 B1
• Filed: 12/22/1998
• Issued: 08/06/2002
• Est. Priority Date: 06/28/1996
• Status: Expired due to Term

• Current Assignee: Caliper Holdings Incorporated
• Original Assignee: Caliper Holdings Incorporated
• Inventors: Kopf-Sill, Anne, Parce, John Wallace, Bousse, Luc J.
• Primary Examiner(s): Chin, Christopher L.
• Application Number:
  US09/214,035
• Time in Patent Office: 1,323 Days
• Field of Search: 436/28-29, 436/62, 436/149, 436/163, 436/806, 436/150, 436/514, 435/4, 435/29, 435/40, 435/291, 435/817, 435/810, 204/150.1, 204/601, 204/451, 204/400, 204/403, 422/68, 422/72, 422/73, 422/81, 422/312, 422/82, 422/57-58, 422/102, 422/99, 422/100
• US Class Current: 436/514
• CPC Class Codes: B01J 19/0093 Microreactors, e.g. miniaturised or microfabricated reactors laboratory containers with capillary fluid transport in microfabricated channels or chambers B01L3/5027
• CPC Class Codes: B01J 2219/00828 Silicon wafers or plates
• CPC Class Codes: B01J 2219/00831 Glass
• CPC Class Codes: B01J 2219/00833 Plastic
• CPC Class Codes: B01J 2219/00853 Employing electrode arrangements
• CPC Class Codes: B01J 2219/00952 Sensing operations
• CPC Class Codes: B01J 2219/00961 Temperature
• CPC Class Codes: B01J 2219/0097 Optical sensors
• CPC Class Codes: B01L 2200/027 for microfluidic devices
• CPC Class Codes: B01L 2200/0605 Metering of fluids
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2200/0673 Handling of plugs of fluid surrounded by immiscible fluid
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0861 Configuration of multiple channels and/or chambers in a single devices
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• 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 2300/0887 Laminated structure
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/0418 : electro-osmotic flow [EOF]
• CPC Class Codes: B01L 2400/0487 : fluid pressure, pneumatics
• CPC Class Codes: B01L 3/5025 : for parallel transport of multiple samples
• CPC Class Codes: B01L 3/502715 : characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• 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 3/502753 : characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation separation in general B01D; microapparatus for analysis using electrophoresis G01N27/44791; sample preparation G01N1/28
• CPC Class Codes: B01L 3/502761 : specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• 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
• CPC Class Codes: G01N 27/44791 : Microapparatus sample containers with integrated microfluidic structures B01L3/5027
• CPC Class Codes: G01N 33/5008 : for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/5011 : for testing antineoplastic activity
• CPC Class Codes: G01N 33/502 : for testing non-proliferative effects
• CPC Class Codes: G01N 33/5064 : Endothelial cells
• CPC Class Codes: G01N 33/5091 : for testing the pathological state of an organism
• CPC Class Codes: G01N 33/5097 : involving plant cells immunoassays of plant cells G01N33/56961
• CPC Class Codes: G01N 33/5304 : Reaction vessels, e.g. agglutination plates for solid-phase systems G01N33/543
• CPC Class Codes: G01N 33/57415 : of breast
• CPC Class Codes: G01N 35/08 : using a stream of discrete samples flowing along a tube system, e.g. flow injection analysis
• CPC Class Codes: Y10S 366/02 : Micromixers: segmented laminar flow with boundary mixing orthogonal to the direction of fluid propagation with or without geometry influences from the pathway
• CPC Class Codes: Y10S 435/81 : Packaged device or kit
• CPC Class Codes: Y10S 435/817 : Enzyme or microbe electrode
• CPC Class Codes: Y10S 436/804 : Radioisotope, e.g. radioimmunoassay
• CPC Class Codes: Y10S 436/805 : Optical property
• CPC Class Codes: Y10S 436/806 : Electrical property or magnetic property
• CPC Class Codes: Y10T 436/11 : Automated chemical analysis
• CPC Class Codes: Y10T 436/110833 : Utilizing a moving indicator strip or tape
• CPC Class Codes: Y10T 436/113332 : with conveyance of sample along a test line in a container or rack
• CPC Class Codes: Y10T 436/117497 : with a continuously flowing sample or carrier stream
• CPC Class Codes: Y10T 436/25 : including sample preparation

Abstract

The present invention provides novel microfluidic devices and methods that are useful for performing high-throughput screening assays. In particular, the devices and methods of the invention are useful in screening large numbers of different compounds for their effects on a variety of chemical, and preferably, biochemical systems.

Claims

1. An apparatus for screening a plurality of test compounds for an effect on at least a first biochemical component, the apparatus comprising:

(i) a body structure comprising at least two intersecting microscale channels disposed therein;

(ii) a source of the plurality of test compounds fluidly coupled to at least one of the at least two microscale channels, which source is coupled to the at least one of the at least two microscale channels via a capillary or microchannel; and

,

(ii) a source of the first biochemical component fluidly coupled to at least one of the at least two microscale channels.

2. The apparatus of claim 1, wherein at least one of the two microscale channels comprises at least one cross-sectional dimension between about 0.1 μ

m and about 500 μ

m.

3. The apparatus of claim 1, wherein the at least two microscale channels comprise transverse channels.

4. The apparatus of claim 1, the apparatus further comprising a plurality of microscale reaction channels fluidly coupled to one or both of the at least two microscale channels.

5. The apparatus of claim 4, wherein at least one member of the plurality of reaction channels comprises at least one cross-sectional dimension between about 0.1 μ

m and about 500 μ

m.

6. The apparatus of claim 4, wherein the plurality of microscale reaction channels comprises a plurality of parallel reaction channels disposed within the body structure and the at least two microscale channels are fluidly coupled to opposite ends of each of the parallel reaction channels.

7. The apparatus of claim 4, wherein the at least two microscale channels are disposed in the body structure in inner and outer concentric channels and the plurality of microscale reaction channels extends radially from the inner concentric channel to the outer concentric channel.

8. The apparatus of claim 4, wherein each of the reaction channels comprises a bead or particle resting well.

9. The apparatus of claim 4, wherein each of the reaction channels comprises a particle retention zone.

10. The apparatus of claim 9, wherein the particle retention zone comprises a particle retention matrix or a microstructural filter.

11. The apparatus of claim 4, wherein the source of the plurality of test compounds is fluidly coupled to each member of the plurality of microscale channels.

12. The apparatus of claim 1, wherein the source of the plurality of test compounds comprises a plurality of reservoirs disposed within the body structure.

13. The apparatus of claim 1, wherein the source of the plurality of test compounds comprises a pipettor element, which pipettor element is fluidly coupled to at least one of the at least two microscale channels during operation of the apparatus.

14. The apparatus of claim 1, wherein the capillary is fluidly connected at a first end to the at least one of the at least two microscale channels and at a second end to the source of the plurality of test compounds.

15. The apparatus of claim 1, wherein the source of the plurality of test compounds comprises a plurality of particles disposed within at least one of the at least two microscale channels.

16. The apparatus of claim 1, wherein the source of the plurality of test compounds comprises the plurality of test compounds.

17. The apparatus of claim 1, wherein the source of the first biochemical component is disposed in a surface of the body structure.

18. The apparatus of claim 1, wherein the body structure is made by heat laminating a first substrate having the at least two microscale channels etched therein to a second substrate which overlays the at least two microscale channels etched into the first substrate.

19. The apparatus of claim 18, wherein the first substrate and the second substrate comprise glass substrates.

20. The apparatus of claim 1, wherein the body structure comprises etched glass, etched silicon, or a molded polymer.

21. The apparatus of claim 20, wherein the body structure comprises etched silicon and a insulating layer disposed over the etched silicon.

22. The apparatus of claim 1, wherein the body structure is made by stamp-molding or injection-molding.

23. The apparatus of claim 1, wherein the at least first biochemical component comprises an enzyme and an enzyme substrate, which enzyme substrate produces a detectable signal when reacted with the enzyme.

24. The apparatus of claim 23, wherein the enzyme substrate is a chromogenic or fluorogenic enzyme substrate.

25. The apparatus of claim 1, wherein the at least first biochemical component comprises a receptor/ligand binding pair, wherein the receptor or the ligand comprises a detectable labeling group.

26. The apparatus of claim 25, wherein binding the receptor to the ligand produces a detectable signal.

27. The apparatus of claim 1, wherein the at least first biochemical component comprises one or both members of an antibody/antigen binding pair, wherein the antibody is specifically immunoreactive with the antigen.

28. The apparatus of claim 27, wherein the antibody or antigen comprises a detectable labeling group.

29. The apparatus of claim 25 or claim 28, wherein the detectable labeling group comprises a fluorescent group.

30. The apparatus of claim 1, wherein the first biochemical component of the plurality of test compounds is blood-derived or derived from a patient.

31. The apparatus of claim 1, further comprising a source of a second biochemical component, which source is fluidly coupled to at least one of the at least two microscale channels.

32. The apparatus of claim 1, further comprising a fluid direction system, which fluid direction system directs the first biochemical component through at least one of the at least two microscale channels and directs the plurality of test compounds from the source of the plurality of test compounds into the at least one of the at least two microscale channels.

33. The apparatus of claim 32, wherein the fluid direction system generates a continuous flow of the first biochemical component along the at least one of the at least two microscale channels and periodically injects a member of the plurality of test compounds into the at least one of the at least two microscale channels.

34. The apparatus of claim 33, wherein the fluid direction system injects a spacer into the at least one of the at least two microscale channels, which spacer separates different members of the plurality of test compounds.

35. The apparatus of claim 32, wherein the fluid direction system comprises:

at least three electrodes, each electrode being in electrical contact with the at least two microscale channels on a different side of an intersection formed by the at least two channels; and

,

a control system for concomitantly applying a variable voltage at each of said electrodes, whereby movement of the test compounds or the first biochemical component in the at least two channels is controlled.

36. The apparatus of claim 32, wherein the fluid direction system comprises a pressure source.

37. The apparatus of claim 36, wherein the pressure source comprises a pump.

38. The apparatus of claim 1, further comprising a fluid direction system and a source of a second biochemical component fluidly coupled to at least one of the two microscale channels, which fluid direction system generates a continuous flow of a mixture of the first biochemical component and the second biochemical component along at least one of the at least two microscale channels and periodically injects a member of the plurality of test compounds into the at least one of the at least two microscale channels.

39. The apparatus of claim 1, further comprising a detection zone in at least one of the at least two microscale channels for detecting an effect on the at least one biochemical component.

40. The apparatus of claim 39, wherein the detection zone comprises a detection window in the at least one of the at least two microscale channels.

41. The apparatus of claim 1, further comprising a detection system for detecting an effect on the at least one biochemical component.

42. The apparatus of claim 41, wherein the detection system comprises a fluorescent detection system.

43. The apparatus of claim 1, further comprising a plurality of electrodes in a plurality of reservoirs fluidly connected to one or more of the at least two microscale channels and a control system for concomitantly applying a voltage to each of the electrodes, whereby movement of the at least first biochemical component or a member of the plurality of test compounds in the at least two microscale channels is controlled.

44. The apparatus of claim 43, wherein the apparatus comprises an electrode interface, which electrode interface minimizes degradation of one or more chemical species present in plurality of reservoirs.

45. The apparatus of claim 44, wherein the apparatus further comprises one or more component for reducing electroosmotic flow selected from:

a frit on one or more member of the plurality of electrodes, which frit reduces electroosmotic flow towards the one or more member of the plurality of electrodes;

a channel between at least two members of the plurality of reservoirs, which channel limits diffusion of the one or more chemical species, the channel having low electroosmotic flow;

a filled channel between at least two members of the plurality of reservoirs, which filled channel comprises a matrix to limit transport of the one or more chemical species through the filled channel, the filled channel thereby having low electroosmotic flow;

a high reservoir having a fluid level higher than at least one low reservoir, which high reservoir is fluidly connected to the low reservoir, which low reservoir comprises an electrode, wherein fluid pressure between the high reservoir and the low reservoir reduces electroosmotic flow towards the electrode; and

,

a dual reservoir system with a first reservoir fluidly coupled through a connecting channel to a narrow diameter second reservoir adapted to receive at least one member of the plurality of electrodes, the narrow diameter second reservoir adapted to draw fluid by capillary electrophoresis towards the at least one member of the plurality of electrodes, thereby countering electroosmotic flow in the connecting channel.

46. A method of screening a plurality of test compounds for an effect on at least one biochemical component, the method comprising:

continuously flowing the at least one biochemical component through at least a first microscale channel;

contacting the at least one biochemical component with at least a first member of the plurality of test compounds; and

,

detecting an effect of the first member of the plurality of test compounds on the at least one biochemical component.

47. The method of claim 46, comprising continuously flowing the at least one biochemical component through the first microscale channel.

48. The method of claim 47, wherein detecting comprises continuously monitoring a detectable signal from the continuously flowing at least one biochemical component, the detectable signal comprising a steady state intensity, wherein the effect of the first member of the plurality of test compounds on the at least one biochemical component comprises a deviation from the steady state intensity of the detectable signal.

49. The method of claim 46, comprising periodically contacting the at least one biochemical component with different members of the plurality of test compounds.

50. The method of claim 46, comprising physically separating the different members of the plurality of test compounds.

51. The method of claim 50, comprising physically separating the different members of the plurality of test compounds with one or more spacer.

52. The method of claim 46, step (i) comprising flowing the at least one biochemical component into a plurality of microscale reaction channels and step (ii) comprising flowing a different member of the plurality of test compounds into each member of the plurality of microscale reaction channels.

53. The method of claim 46, wherein the at least one biochemical component comprises one or both member of an antibody/antigen binding pair, which antibody is specifically immunoreactive with the antigen.

54. The method of claim 53, wherein the antibody or the antigen comprises a detectable labeling group.

55. The method of claim 46, wherein the at least one biochemical component comprises one or both member of a receptor/ligand binding pair, which receptor specifically binds the ligand.

56. The method of claim 55, wherein the receptor or the ligand comprises a detectable labeling group.

57. The method of claim 54 or claim 56, wherein the detectable labeling group comprises a fluorescent group.

58. The method of claim 46, wherein the at least one biochemical component comprises an enzyme and an enzyme substrate, wherein action of the enzyme on the enzyme substrate produces a detectable signal.

59. The method of claim 46, wherein the at least one biochemical component comprises a biological barrier and the effect of the first member of the plurality of test compounds comprises an ability of the first member of the plurality of test compounds to traverse the barrier.

60. The method of claim 59, wherein the biological barrier comprises an epithelial or an endothelial layer.

61. The method of claim 46, wherein the at least one biochemical component comprises one or more cell and detecting comprises determining an effect of the first member of the plurality of test compounds on the one or more cell.

62. The method of claim 61, wherein the one or more cell produces a detectable signal corresponding to a cellular function and detecting comprises detecting an effect of the first member of the plurality of test compounds on the cellular function by detecting a level of the detectable signal.

63. The method of claim 61, wherein determining the effect of the first member of the plurality of test compounds on the one or more cell comprises detecting an effect on viability of the one or more cell.

64. The method of claim 46, wherein the at least one biochemical component or the plurality of test compounds is blood-derived or is derived from a patient.

65. The method of claim 46, wherein detecting comprises measuring a parameter of a biochemical system comprising the at least one biochemical component in the presence and absence of the first member of the plurality of test compounds, a change in the parameter indicating an effect on the biochemical system or biochemical component.

66. The method of claim 46, wherein the at least one biochemical component produces a detectable signal representative of a function of the biochemical component.

67. The method of claim 66, wherein the detectable signal comprises a fluorescent or colorimetric signal.

68. The method of claim 46, wherein the at least one biochemical component comprises an indicator compound which interacts with the first biochemical component thereby producing a detectable signal representative of a functioning biochemical component.