Apparatus and methods for conducting assays and high throughput screening

• US 7,378,280 B2
• Filed: 11/16/2001
• Issued: 05/27/2008
• Est. Priority Date: 11/16/2000
• Status: Expired due to Fees

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Unger, Marc A., Chou, Hou-Pu, Scherer, Axel, Quake, Stephen R., Thorsen, Todd A.
• Primary Examiner(s): SINES, BRIAN J
• Application Number:
  US10/416,418
• Publication Number:
  US 20040115838A1
• Time in Patent Office: 2,384 Days
• Field of Search: 422/50, 422/68.1, 422/81, 422/82, 422/100, 422/101, 422/103, 436/43, 436/63, 436/174, 436/177, 436/180, 435/4, 435/325
• US Class Current: 436/63
• CPC Class Codes: B01D 2313/08 Flow guidance means within the module or the apparatus
• CPC Class Codes: B01D 2313/60 Specific sensors or sensor arrangements
• CPC Class Codes: B01D 57/02 by electrophoresis treatment of water, waste water, sewage or sludge by electrophoresis C02F1/469; electrophoretic production of compounds or non-metals C25B7/00; investigating or analysing materials by using electrophoresis G01N27/26
• CPC Class Codes: B01D 61/18 Apparatus therefor
• CPC Class Codes: B01D 61/28 Apparatus therefor
• CPC Class Codes: B01D 63/081 Manufacturing thereof
• CPC Class Codes: B01D 63/088 Microfluidic devices comprising semi-permeable flat membranes
• CPC Class Codes: B01F 25/433 Mixing tubes wherein the shape of the tube influences the mixing, e.g. mixing tubes with varying cross-section or provided with inwardly extending profiles
• CPC Class Codes: B01F 25/4331 Mixers with bended, curved, coiled, wounded mixing tubes or comprising elements for bending the flow
• CPC Class Codes: B01F 27/00 Mixers with rotary stirring devices in fixed receptacles magnetic mixers B01F33/45; Kneaders
• CPC Class Codes: B01F 31/65 the materials to be mixed being directly submitted to a pulsating movement, e.g. by means of an oscillating piston or air column
• CPC Class Codes: B01F 33/30 Micromixers
• CPC Class Codes: B01F 33/81 Combinations of similar mixers, e.g. with rotary stirring devices in two or more receptacles
• CPC Class Codes: B01F 33/813 mixing simultaneously in two or more mixing receptacles
• CPC Class Codes: B01L 2200/0631 Purification arrangements, e.g. solid phase extraction [SPE]
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2300/0627 Sensor or part of a sensor is integrated
• CPC Class Codes: B01L 2300/0645 Electrodes
• CPC Class Codes: B01L 2300/0681 Filter
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• 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 2300/123 : Flexible; Elastomeric
• CPC Class Codes: B01L 2300/1822 : using Peltier elements
• CPC Class Codes: B01L 2300/1827 : using resistive heater
• CPC Class Codes: B01L 2400/0415 : electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/0481 : squeezing of channels or chambers
• CPC Class Codes: B01L 2400/0487 : fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/0633 : with moving parts
• CPC Class Codes: B01L 2400/0655 : pinch valves
• CPC Class Codes: B01L 2400/082 : Active control of flow resistance, e.g. flow controllers
• CPC Class Codes: B01L 3/502707 : characterised by the manufacture of the container or its components by shaping or joining plastic parts B29C59/00, B29C65/00; by laminating B32B37/00; manufacture of microstructural devices in general B81C
• 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 3/502738 : 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 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: C12M 23/16 : Microfluidic devices; Capillary tubes integrated microfluidic structures B01L3/5027; microreactors B01J19/0093
• CPC Class Codes: C12M 41/46 : of cellular or enzymatic activity or functionality, e.g. cell viability
• CPC Class Codes: F04B 43/043 : Micropumps
• CPC Class Codes: F04B 43/14 : having plate-like flexible members
• CPC Class Codes: F16K 2099/0074 : using photolithography, e.g. etching
• CPC Class Codes: F16K 2099/008 : Multi-layer fabrications
• CPC Class Codes: F16K 2099/0084 : Chemistry or biology, e.g. "lab-on-a-chip" technology
• CPC Class Codes: F16K 99/0001 : Microvalves microdevices B81B1/00; manufacture or treatment of devices or systems in or on a substrate B81C1/00; microfluidic structures B01L3/5027; micropumps F04B19/006
• CPC Class Codes: F16K 99/0009 : the valve element held by multiple arms
• CPC Class Codes: F16K 99/0015 : Diaphragm or membrane valves
• CPC Class Codes: F16K 99/0046 : using magnets
• CPC Class Codes: F16K 99/0048 : using piezoelectric means
• CPC Class Codes: F16K 99/0051 : using electrostatic means
• CPC Class Codes: F16K 99/0059 : actuated by a pilot fluid
• CPC Class Codes: G01N 15/06 : Investigating concentration of particle suspensions by weighing G01N5/00; investigating sedimentation of particle suspensions G01N15/04; investigating individual particles G01N15/10
• CPC Class Codes: G01N 27/44743 : Introducing samples
• CPC Class Codes: G01N 27/44791 : Microapparatus sample containers with integrated microfluidic structures B01L3/5027
• CPC Class Codes: G01N 33/00 : Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
• CPC Class Codes: G01N 33/5005 : involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025
• 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 33/56966 : Animal cells
• CPC Class Codes: Y10T 436/11 : Automated chemical analysis
• CPC Class Codes: Y10T 436/117497 : with a continuously flowing sample or carrier stream
• CPC Class Codes: Y10T 436/25 : including sample preparation
• CPC Class Codes: Y10T 436/25375 : Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
• CPC Class Codes: Y10T 436/2575 : Volumetric liquid transfer

Abstract

The present invention provides microfluidic devices and methods for using the same. In particular, microfluidic devices of the present invention are useful in conducting a variety of assays and high throughput screening. Microfluidic devices of the present invention include elastomeric components and comprise a main flow channel; a plurality of branch flow channels; a plurality of control channels; and a plurality of valves. Preferably, each of the valves comprises one of the control channels and an elastomeric segment that is deflectable into or retractable from the main or branch flow channel upon which the valve operates in response to an actuation force applied to the control channel.

Claims

1. A method of treating a cell, comprising:

(a) providing a microfluidic device comprising;

(i) a flow channel comprising a detection region;

(ii) a plurality of control channels; and

(iii) a pair of storage valves operatively disposed with respect to the flow channel, wherein each storage valve comprises one of the control channels, which control channel is separated from the flow channel by an elastomeric membrane, the elastomeric membrane being deflectable into the flow channel in response to an actuation force applied to the control channel, the two control channels of the storage valves being disposed relative to one another such that deflection of the elastomeric membrane of the respective control channels into the flow channel forms a holding space within the flow channel;

(b) introducing a sample containing one or more cells into the flow channel;

(c) actuating the storage valves to hold at least one of the cells within the holding space; and

(d) contacting the cell or cells within the holding space with a solution containing one or more agents.

2. The method of claim 1, wherein the elastomeric membranes of the storage valves each comprise one or more protrusions, the protrusions adapted to allow for solution to flow through the holding space once the elastomeric membranes are deflected into the flow channel while retaining the cell(s) within the holding space.

3. The method of claim 1, wherein the flow channel is formed within an elastomeric material and a segment of the flow channel opposite the elastomeric membrane of the storage valves comprises one or more elastomeric protrusions, the protrusions adapted to allow for solution to flow through the holding space once the elastomeric membranes are deflected into the flow channel while retaining the cell(s) within the holding space.

4. The method of claim 1, wherein the flow channel is formed within an elastomeric material;

the elastomeric segments of the storage valves comprise one or more protrusions;

a segment of the flow channel opposite the elastomeric membrane of each storage valve comprises one or more protrusions, and wherein the protrusions of the elastomeric membrane and the protrusions of the branch flow channel are adapted to allow for solution to flow through the holding space once the elastomeric membranes are deflected into the branch flow channel while retaining the cell(s) within the holding space.

5. The method of claim 1, wherein the microfluidic device further comprises a chamber adapted for storing solution in fluid communication with the flow channel and the method further comprises transporting solution and/or cells back and forth between the chamber and the holding space.

6. The method of claim 1, wherein the flow channel is in communication with a pump, and wherein the sample is transported through the flow channel under the action of the pump.

7. The method of claim 6, wherein the pump comprises at least three of the control channels, each formed within an elastomeric material and separated from the flow channel by an elastomeric membrane, each membrane being deflectable into the flow channel in response to an actuation force, whereby sample is transported along the flow channel.

8. The method of claim 1, wherein the flow channel comprises a cell enrichment section that selectively retains cells of interest, and the method further comprises transporting the sample through the enrichment section.

9. The method of claim 8, wherein the enrichment section contains a ligand that specifically binds to a receptor on target cells.

10. The method of claim 1, further comprising detecting an event and/or agent within the detection region with a detector.

11. The method of claim 10, wherein the detection region includes the holding space.

12. The method of claim 10, wherein the detection region is located at a section of the flow channel other than the holding space.

13. The method of claim 10, wherein the detector detects an optical signal within the detection region.

14. The method of claim 13, wherein the detector detects a fluorescence emission, fluorescence polarization or fluorescence resonance energy transfer.

15. The method of claim 13, wherein the detector is an optical microscope, a confocal microscope or a laser scanning confocal microscope.

16. The method of claim 10, wherein the detector is a non-optical sensor selected from the group consisting of a temperature sensor, a conductivity sensor, a potentiometric sensor and an amperometric sensor.

17. The method of claim 1, wherein the assay step comprises flowing a solution through the holding spaces after the storage valves have been actuated.

18. The method of claim 17 wherein the solution flowed through the holding spaces is culture medium for the cells within the holding space.

19. The method of claim 17, wherein the solution comprises a cellular stain.

20. The method of claim 1, further comprising detecting cellular morphology.

21. The method of claim 1, further comprising conducting a cell reporter assay.

22. The method of claim 1, further comprising detecting binding between a ligand and a cell receptor.

23. The method of claim 1, further comprising measuring cell membrane potential.

24. The method of claim 1, further comprising detecting a toxic effect on cells.

25. The method of claim 24, wherein the effect is cell proliferation, dysfunction of mitochondrial membrane potential, caspase activation, or cytochrome c release from cells.

26. The method of claim 1, wherein the treatment is cell lysis.

27. The method of claim 1, further comprising conducting an antimicrobial assay.

28. A method of treating a cell, comprising:

(a) providing a microfluidic device comprising;

(i) a main flow channel;

(ii) a plurality of branch flow channels, each branch flow channel being in fluid communication with the main flow channel;

(iii) a plurality of control channels; and

(iv) a plurality of storage valves, wherein a pair of storage valves are operatively disposed with respect to each of the branch flow channels, and wherein, each of the storage valves comprises an elastomeric segment that separates one of the branch flow channels and one of the control channels and that is deflectable, into or retractable from the flow channel upon which the valve operates in response to an actuation force applied to the control channel, the storage valves of a pair being disposed with respect to one another such that when the elastomeric segment of each storage valve of the pair extends into the branch flow channel a holding space is formed between the segments in which one or more cells can be retained;

(b) introducing a solution containing one or more cells into each of the branch flow channels;

(c) actuating the storage valves within each of the branch flow channels to form the holding space to hold at least one of the cells, within the sample introduced into the branch flow channel; and

(d) contacting the cell or cells within the holding space with a solution containing one or more assay agents.

29. The method of claim 28, wherein different cells are introduced into different branch flow channels and the contacting step comprises contacting the cells within each branch flow-channel with the same solution.

30. The method of claim 28, wherein cells of the same type are introduced into each of the flow channels and the contacting step comprises contacting the cells within different branch flow channels with different assay solutions.

31. A method for conducting an assay, comprising:

(a) providing a microfluidic device comprising;

(i) a main flow channel;

(ii) a plurality of branch flow channels, each in fluid communication with the main flow channel and comprising a detection region;

(iii) a plurality of control channels; and

(iv) a plurality of valves operatively disposed with respect to the main flow channel and/or the plurality of branch flow channels to regulate flow of the first and second solutions therethrough, wherein each of the valves comprises one of the control channels and an elastomeric segment that is deflectable into or retractable from the main or branch flow channel upon which the valve operates in response to an actuation force applied to the control channel, the elastomeric segment when positioned in the flow channel restricting solution flow therethrough;

(b) introducing a first assay solution into the main flow channel and a second assay solution into each of the plurality of branch flow channels;

(c) actuating one or more of the valves to control flow of the first and second assay solutions through the main and branch flow channels, whereby the first and second assay solutions are brought into contact with one another to form an assay solution mixture; and

(d) monitoring each of the assay solution mixtures for a desired property within the detection region.

32. The method of claim 31, wherein the control channel of each valve is separated from the flow channel in which the valve operates by the elastomeric segment.

33. The method of claim 32, wherein:

the method is a method for screening a plurality of target agents for the desired property;

the first assay solution is a solution comprising one or more assay agents and the second assay solution is a solution composing one of the test compounds;

the introducing step comprises introducing a different test compound into each of the plurality of branch flow channels; and

the monitoring step comprises identifying at least one test compound having the desired activity.

34. The method of claim 32, wherein the microfluidic device further comprises a first inlet in fluid communication with the main flow channel and a second inlet for each branch flow channel, and wherein the first assay solution is introduced via the first inlet and the second assay solution introduced via the second inlet.

35. The method of claim 32, wherein the microfluidic device further comprises a plurality of pumps, each pump being operatively disposed with respect to one of the branch flow channels, and wherein the method further comprises actuating the pump of each branch channel to transport the first and/or second assay solution through the branch flow channel.

36. The method of claim 35, wherein each of the plurality of pumps comprises a at least three control channels each formed within an elastomeric material and separated from one of the branch flow channels by a section of an elastomeric membrane, the membrane being deflectable into the branch flow channel from which it is separated in response to an actuation force.

37. The method of claim 33, wherein the microfluidic device further comprises a plurality of mixers, each mixer being operatively disposed with respect to one of the branch flow channels, and wherein the method further comprises mixing the first assay solution and the second assay solution with the mixer to form the assay solution mixture prior to the assaying step.

38. The method of claim 37, wherein the mixer comprises

(i) an inlet section and an outlet section

(ii) a looped flow channel in fluid communication with the inlet and outlet section;

and

(iii) two sets of control channels, each set comprising at least three control channels, and each control channel separated from the looped flow channel by an elastomeric membrane that is deflectable into the looped flow channel in response to an actuation force, and wherein the mixing step comprises transporting the first and second assay solution into the mixer via the inlet section and mixing the first and second solution in the looped flow channel by applying the actuation force to the control channels within a set as part of a repeating sequence.

39. The method of claim 33, wherein the microfluidic device further comprises a plurality of temperature controllers, each temperature controller being operatively disposed with respect to one of the branch channels, and the method further comprises actuating the temperature controller to regulate the temperature of the first assay solution, the second assay solution and/or the assay solution mixture.

40. The method of claim 39, wherein the temperature controller is disposed to regulate the temperature of the assay solution mixture within the detection region.

41. The method of claim 39, wherein the microfluidic device further comprises a plurality of mixers, each mixer being operatively disposed with respect to one of the branch flow channels, and wherein the method further comprises mixing the first assay solution and the second assay solution with the mixer to form the assay solution mixture prior to the assaying step and wherein the temperature controller is disposed to regulate the temperature of the assay solution mixture within the mixer.

42. The method of claim 33, wherein the microfluidic device further comprises a plurality of separation units, each separation unit being positioned within one of the branch flow channels, and wherein the method further comprises

(e) transporting a mixture of the first and second assay solutions through the separation unit whereby one or more unwanted compounds are removed to form the assay solution mixture;

and

(f) transporting the assay solution mixture to the detection region.

43. The method of claim 33, wherein the microfluidic device further comprises a plurality of mixers and a plurality of separation units, each mixer being operatively disposed with respect to one of the branch flow channels, and each separation unit being positioned within one of the branch flow channels, and wherein the method further comprises (e) mixing the first assay solution and the second assay solution with the mixer;

(f) transporting the mixture of the first and second assay solutions through the separation unit whereby one or more unwanted compounds are removed to form the assay solution mixture; and

(g) transporting the assay solution mixture to the detection region.

44. The method of claim 43, wherein each separation unit comprises a semi-permeable membrane that separates one of the branch flow channels and a collection channel, the membrane allowing passage of certain agents in the first or second assay solution or a mixture thereof flowing through the branch flow channel to pass through the membrane and into the collection flow channel.

45. The method of claim 43, wherein the separation unit comprises a separation material that separates molecules on the basis of affinity, size, charge or mobility.

46. The method of claim 33, wherein the microfluidic device further comprises a detector operatively disposed with respect to at least one of the detection regions, and the monitoring step comprises detecting an event or agent within the detection region.

47. The method of claim 46, wherein the detection step comprises detecting an optical signal.

48. The method of claim 47, wherein the detector detects a fluorescence emission, fluorescence polarization or fluorescence resonance energy transfer.

49. The method of claim 48, wherein the detector performs time-resolved fluorescence measurements.

50. The method of claim 48, wherein the detector performs fluorescence correlation spectroscopy.

51. The method of claim 47, wherein the detector is an optical microscope, a confocal microscope or a laser scanning confocal microscope.

52. The method of claim 46, wherein the detector is a non-optical sensor selected from the group consisting of a temperature sensor, a conductivity sensor, a potentiometric sensor and an amperometric sensor.

53. The method of claim 33, wherein the desired property is the ability of the at least one test agent to promote or inhibit binding between a ligand and an antiligand.

54. The method of claim 53 wherein the antiligand is a protein receptor and the ligand is the receptor'"'"'s cognate ligand.

55. The method of claim 53, wherein the ligand is an antigen and the antiligand is an antibody that specifically binds to the antigen.

56. The method of claim 53, wherein the antiligand is an enzyme and the ligand is an enzyme substrate or inhibitor of the enzyme.

57. The method of claim 53, wherein the ligand is a nucleic acid and the antiligand is a nucleic acid binding protein.

58. The method of claim 33, wherein the desired property is the ability of the at least one test agent to trigger a signal transduction pathway.

59. The method of claim 33, wherein the desired property is the ability of the at least one test agent to inhibit microbial growth.

60. The method of claim 33, wherein the desired property is the ability of the at least one test agent to trigger cell death.

61. The method of claim 33, wherein the desired property is the ability of the at least one test agent to trigger apoptosis in a cell.

62. The method of claim 55, where the monitoring comprises monitoring inhibition of cell proliferation, monitoring mitochondrial membrane function, monitoring caspase activation, or monitoring cytochrome c release from a cell.

63. The method of claim 33, wherein the monitoring step is in a homogenous format.

64. The method of claim 33, wherein the monitoring step is in a heterogenous format.