Recirculating fluidic network and methods for using the same

• US 8,168,139 B2
• Filed: 06/23/2003
• Issued: 05/01/2012
• Est. Priority Date: 06/24/2002
• Status: Expired due to Fees

• Current Assignee: Fluidigm Corporation
• Original Assignee: Fluidigm Corporation
• Inventors: Manger, Ian David, Barco, Joseph W., Nassef, Hany Ramez
• Primary Examiner(s): Bullock, In Suk
• Assistant Examiner(s): HYUN, PAUL SANG HWA
• Application Number:
  US10/602,489
• Publication Number:
  US 20040180377A1
• Time in Patent Office: 3,235 Days
• Field of Search: 422502-505, 436/180, 435/287.2
• US Class Current: 422/503
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0645 Electrodes
• 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 2300/088 Channel loops
• CPC Class Codes: B01L 2300/0887 Laminated structure
• 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 2400/0655 pinch valves
• CPC Class Codes: B01L 3/5025 for parallel transport of multiple samples
• 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 3/50273 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: B81B 3/00 Devices comprising flexible or deformable elements, e.g. comprising elastic tongues or membranes B81B5/00 takes precedence
• CPC Class Codes: F04B 19/006 Micropumps F04B43/043 and F04B43/095 take precedence
• CPC Class Codes: F04B 43/02 having plate-like flexible members, e.g. diaphragms F04B43/14 takes precedence
• 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 2099/0078 using moulding or stamping
• 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 99/0003 : Constructional types of microvalves; Details of the cutting-off member
• CPC Class Codes: F16K 99/0026 : Valves using channel deformation
• CPC Class Codes: F16K 99/0036 : operated by temperature variations
• CPC Class Codes: F16K 99/0046 : using magnets
• CPC Class Codes: F16K 99/0051 : using electrostatic means
• CPC Class Codes: F16K 99/0059 : actuated by a pilot fluid
• CPC Class Codes: G01N 33/5304 : Reaction vessels, e.g. agglutination plates for solid-phase systems G01N33/543
• CPC Class Codes: G01N 33/54366 : Apparatus specially adapted for solid-phase testing
• CPC Class Codes: Y10T 436/2575 : Volumetric liquid transfer

Abstract

The present invention provides a variety of microfluidic devices and methods for conducting assays and syntheses. The devices include a solid substrate layer having a surface that is capable of attaching ligand and or anti-ligand, and an elastomeric layer attached to said surface. Preferred embodiments have deflectable membrane valves and pumps, for example, rotary pumps associated therewith.

Claims

1. A method of conducting a binding assay, comprising:

(a) providing a microfluidic device comprising a solid substrate layer having a surface that is capable of attaching ligand and/or anti-ligand, and an elastomeric layer attached to said solid substrate surface, wherein said elastomeric layer comprises;

(i) a plurality of first flow channels;

(ii) a plurality of second flow channels each intersecting and crossing each of the first flow channels thereby providing a plurality of intersecting areas formed at intersections between the first flow channels and the second flow channels, wherein the plurality of first flow channels and the plurality of second flow channels are adapted to allow the flow of a solution therethrough, wherein the solid substrate surface is in fluid communication with at least the intersecting areas of the plurality of first flow channels and the plurality of second flow channels, and wherein the plurality of first flow channels and/or the plurality of second flow channels are capable of forming a plurality of closed looped flow channels, wherein the plurality of second flow channels are in communication with a pump, and wherein a sample solution is transported through the plurality of second flow channels under the action of the pump, wherein the pump comprises more than one control channel each formed within the elastomeric layer and separated from the plurality of second flow channels by an elastomeric segment that is deflectable into the plurality of second flow channels in response to an actuation force, whereby the sample solution is transported along the plurality of second flow channels upon independent, sequential actuation of the more than one control channels;

(iii) a plurality of control channels;

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

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

(vi) a plurality of sets of closed loop forming control valves each set having a first valve operatively disposed with respect to an inlet of one of each of the first and/or the second flow channels and a second valve operatively disposed with respect to an outlet of one of each of the first and/or second flow channels to form the plurality of closed looped flow channels, wherein each of the loop forming control valves comprises a loop forming control channel and an elastomeric segment that is deflectable into or retractable from the first and/or the second flow channels upon which the loop forming control valve operates in response to an actuation force applied to the loop forming control channel, the elastomeric segment when positioned in the first and/or the second flow channels restricting solution flow therethrough thereby forming the looped flow channel, wherein the first valve of the set of loop forming control valves comprises a control channel of the pump that is independently actuated with respect to other control channels of the pump; and

(vii) a plurality of recirculating pumps, and wherein each recirculating pump is operatively disposed with respect to one of the closed looped flow channels such that circulation of solution through each of the closed looped flow channels can be regulated by one of the recirculating pumps;

(b) applying an actuating force to the second control valves to restrict solution flow through each of the second flow channels;

(c) introducing a reagent comprising a ligand into at least one of the first flow channels under conditions sufficient to attach the ligand to the solid substrate surface;

(d) removing the actuation force to the second flow channel control channel and applying an actuation force to the first control channel such that solution flow through the first flow channel is restricted; and

(e) performing a binding assay by introducing the sample solution into the second flow channel;

(f) applying an actuating force from two control lines to the plurality of sets of closed loop forming control valves to form the plurality of closed looped flow channels such that each closed looped flow channel comprises a closed loop spanning multiple rows and multiple columns; and

recirculating the sample solution within the closed loop each of the closed looped flow channels using the recirculating pump under conditions sufficient to specifically bind an anti-ligand that may be present in the sample solution to the ligand that is attached to the solid substrate surface; and

(g) detecting the binding of the anti-ligand in the sample to the ligand.

2. The method of claim 1 further comprising removing any ligand that is not attached to the solid substrate surface from the first flow channel prior to introducing the sample solution into the second flow channel.

3. The method of claim 1, wherein the plurality of first flow channels and the plurality of second flow channels are located within the elastomeric layer, and wherein each of the intersecting areas formed at intersections between the first flow channels and the second flow channels comprises a via which is in fluid communication with the solid substrate surface thereby forming a well that is adapted to collect a fluid therein.

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

5. The method of claim 4, wherein the pump comprises more than one control channels each formed within the elastomeric layer and separated from the first flow channel by an elastomeric segment that is deflectable into the first flow channel in response to an actuation force, whereby the reagent is transported along the first flow channel.

6. The method of claim 1, wherein said step (e) of performing binding assay comprises removing the elastomeric layer from the solid substrate surface and determining ligand/antiligand binding at each of the intersecting areas with a detector.

7. The method of claim 6, wherein the detector detects an optical signal within the intersecting areas.

8. The method of claim 7, wherein the detector detects a fluorescence emission, fluorescence polarization or fluorescence resonance energy transfer.

9. The method of claim 7, wherein the detector is an optical microscope, a confocal microscope or a laser scanning confocal microscope.

10. The method of claim 6, wherein the detector is a non-optical sensor selected from the group consisting of a radioactivity sensor, and an electrical potential difference sensor.

11. The method of claim 1, wherein the assay comprises detecting binding between a substrate and a cell receptor;

a substrate and an enzyme;

an antibody and an antigen;

a nucleic acid and a nucleic acid binding protein;

a protein and a protein;

a small molecule and a protein;

a small molecule and an oligonucleotide;

or a protein affinity tag and a metal ion.

12. The method of claim 1, wherein the assay is an assay for detecting a toxic effect on cells or a cell death assay, or a cell proliferation assay.

13. The method of claim 1, wherein the assay is an oligonucleotide binding assay or a peptide binding assay.

14. The method of claim 1, wherein the assay is an antimicrobial assay.

15. A method of conducting a binding assay, comprising:

(a) providing a microfluidic device comprising a solid substrate layer having a surface that is capable of attaching ligand and/or anti-ligand, and an elastomeric layer attached to said solid substrate surface, wherein said elastomeric layer comprises;

(i) a first flow channel;

(ii) a second flow channel intersecting and crossing the first flow channel thereby providing an intersecting area formed at an intersection between the first flow channel and the second flow channel, wherein the first flow channel and the second flow channel are adapted to allow the flow of a solution therethrough, wherein the solid substrate surface is in fluid communication with at least the intersecting area of the first flow channel and the second flow channel, and wherein the first flow channel and/or the second flow channel are capable of forming a closed looped flow channel, wherein the second flow channel is in communication with a pump, and wherein a sample solution is transported through the second flow channel under the action of the pump, wherein the pump comprises more than one control channel each formed within the elastomeric layer and separated from the second flow channel by an elastomeric segment that is deflectable into the second flow channel in response to an actuation force, whereby the sample solution is transported along the second flow channel upon independent, sequential actuation of the more than one control channels;

(iii) a plurality of control channels;

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

(v) a second control valve operatively disposed with respect to the second flow channel to regulate flow of the solution through the second flow channel, wherein the second control valve comprises a second control channel and an elastomeric segment that is deflectable into or retractable from the second flow channel upon which the second control valve operates in response to an actuation force applied to the second control channel, the elastomeric segment when positioned in the second flow channel restricting solution flow therethrough;

(vi) a first loop forming control valve operatively disposed with respect to an inlet of one of the first and/or the second flow channels and a second loop forming control valve operatively disposed with respect to an outlet of one of the first and/or second flow channels to form the closed looped flow channel, wherein each of the first and second loop forming control valves comprises a loop forming control channel and an elastomeric segment that is deflectable into or retractable from the first and/or the second flow channels upon which the loop forming control valve operates in response to an actuation force applied to the loop forming control channel, the elastomeric segment when positioned in the first and/or the second flow channels restricting solution flow therethrough thereby forming the closed looped flow channel, wherein the first loop forming control valve comprises a control channel of the pump that is independently actuated with respect to other control channels of the pump; and

(vii) a recirculating pump operatively disposed with respect to the closed looped flow channel such that circulation of solution through the closed looped flow channel can be regulated by the recirculating pump;

(b) applying an actuating force to the second control valve to restrict solution flow through the second flow channel;

(c) introducing a reagent comprising a ligand into the first flow channel under conditions sufficient to attach the ligand to the solid substrate surface;

(d) removing the actuation force to the second flow channel control channel and applying an actuation force to the first control channel such that solution flow through the first flow channel is restricted; and

(e) performing a binding assay by introducing a sample solution into the second flow channel;

(f) applying an actuating force to the first loop forming control valve using a first control line and to the second loop forming control valve using a second control line to form the closed looped flow channel such that the closed looped flow channel comprises a closed loop; and

recirculating the sample solution within the closed loop of the closed looped flow channel using the recirculating pump under conditions sufficient to specifically bind an anti-ligand that may be present in the sample solution to the ligand that is attached to the solid substrate surface; and

(g) detecting the binding of the anti-ligand in the sample to the ligand.

16. The method of claim 15, wherein the first flow channel and the second flow channel are located within the elastomeric layer, and wherein the intersecting area formed at the intersection between the first flow channel and the second flow channel comprises a via which is in fluid communication with the solid substrate surface thereby forming a well that is adapted to collect a fluid therein.

17. The method of claim 15, wherein said step (e) of performing binding assay comprises removing the elastomeric layer from the solid substrate surface and determining ligand/antiligand binding at the intersecting area with a detector, wherein the detector detects an optical signal within the intersecting areas, and wherein the detector detects a fluorescence emission, fluorescence polarization, or fluorescence resonance energy transfer.

18. The method of claim 15, wherein said step (e) of performing binding assay comprises removing the elastomeric layer from the solid substrate surface and determining ligand/antiligand binding at the intersecting area with a detector, wherein the detector detects an optical signal within the intersecting areas, and wherein the detector is an optical microscope, a confocal microscope, or a laser scanning confocal microscope.

19. The method of claim 15, wherein the second flow channel is in communication with a pump comprising a plurality of sequentially actuated control channels, and wherein the sample solution is transported through the second flow channel under the action of the pump.

20. The method of claim 19, wherein the first loop forming control valve comprises a control channel of the pump.