Methods and apparatus for pathogen detection and analysis

• US 20060073484A1
• Filed: 12/29/2003
• Published: 04/06/2006
• Est. Priority Date: 12/30/2002
• Status: Abandoned Application

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Lagally, Eric, Paegel, Brian, Skelley, Alison, Blazej, Robert, Grover, William H, Liu, Chung N, Mathies, Richard A
• Application Number:
  US10/540,658
• Publication Number:
  US 20060073484A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01F 25/50 Circulation mixers, e.g. wherein at least part of the mixture is discharged from and reintroduced into a receptacle
• 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: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0803 Disc shape
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0819 Microarrays; Biochips
• CPC Class Codes: B01L 2300/0861 Configuration of multiple channels and/or chambers in a single devices
• CPC Class Codes: B01L 2300/087 Multiple sequential chambers
• CPC Class Codes: B01L 2300/088 Channel loops
• CPC Class Codes: B01L 2300/0887 Laminated structure
• CPC Class Codes: B01L 2300/123 Flexible; Elastomeric
• CPC Class Codes: B01L 2300/1827 using resistive heater
• CPC Class Codes: B01L 2400/0409 centrifugal forces
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/0638 membrane valves, flap valves
• 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: B01L 7/52 : with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: C12Q 1/6806 : Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/689 : for bacteria
• CPC Class Codes: C12Q 2565/629 : being a microfluidic device
• CPC Class Codes: F04B 43/06 : Pumps having fluid drive
• 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: G01N 35/00069 : whereby the sample substrate is of the bio-disk type, i.e. having the format of an optical disk
• CPC Class Codes: Y10T 137/0318 : Processes
• CPC Class Codes: Y10T 137/2224 : Structure of body of device
• CPC Class Codes: Y10T 137/87716 : For valve having a flexible diaphragm valving member

Abstract

Methods and apparatus for implementing microfluidic analysis devices are provided. A monolithic elastomer membrane associated with an integrated pneumatic manifold allows the placement and actuation of dense arrays of a variety of fluid control structures, such as structures for isolating, routing, merging, splitting, and storing volumes of fluid. The fluid control structures can be used to implement a pathogen detection and analysis system including integrated immunoaffinity capture and analysis, such as polymerase chain reaction (PCR) and capillary electrophoresis (CE) analysis. An analyte solution can be input into the device and pumped through a series of immunoaffinity capture matrices in microfabricated chambers having antibodies targeted to the various classes of microbiological organisms such as bacteria, viruses and bacterial spores. The immunoaffinity chambers can capture, purify, and concentrate the target for further analysis steps.

Claims

1. A pathogen detection system, the system comprising:

an immunocapture chamber integrated on a microfluidic device, the immunocapture chamber operable to capture a target provided to the immunocapture chamber through a microfluidic channel;

a DNA analysis mechanism associated with the immunocapture chamber, the DNA analysis mechanism integrated on the microfluidic device, the DNA analysis mechanism operable to perform DNA analysis on the target.

2. The pathogen detection system of claim 1, wherein the DNA analysis to mechanism comprises PCR and CE.

3. The pathogen detection system of claim 2, wherein PCR is included in a chamber separate from the immunocapture chamber.

4. The pathogen detection system of claim 2, wherein PCR is included in the immunocapture chamber.

5. The pathogen detection system of claim 2, wherein the chamber for PCR is used for amplification of DNA obtained from lysing the target of interest.

6. The pathogen detection system of claim 3, further comprising an etched capillary electrophoresis microchannel for separation and detection of PCR amplicons.

7. The pathogen detection system of claim 6, further comprising DNA preconcentration and clean-up chambers for purification of released pathogen genomic DNA or for desalting and preconcentration of amplified DNA before injection onto the CE microchannel.

8. A pathogen detection system, the system comprising:

immunocapture means integrated on a microfluidic device, the immunocapture means operable to capture a target provided through a microfluidic channel;

DNA analysis means associated with the immunocapture means, the DNA analysis means integrated on the microfluidic device, the DNA analysis means operable to perform DNA analysis on the target.

9. The pathogen detection system of claim 8, wherein the DNA analysis means comprises a PCR chamber separate from the immunocapture means.

10. The pathogen detection system of claim 9, wherein the PCR chamber is used for amplification of DNA obtained from lysing the target of interest.

11. A pathogen detection system on a monolithic device, the system comprising:

a plurality of immunocapture chambers integrated on the monolithic device, the immunocapture chambers operable to capture a target provided to the immunocapture chambers through microfluidic channels;

a plurality of DNA analysis mechanisms associated with the immunocapture chambers, the plurality of DNA analysis mechanisms integrated on the monolithic device, the plurality of DNA analysis mechanisms operable to perform DNA analysis on the target.

12. The pathogen detection system of claim 11, wherein the plurality of DNA analysis mechanisms comprise PCR and CE.

13. The pathogen detection system of claim 11, wherein PCR is performed in chambers separate from the plurality of immunocapture chambers.

14. The pathogen detection system of claim 13, further comprising a plurality of etched capillary electrophoresis microchannels for separation and detection of PCR amplicons.

15. The pathogen detection system of claim 14, further comprising a plurality of integrated DNA preconcentration and clean-up chambers for purification of released pathogen genomic DNA or for desalting and preconcentration of amplified DNA before injection onto the CE microchannel.

16. The pathogen detection system of claim 11, wherein the immunocapture chambers are further operable to purify and concentrate target.

17. The pathogen detection system of claim 11, wherein the plurality of microfabricated immunocapture chambers are configured to hold selected antibodies.

18. The pathogen detection system of claim 17, wherein the selected antibodies are held with beads, frits, sol-gels, gels, or polymer monoliths.

19. The pathogen detection system of claim 17, wherein the selected antibodies are held with molded blocks of porous, surface functionalized polymer formed directly within the capture chambers.

20. The pathogen detection system of claim 19, wherein the molded blocks are formed by photo polymerization of a precursor mixture including monomers and porogenic solvents.

21. The pathogen detection system of claim 17, wherein the plurality of 5 immunocapture chambers are configured in a radially parallel manner.

22. The pathogen detection system of claim 21, further comprising ring heaters coupled to the plurality of immunocapture chambers, the ring heaters operable to heat the plurality of immunocapture chambers to release the captured target.

23. The pathogen detection system of claim 17, wherein the plurality of immunocapture chambers are configured on a glass layer.

24. The pathogen detection system of claim 23, wherein the glass layer is coupled to a monolithic membrane layer.

25. The pathogen detection system of claim 23, wherein the glass layer includes a plurality of etched channels, the etched channels operable to provide paths for fluid flow.

26. The pathogen detection system of claim 25, wherein the glass layer and a pneumatic layer sandwich the membrane layer.

27. A method for pathogen analysis, the method comprising:

providing a fluid analyte to a plurality of immunocapture chambers through microfluidic channels integrated on a monolithic device;

capturing a target associated with the fluid analyte at the immunocapture chambers; and

performing DNA analysis on the target using a plurality of DNA analysis mechanisms associated with the plurality of immunocapture chambers, the plurality of DNA analysis mechanisms integrated on the monolithic device.

28. The method of claim 27, wherein the plurality of DNA analysis mechanisms comprise PCR and CE.

29. The method of claim 27, wherein PCR mechanisms are included in chambers separate from the plurality of immunocapture chambers.

30. The method of claim 29, wherein the plurality of DNA analysis mechanisms include PCR chambers for amplification of DNA obtained from lysing the target of interest.

31. The method of claim 29, further comprising a plurality of etched capillary electrophoresis microchannels for separation and detection of PCR amplicons.

32. The method of claim 31, further comprising a plurality of integrated DNA preconcentration and clean-up chambers for purification of released pathogen genomic DNA or for desalting and preconcentration of amplified DNA before injection onto the CE microchannel.

33. The method of claim 27, wherein the immunocapture chambers are further operable to purify and concentrate target.

34. The method of claim 27, wherein the plurality of microfabricated immunocapture chambers are configured to hold selected antibodies.

35. The method of claim 34, wherein the selected antibodies are held with beads, sol-gels, gels, or polymer monoliths.

36. The method of claim 34, wherein the selected antibodies are held with molded blocks of porous, surface functionalized polymer formed directly within the capture chambers.

37. An apparatus for detecting pathogens, the apparatus comprising:

means for providing a fluid analyte to a plurality of immunocapture chambers through microfluidic channels integrated on a monolithic device;

means for capturing a target associated with the fluid analyte; and

means for performing DNA analysis on the target using a plurality of DNA analysis mechanisms associated with the plurality of immunocapture chambers, the plurality of DNA analysis mechanisms integrated on the monolithic device.