STOCHASTIC CONFINEMENT TO DETECT, MANIPULATE, AND UTILIZE MOLECULES AND ORGANISMS

• US 20100227767A1
• Filed: 07/28/2008
• Published: 09/09/2010
• Est. Priority Date: 07/26/2007
• Status: Abandoned Application

• Current Assignee: University of Chicago
• Original Assignee: University of Chicago
• Inventors: Kim, Hyun Jung, Kastrup, Christian, Shen, Feng, Huynh, Toan, Runyon, Matthew K., Gerdts, Cory, Boedicker, James Q., Ismagilov, Rustem F.
• Application Number:
  US12/670,739
• Publication Number:
  US 20100227767A1
• Time in Patent Office: Days
• US Class Current: 506/7
• CPC Class Codes: C12M 1/14 with means providing thin layers or with multi-level trays
• CPC Class Codes: C12M 23/34 Internal compartments or partitions
• CPC Class Codes: C12M 25/01 Drops
• CPC Class Codes: C12M 35/08 Chemical, biochemical or biological means, e.g. plasma jet, co-culture
• CPC Class Codes: C12N 1/20 Bacteria; Culture media therefor
• CPC Class Codes: C12N 1/38 Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound C12N1/34 takes precedence
• CPC Class Codes: C12N 11/04 entrapped within the carrier, e.g. gel or hollow fibres
• CPC Class Codes: C12P 39/00 Processes involving microorganisms of different genera in the same process, simultaneously
• CPC Class Codes: C12Q 1/18 Testing for antimicrobial activity of a material
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/54313 the carrier being characterised by its particulate form

Abstract

Methods of detecting organisms e.g. bacteria using stochastic confinement effects with microfluidic technologies involving plugs are provided. Signal amplification methods for the detection of molecules are also disclosed.

Claims

1. A method of detecting bacteria in a sample, comprising:

creating an array of plugs by introducing a first plug fluid into a flow of carrier fluid in a microchannel;

wherein the majority of plugs in the array do not contain a bacterium;

wherein the first plug fluid is substantially immiscible with the carrier fluid and comprises a concentration of the sample diluted such that at most 2 bacteria are present in any plug; and

analyzing the array for the presence of bacteria.

2. The method of claim 1, wherein the array is analyzed for a detectable signal produced by the bacteria, wherein the detectable signal is a substance produced by the bacteria or is produced when the bacteria consumes a substance in the plug.

3. The method of claim 2, wherein the substance is selected from the group consisting of oxygen, carbon, a protein produced by the bacteria, a molecule produced by a bacterial enzymatic reaction, and a redox/potential sensitive indicator.

6. The method of claim 1, wherein the sample is from human, soil or marine.

8. The method of claim 1, wherein the bacteria are at a higher concentration in the plugs than in the sample.

9. The method of claim 1, wherein the plugs contain different species of bacteria.

10. The method of claim 1, further comprising introducing a plug fluid comprising media capable of supporting bacterial growth into the plug.

11. The method of claim 10, wherein the detectable signal is produced by growth of the bacteria.

12. The method of claim 1, wherein the plugs comprise a substance capable of inducing virulence in the bacteria.

13. The method of claim 1, wherein the at least two plug comprise a substance capable of lysing the bacteria.

15. The method of claim 1, further comprising assaying the bacteria detected for a biological activity.

16. The method of claim 1, further comprising identifying the bacteria.

17. The method of claim 1, further comprising splitting a plug that has been determined to contain bacteria into multiple plugs, each containing at least one of the bacteria.

18. The method of claim 1, further comprising conducting a polymerase chain reaction on the contents of the at least two plugs prior to analyzing the at least two plugs for the detectable signal.

4-5. -5. (canceled)

7. (canceled)

14. (canceled)

19. A method of detecting bacteria comprising:

flowing at least two plugs in a carrier fluid through a microchannel;

wherein each plug comprises a plug fluid substantially immiscible with the carrier fluid;

wherein a first plug comprises a means for detecting a first species of bacteria;

wherein a second plug comprises a means for detecting a second species of bacteria different from the first species of bacteria;

introducing a sample optionally comprising bacteria into the first and second plugs, wherein the bacteria produce a detectable signal; and

analyzing the plugs for the detectable signal.

20-28. -28. (canceled)

29. A method of screening for antibiotic activity comprising:

flowing at least two plugs in a carrier fluid through a microchannel;

wherein each plug comprises a plug fluid immiscible with the carrier fluid and media capable of supporting bacterial growth;

wherein the first plug comprises a first antibiotic candidate;

wherein the second plug comprises a second antibiotic candidate;

introducing a sample which comprises bacteria into the first and second plugs; and

detecting the presence of bacterial growth.

30-44. -44. (canceled)

45. A method of detecting bacteria in an aqueous sample, comprising:

dividing a sample into a plurality of subsamples in a microfluidic device,

where each subsample is separated from another by a fluorinated liquid, each subsample has a volume that is a nanoliter or less, the majority of the subsamples do not contain a bacterium, and each subsample has at most 2 bacteria; and

analyzing the volumes for the presence of bacteria.