Methods and arrays for detecting cells and cellular components in small defined volumes

• US 8,460,878 B2
• Filed: 02/16/2007
• Issued: 06/11/2013
• Est. Priority Date: 02/21/2006
• Status: Active Grant

• Current Assignee: Trustees of Tufts College
• Original Assignee: Trustees of Tufts College
• Inventors: Walt, David R., Rissin, David M.
• Primary Examiner(s): Hines, Ja'na
• Application Number:
  US11/707,383
• Publication Number:
  US 20070259385A1
• Time in Patent Office: 2,307 Days
• Field of Search: None
• US Class Current: 435/7.1
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2527/143 Concentration of primer or probe
• CPC Class Codes: G01N 2333/938 acting on beta-galactose-glycoside bonds, e.g. beta-galactosidase
• CPC Class Codes: G01N 33/543 with an insoluble carrier for immobilising immunochemicals
• CPC Class Codes: G01N 33/54346 Nanoparticles
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing
• CPC Class Codes: G01N 33/54386 Analytical elements
• CPC Class Codes: G01N 33/581 with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)

Abstract

Arrays of single cells and methods of producing an array of single cells are described. Arrays with defined volumes between 10 attoliters and 50 picoliters enable single cell capture, detection and quantitation.

Claims

1. A method of detecting a target analyte of a cell in a sample, the method comprising:

a) providing a sample and a solid support said sample comprising cells in a first concentration, each cell comprising a target analyte, said solid support comprising at least 1000 assay sites;

b) contacting said solid support with said sample such that the ratio of a total number of cells contained within the assay sites to the total number of assay sites contacted with said sample is less than 1;

5; and

c) determining the number of assay sites of said solid support which contain a target analyte of said cells.

2. The method of claim 1, wherein said cells are prokaryotic.

3. The method of claim 1, wherein said cells are eukaryotic.

4. The method of claim 1, further comprising, after step b), sealing the assay sites such that the contents of each assay site cannot escape said assay site.

5. The method of claim 1, wherein said ratio is less than 1:

10.

6. The method of claim 1, wherein said solid support comprises between 20,000 and 30,000 assay sites.

7. The method of claim 1, wherein each site of said solid support comprises a capture component.

8. The method of claim 7, wherein said capture component immobilizes said cells.

9. The method of claim 1, further comprising prior to step b) a step of diluting said sample to create a diluted sample, said diluted sample comprising cells in a second concentration, and wherein in step b), said sample contacted with said solid support is said diluted sample.

10. The method of claim 1, further comprising a step of treating said cells in said solid support under conditions such that said cells are lysed prior to step (c).

11. The method of claim 1, wherein said ratio is between 1:

5 and 1;

500.

12. The method of claim 1, wherein said determining comprises detecting a change in an optical property at said assay sites as an indication of the presence of said target analyte.

13. The method of claim 1, further comprising determining the percentage of assay sites containing a target analyte of said cells.

14. The method of claim 1, wherein said solid support comprises between 100,000 and 10,000,000 assay sites.

15. The method of claim 7, wherein the capture component is a receptor.

16. The method of claim 7, wherein the capture component is fibronectin.

17. The method of claim 7, wherein the capture component is an antibody.

18. The method of claim 13, further comprising determining a measure of the concentration of target analyte of said cells in said sample from said percentage.