Direct Clone Analysis and Selection Technology

• US 20130190206A1
• Filed: 07/13/2011
• Published: 07/25/2013
• Est. Priority Date: 07/13/2010
• Status: Active Grant

• Current Assignee: Dublin City University
• Original Assignee: Dublin City University
• Inventors: Dimov, Ivan, Leonard, Paul, O'Kennedy, Richard, Fitzgerald, Valerie
• Granted Patent: US 9,291,628 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0819 Microarrays; Biochips
• CPC Class Codes: B01L 2300/12 Specific details about materials
• CPC Class Codes: B01L 2300/16 Surface properties and coatings
• CPC Class Codes: B01L 3/5085 for multiple samples, e.g. microtitration plates
• CPC Class Codes: B01L 3/50857 using arrays or bundles of open capillaries for holding samples
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: G01N 33/53 Immunoassay; Biospecific binding assay; Materials therefor
• CPC Class Codes: G01N 33/5302 Apparatus specially adapted for immunological test procedures
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/545 Synthetic resin
• CPC Class Codes: G01N 33/569 for microorganisms, e.g. protozoa, bacteria, viruses
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92

Abstract

The present invention describes a spatial addressing technique that uses a very high-density micro-pore array for high-throughput screening of biological interactions. The therapeutic, diagnostic and drug-discovery implications of being able to identify, select and characterize specific protein-protein, protein-DNA and/or protein-carbohydrate interactions from heterogeneous populations of millions (to billions) of cells is discussed. Importantly, this technique possesses the screening and selection capacity of current display-based screening systems (i.e., millions-billions) but with greater efficiency and shorter time.

Claims

1. A device comprising an array of micro-pores, the micro-pore array being reversibly attached to a solid substrate, wherein at least one binding partner is attached to said solid substrate, and wherein the internal diameter of the micro-pores ranges between approximately 1.0 micrometers and 500 micrometers.

2. The device of claim 1, wherein the micro-pores are not coated with at least one binding partner.

5. The device of claim 1, wherein said device further comprises a polymeric film, wherein said array is covered by said polymeric film, wherein said polymeric film further comprises at least one hole and wherein said hole is positioned over at least one of said micro-pores.

6. The device of claim 5, wherein said device further comprises a pressure source or electrolytic expulsion source configured proximal to said at least one hole.

8. The device of claim 1, wherein said micro-pores further comprise at least one biological cell.

10. The device of claim 1, wherein said device comprises a micro-pore testbed array, said array comprising a plurality of longitudinally fused fibers reversibly bonded to a single gas permeable solid substrate, wherein said solid substrate is degassed and wherein said solid substrate is attached to at least one binding partner.

13. The device of claim 10, comprising between approximately 300 to 11,500,000 of said fused fibers, per cm² of the array.

14. The device of claim 1, wherein said solid substrate comprises a gas permeable material.

15. The device of claim 1, wherein said gas permeable material comprises poly(dimethylsiloxane) (PDMS).

16. The device of claim 10 comprising said plurality of longitudinally fused fibers and a polymeric film, wherein said fused fibers are covered by said polymeric film, wherein said polymeric film further comprises at least one hole and wherein said hole is positioned over at least one of said fused fibers.

17. The device of claim 15, wherein said device further comprises a pressure source configured proximal to said at least one hole.

3-4. -4. (canceled)

7. (canceled)

9. (canceled)

11-12. -12. (canceled)

18-19. -19. (canceled)

20. A method for identifying a sub-population of cells from a heterologous population of biological cells, the method comprising:

a) providing;

i) an array of micro-pores, wherein the internal diameter of micro-pores ranges between approximately 1.0 micrometers and 500 micrometers;

ii) said heterologous population of cells;

iii) at least one binding partner;

b) contacting said array with said heterologous population of cells and said at least one binding partner such that a sub-population comprising at least one of said biological cells settles into at least one of said micro-pores of said array;

c) incubating said array under conditions to promote the secretion of molecules from said biological cells; and

d) detecting desired secreted molecules in at least one of said micro-pores of said array, thereby identifying said sub-population of cells.

21. The method of claim 20, wherein the providing step comprises providing an array of micro-pores not being coated with at least one binding partner.

22. The method of claim 20, wherein the detecting step comprises detecting the desired secreted molecules in association with at least one binding partner.

23. The method of claim 20, the method comprising:

a) providing;

i) said array reversibly attached to a solid substrate, wherein the at least one binding partner is attached to said solid substrate;

ii) said heterologous population of cells;

b) contacting said array with said solution such that at least one of said cells settles into at least one of said micro-pores of said array;

c) incubating said array under conditions to promote the secretion of molecules from said biological cells;

d) removing said array from said solid support; and

e) detecting whether secreted molecules bound said binding partner on said solid support.

24. The method of claim 20, further comprising collecting said sub-population of cells from said at least one of said micro-pores of said array;

wherein, optionally, said collecting comprises cultivating said sub-population of cells.

25. The method of claim 22, comprising:

a) providing;

i) a plurality of longitudinally fused fibers reversibly attached to a solid support, each fiber defining a well, the solid surface comprising at least one type of binding partner and serving as the bottom of the well, the wells collectively comprising an array;

ii) a solution comprising a heterologous population of biological cells;

b) contacting said array with said solution such that at least one of said cells settles into at least one of said wells of said array;

c) incubating said array under conditions to promote the secretion of molecules from said cells;

d) removing said plurality of fused fibers from said solid support; and

e) detecting whether secreted molecules bound said binding partner on said solid support.

26. The method of claim 22 comprising:

a) providing;

i) a degassed solid substrate comprising at least one binding partner, wherein said solid substrate is attached to a plurality of longitudinally fused fibers thereby creating a micro-pore testbed array;

ii) a solution comprising a plurality of biological cells; and

iii) a polymeric film comprising at least one hole, such that said film is configured to bond to said fused fibers;

b) contacting said micro-pore testbed array with said solution such that at least one of said plurality of biological cells settles into at least one of said fused fibers;

c) bonding said polymeric film to said fused fibers such that said hole is positioned over at least one of said fibers;

d) removing said fused fiber-polymeric film array from said solid substrate.

27. The method of claim 20, further comprising providing a pressure source or electrolytic expulsion source configured proximal to said at least one hole.

31. The method of claim 26, wherein said pressure source is a source of positive or negative pressure.

32. The method of claim 24, further comprising collecting said plurality of biological cells from said at least one fused fiber;

wherein, optionally, said collecting comprises cultivating said at least one biological cell.

33. The method of claim 20, wherein said plurality of biological cells release at least one biological compound having affinity for said at least one binding partner.

34. The method of claim 20, wherein said at least one binding partner may be selected from the group comprising antigens, antibodies, proteins, peptides, nucleic acids, deoxyribonucleic acids, ribonucleic acids, lipids, and/or carbohydrates.

35. The method of claim 20, wherein said detecting comprises a labeled reagent having affinity for said binding/partner/biological compound complex.

28-30. -30. (canceled)

36-45. -45. (canceled)