Method for conducting chemical or biochemical reactions on a solid surface within an enclosed chamber

• US 6,911,343 B2
• Filed: 07/06/2001
• Issued: 06/28/2005
• Est. Priority Date: 06/30/1999
• Status: Expired due to Term

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Schembri, Carol T., Hotz, Charles Z., Overman, Leslie B.
• Primary Examiner(s): Sisson, Bradley L.
• Application Number:
  US09/900,294
• Publication Number:
  US 20020001839A1
• Time in Patent Office: 1,453 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/183, 435/287.2, 536/23.1, 536/24.3, 536/24.31, 536/24.33, 436/94
• US Class Current: 436/94
• CPC Class Codes: B01F 29/321 of test-tubes or the like
• CPC Class Codes: B01F 33/25 Mixers with loose mixing elements, e.g. loose balls in a receptacle
• CPC Class Codes: B01F 33/252 using bubbles as loose mixing element
• CPC Class Codes: B01F 33/30 Micromixers
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: G01N 33/54393 Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The invention provides an apparatus and method for conducting chemical or biochemical reactions on a solid surface within an enclosed chamber. The invention may be used in conducting hybridization reactions, as of biopolymers such as DNA, RNA, oligonucleotides, peptides, polypeptides, proteins, antibodies, and the like. In another aspect, the invention provides an improved method for mixing a thin film of solution, as in a hybridization chamber. The invention further provides a kit for carrying out the methods of the invention.

Claims

1. A method for conducting a hybridization assay within an enclosed hybridization chamber, comprising:

(a) providing a device comprised of a (i) a substrate having a surface with at least a portion of said surface representing a hybridization region, wherein a plurality of oligonucleotide probes are bound to the substrate surface within the hybridization region and arranged in a spatially defined and physically addressable manner, and (ii) a cover which sealingly contacts the substrate surface, wherein the cover and the hybridization region form an enclosure having an interior space comprising a hybridization chamber having a height ranging from about 50 μ

m to about 500 μ

m; and

(b) introducing into the hybridization chamber a sample fluid comprising (i) a target molecule which may hybridize to a surface-bound molecular probe within the hybridization region, (ii) a hybridization buffer, and (iii) a surfactant of a type and present at a concentration effective to substantially reduce nonspecific binding and promote mixing of components within the sample fluid; and

(c) mixing the sample fluid by moving a bubble within the hybridization chamber to displace the sample fluid ad maintaining hybridization conditions within the hybridization chamber for a period of time sufficient to allow hybridization between the target molecule and a surface-bound molecular probe to occur.

2. The method of claim 1, wherein the hybridization chamber has a volume in the range of about 0.2 μ

l to about 312 μ

l.

3. The method of claim 1, wherein the surfactant additionally comprises a surfactant selected from the group consisting of anionic surfactants, cationic surfactants, amphoteric surfactants, nonionic surfactants, and combinations thereof.

4. The method of claim 1, wherein the surfactant represents in the range of approximately 0.1 wt. % to 10 wt. % of the sample fluid.

5. The method of claim 1, wherein the surfactant comprises a combination of polyethylene oxide and lithium lauryl sulfate, and further wherein the polyethylene oxide represents up to about 1 wt. % of the sample fluid and the lithium lauryl sulfate represents up to about 0.5 wt. % of the sample fluid.

6. The method of claim 1, wherein an air bubble is present within the hybridization chamber.

7. A method according to claim 1 wherein the surface is a silane functionalized surface.

8. The method of claim 1, wherein said cover comprises a peripheral lip.

9. The method of claim 2, wherein the hybridization chamber has a volume in the range of about 1 μ

l to about 200 μ

l.

10. The method of claim 2, wherein the hybridization region has an area in the range of about 4 mm² to about 500 mm².

11. The method of claim 3, wherein the surfactant is an anionic surfactant.

12. The method of claim 3, wherein the surfactant is a nonionic surfactant.

13. A method according to claim 3 wherein the surface is a silane functionalized surface.

14. The method of claim 4, wherein the surfactant represents in the range of approximately 0.5 wt. % to 5 wt. % of the sample fluid.

15. The method of claim 9, wherein the hybridization region has an area in the range of about 20 mm² to about 350 mm².

16. The method of claim 11, wherein the anionic surfactant is a sodium, potassium, ammonium or lithium salt of lauryl sulfate.

17. The method of claim 12, wherein the nonionic surfactant is polymeric.

18. The method of claim 14, wherein the surfactant represents in the range of approximately 0.75 wt. % to 5 wt. % of the sample fluid.

19. The method of claim 16, wherein the anionic surfactant is lithium lauryl sulfate.

20. A method according to claim 16 wherein the surface is a silane functionalized surface.

21. The method of claim 17, wherein the nonionic surfactant is polyethylene oxide.

22. A method comprising:

(a) scalingly contacting a cover to a first substrate having a plurality of molecular probes bound to the surface of the first substrate to form a first sealed hybridization chamber having a height ranging from about 50 μ

m to about 500 μ

m about the substrate surface-bound molecular probes,

(b) performing a hybridization assay with the first sealed hybridization chamber and a sample comprising a target molecule which may hybridize to a surface-bound molecular probe,

(c) opening the hybridization chamber and removing the first substrate,

(d) reusing the cover by sealingly contacting the cover to a second substrate having a plurality of molecular probes bound to the surface of the second substrate, wherein the cover and substrate surface form a second sealed hybridization chamber about the substrate surface-bound molecular probes,

(e) performing a hybridization assay with the second sealed hybridization chamber and a sample comprising a target molecule which may hybridize to a surface-bound molecular probe.

23. The method of claim 22, further comprising compressing together at least one of the first substrate and cover and the second substrate and cover.

24. The method of claim 23, wherein said compressing is accomplished by tightening screws.