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

• US 6,258,593 B1
• Filed: 06/30/1999
• Issued: 07/10/2001
• Est. Priority Date: 06/30/1999
• Status: Expired due to Term

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Overman, Leslie B., Schembri, Carol T., Hotz, Charles Z.
• Primary Examiner(s): Sisson, Bradley L.
• Application Number:
  US09/343,372
• Time in Patent Office: 741 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/287.2, 436/501, 436/94, 536/23.1, 536/24.3, 536/24.33
• US Class Current: 435/287.2
• 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 device for conducting hybridization assays within an enclosed hybridization chamber, comprising:

a substrate having a surface with at least a portion of said surface representing a hybridization region, having an area of about 4 mm² to about 500 mm² wherein the substrate surface is functionalized with a mixture of a first silane providing surface —

Si—

R¹ groups where R¹ is a chemically inert moiety and a second silane providing surface —

Si—

(L)_n—

R² where L is a linking group, n is 0 or 1, and R² is a functional group enabling binding of the oligonucleotide probes;

a plurality of oligonucleotide probes bound to the substrate surface within the hybridization region and arranged in a spatially defined and physically addressable manner;

a cover having a peripheral lip which sealingly contacts the substrate surface about the hybridization region, wherein the cover and the hybridization region form an enclosure having an interior space comprising a hybridization chamber having a height of about 50 μ

m to about 500 μ

m; and

,

containing within the hybridization chamber, a sample fluid comprising a target molecule which may hybridize to a surface-bound molecular probe within the hybridization region, and wherein the sample fluid additionally comprises a surfactant of a type and present at a concentration effective to substantially reduce non-specific binding and promote mixing of components within the sample fluid.

2. The device of claim 1, wherein the cover is comprised of a material that is thermally stable at temperatures of at least about 50°

C.

3. The device of claim 1, wherein the cover is comprised of a material that is chemically inert.

4. The device of claim 1, wherein the cover is comprised of a material that is non-stick.

5. The device of claim 1, further comprising means for introducing fluid into the hybridization chamber comprised of at least one port in the cover.

6. The device of claim 1, wherein the cover is a plastic cover having a peripheral lip that contacts the substrate surface about the hybridization region.

7. The device of claim 1, wherein the cover is comprised of a material selected from the group consisting of polypropylene, polyethylene and acrylonitrile-butadiene-styrene.

8. The device of claim 1, further including a fastening means for immobilizing the cover on the substrate surface and providing a temporary, watertight seal between the cover and the hybridization chamber.

9. The device of claim 1, wherein the fluid further comprises a hybridization buffer.

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

11. The device 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.

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

13. The device of claim 1, further including a fastening means for immobilizing the cover on the substrate surface and providing a temporary, watertight seal between the cover and the hybridization region.

14. The device of claim 7, wherein the cover is comprised of polypropylene.

15. The device of claim 7, wherein the substrate is comprised of glass.

16. The device of claim 7, wherein the substrate is comprised of plastic.

17. The device of claim 7, wherein the substrate is comprised of silicon.

18. The device of claim 7, wherein the substrate is comprised of fused silica.

19. The device of claim 8, wherein the surfactant is an anionic surfactant.

20. The device of claim 8, wherein the surfactant is a nonionic surfactant.

21. The device of claim 8, wherein the surfactant is selected from the group consisting of anionic surfactants, cationic surfactants, amphoteric surfactants, nonionic surfactants, and combinations thereof.

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

23. The device of claim 10, wherein the surfactant represents in the range of approximately 0.5 wt. % to 5 wt. % of the sample fluid.

24. The device of claim 13, further including a means for introducing fluid into the hybridization chamber.

25. The device of claim 22, wherein the anionic surfactant is a sodium, potassium, ammonium or lithium salt of lauryl sulfate.

26. The device of claim 22, wherein the anionic surfactant is lithium lauryl sulfate.

27. The device of claim 23, wherein the surfactant represents in the range of approximately 0.75 wt. % to 5 wt. % of the sample fluid.

28. The device of claim 25, wherein the nonionic surfactant is polymeric.

29. The device of claim 28, wherein the nonionic surfactant is polyethylene oxide.

30. A kit for carrying out hybridization in an enclosed hybridization chamber comprising:

a substrate having a surface with at least a portion of said surface representing a hybridization region having an area of about 4mm² to about 500 mm², wherein the substrate surface is functionalized with a mixture of a first silane providing surface —

Si—

R¹ groups where R¹ is a chemically inert moiety and a second silane providing surface —

Si—

(L)_n—

R² groups where L is a linking group, n is 0 or 1, and R² is a functional group enabling binding of the oligonucleotide probes;

a plurality of oligonucleotide probes bound to the substrate surface within the hybridization region;

a cover having a peripheral lip adapted to provide a sealed, enclosed chamber upon placement on the substrate surface over the hybridization region whereby the chamber has a height of about 50 μ

m to about 500 μ

m; and

surfactant effective to promote mixing and substantially reduce nonspecific binding in a hybridization assay conducted within the chamber.