Substrates having low density reactive groups for monitoring enzyme activity

• US 7,763,423 B2
• Filed: 09/30/2005
• Issued: 07/27/2010
• Est. Priority Date: 09/30/2005
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Peluso, Paul, Roitman, Daniel, Foquet, Mathieu
• Primary Examiner(s): Naff; David M
• Application Number:
  US11/240,662
• Publication Number:
  US 20070077564A1
• Time in Patent Office: 1,761 Days
• Field of Search: None
• US Class Current: 435/6.11
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00628 Ionic
• CPC Class Codes: B01J 2219/0063 Other, e.g. van der Waals forces, hydrogen bonding
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01J 2219/00725 Peptides
• CPC Class Codes: G01N 33/543 with an insoluble carrier for immobilising immunochemicals
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing
• 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

Abstract

Reactive surfaces, substrates and methods of producing and using such substrates and surfaces are provided. The substrates and surfaces provide low density reactive groups preferably on an otherwise non-reactive surface for use in different applications including single molecule analyses.

Claims

1. A substrate for monitoring enzyme activity, comprising a surface having first and second surface modifying agents coupled thereto, wherein the first surface modifying agent comprises a reactive moiety coupled thereto, and wherein the second surface modifying agent does not have the reactive moiety coupled thereto, wherein the first and second surface modifying agents are coupled to the surface by contacting the surface with a mixture of first and second surface modifying agents at a ratio of first surface modifying agent to second surface modifying agent that provides the reactive moiety coupled to the surface at a density of less than 1 reactive moiety per 100 nm², wherein the reactive moiety comprises an enzyme.

2. The substrate of claim 1 wherein the reactive moiety is coupled to the surface at a density of less than 1 reactive moiety per 1000 nm².

3. The substrate of claim 1 wherein the reactive moiety is coupled to the surface at a density of less than 1 reactive moiety per 10000 nm².

12. The substrate of claim 1, wherein the enzyme is selected from a nucleic acid polymerase, a ligase, a nuclease, a protease, a kinase and a phosphatase.

13. The substrate of claim 1, wherein the enzyme comprises a DNA polymerase.

14. The substrate of claim 1, wherein the surface comprises an observation area.

15. The substrate of claim 1, wherein the surface comprises an observation surface of an optical confinement.

16. The substrate of claim 14, wherein the observation area comprises the observation surface of a zero mode waveguide.

17. The substrate of claim 1, wherein the surface of the substrate is selected from glass, quartz, fused silica, and silicon.

4. A substrate for monitoring enzyme activity comprising:

a surface comprising first and second surface modifying agents coupled thereto, wherein the first surface modifying agent comprises a reactive moiety coupled thereto, and the second surface modifying agent does not comprise the reactive moiety coupled thereto; and

wherein the first and second surface modifying agents are coupled to the surface by contacting the surface with a mixture of the first and second surface modifying agents at a ratio of the first and second surface modifying agents that provides the reactive moiety coupled to the surface at a density of between about 1 reactive moiety per 50,000 nm² and 1 reactive moiety per 100 nm², wherein the reactive moiety comprises an enzyme.

5. The substrate of claim 4, wherein the reactive moiety is coupled to the surface at a density of between about 1 reactive moiety per 8000 nm² and 1 reactive moiety per 300 nm².

6. The substrate of claim 4, wherein the reactive moiety is coupled to the surface at a density of between about 1 reactive moiety per 8000 nm² and 1 reactive moiety per 1000 nm².

7. The substrate of claim 4, wherein a remainder of the surface other than the reactive moieties is a substantially non-reactive surface.

8. The substrate of claim 4, wherein the reactive moiety is coupled to the surface through a linker group.

9. The substrate of claim 8, wherein the linker group comprises a polymer.

10. The substrate of claim 8, wherein the linker group comprises polyethylene glycol.

11. The substrate of claim 7, wherein the reactive moiety is coupled to the surface through a linker group, and the substantially non-reactive surface comprises the linker group without the reactive moiety.

18. A substrate for monitoring enzyme activity, comprising:

a surface having at least a first observation area provided therein, the observation area having an area of from about 100 nm² to 50000 nm²; and

first and second surface modifying agents coupled to the surface by contacting the surface with a mixture of the first and second surface modifying agents, wherein the first surface modifying agent comprises a reactive moiety coupled thereto, and the second surface modifying agent does not comprise the reactive moiety coupled thereto, wherein the mixture of first and second surface modifying agents comprises a ratio of first surface modifying agent to second surface modifying agent such that from 1 reactive moiety to 3 reactive moieties are coupled to the surface within the observation area, wherein the reactive moiety comprises an enzyme.

19. The substrate of claim 18, wherein the observation area is bounded by an optically opaque cladding.

20. The substrate of claim 18, wherein the enzyme comprises a DNA polymerase.

21. A method of preparing a modified surface for monitoring enzyme activity, comprising:

providing a surface to be modified;

contacting the surface to be modified with a surface modifying composition, wherein the surface modifying composition comprises;

a first surface modifying agent coupled to a desired reactive moiety, and a second surface modifying agent not coupled to the desired reactive moiety; and

the first surface modifying agent and second surface modifying agent are present in the surface modifying composition at a ratio that produces the modified surface, wherein the reactive moieties are present on the modified surface at a density of between about 1 reactive moiety per 50,000 nm² and 1 reactive moiety per 100 nm², wherein the reactive moiety comprises an enzyme.