Reversible surface functionalization

• US 9,982,250 B2
• Filed: 08/20/2015
• Issued: 05/29/2018
• Est. Priority Date: 08/21/2014
• Status: Active Grant

• Current Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Inventors: von Hatten, Xavier, George, Wayne N., Richez, Alexandre, Dingwall, Anne-Cecile, Brown, Andrew A.
• Primary Examiner(s): Forman, Betty J
• Application Number:
  US14/831,062
• Publication Number:
  US 20160053252A1
• Time in Patent Office: 1,013 Days
• Field of Search: None
• CPC Class Codes: C09D 183/04 Polysiloxanes
• CPC Class Codes: C12N 11/10 the carrier being a carbohydrate
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2565/543 characterised by the use of two or more capture oligonucleotide primers in concert, e.g. bridge amplification
• CPC Class Codes: G01N 33/543 with an insoluble carrier for immobilising immunochemicals
• CPC Class Codes: G01N 33/54353 with ligand attached to the carrier via a chemical coupling agent coatings G01N33/54393
• 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: G01N 33/551 the carrier being inorganic

Abstract

Some embodiments described herein relate to a substrate comprising a silane functionalized surface for reversibly immobilizing a biological molecule of interest, such as oligonucleotides, polynucleotides, or protein. Methods for immobilizing the biological molecule and the use in DNA sequencing and other diagnostic applications are also disclosed.

Claims

1. A method for reversibly immobilizing a biological molecule to a surface of a substrate, comprising:

providing a biological molecule covalently bonded to a host molecule, said host molecule comprises a hydrophilic portion and a hydrophobic portion, wherein the biological molecule comprises a primer oligonucleotide and wherein said host molecule comprises an optionally substituted cyclodextrin or a derivative thereof;

providing a substrate having a surface comprising functionalized silane covalently attached thereto, wherein the functionalized silane comprises a guest moiety that can form a host-guest complex with the host molecule;

contacting the biological molecule with the functionalized silane such that the biological molecule is immobilized to the surface through the host-guest reversible interaction between the functionalized silane and the host molecule; and

generating a clustered array of polynucleotides from the primer oligonucleotide.

2. The method of claim 1, wherein the host-guest interaction is a non-covalent interaction between the guest moiety of the functionalized silane and the host molecule.

3. The method of claim 1, wherein the guest moiety of the functionalized silane comprises a hydrophobic moiety.

4. The method of claim 3, wherein the host-guest non-covalent interaction is between the hydrophobic moiety of the functionalized silane and the hydrophobic portion of the host molecule.

5. The method of claim 1, wherein the biological molecule is covalently bonded to the hydrophilic portion of the host molecule.

6. The method of claim 1, further comprising dissociating the host molecule from the surface of the substrate.

7. The method of claim 6, wherein the dissociation is achieved by adding a molecule of greater affinity to the host molecule than the guest moiety of the functionalized silane.

8. The method of claim 7, wherein the dissociation is achieved by adding a molecule of greater affinity to the hydrophobic portion of the host molecule than the hydrophobic moiety of the functionalized silane.

9. The method of claim 8, wherein said molecule of greater affinity to the hydrophobic portion of the host molecule is 1,8-octanediol.

10. The method of claim 1, wherein the host molecule is β

-cyclodextrin.

11. The method of claim 1, further comprising a washing step to recycle the substrate.

12. The method of claim 1, wherein the guest moiety of the functionalized silane comprises aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, or cycloalkynyl, or optionally substituted variants thereof with one or more functional groups capable of forming covalent bonding with a functionalized polymer or hydrogel coating for surface functionalization.

13. The method of claim 12, wherein the guest moiety comprises aryl.

14. The method of claim 12, wherein the guest moiety comprises cycloalkenyl.

15. The method of claim 14, wherein the cycloalkenyl is norbornene.

16. The method of claim 1, wherein the guest moiety is substituted with one or more fluoro groups.

17. A method for reversibly immobilizing a biological molecule to a surface of a substrate, comprising:

providing a biological molecule covalently bonded to a host molecule, said host molecule comprises a hydrophilic portion and a hydrophobic portion;

providing a substrate having a surface comprising functionalized silane covalently attached thereto, wherein the functionalized silane comprises a guest moiety that can form a host-guest complex with the host molecule;

contacting the biological molecule with the functionalized silane such that the biological molecule is immobilized to the surface through the host-guest reversible interaction between the functionalized silane and the host molecule,

wherein the guest moiety of the functionalized silane comprises norbornene.

18. The method of claim 17, wherein said host molecule comprises an optionally substituted cyclodextrin or a derivative thereof.

19. The method of claim 17, wherein the biological molecule comprises a primer oligonucleotide.