Catalyst-free surface functionalization and polymer grafting
Some embodiments described herein relate to a substrate with a surface comprising a silane or a silane derivative covalently attached to optionally substituted cycloalkene or optionally substituted heterocycloalkene for direct conjugation with a functionalized molecule of interest, such as a polymer, a hydrogel, an amino acid, a nucleoside, a nucleotide, a peptide, a polynucleotide, or a protein. In some embodiments, the silane or silane derivative contains optionally substituted norbornene or norbornene derivatives. Method for preparing a functionalized surface and the use in DNA sequencing and other diagnostic applications are also disclosed.
1. A substrate comprising a first surface comprising silane or a silane derivative covalently bound, optionally through a linker, to a functionalized molecule through reaction of the functionalized molecule with a first plurality of unsaturated moieties selected from the group consisting of cycloalkenes, heterocycloalkenes, and optionally substituted variants and combination thereof covalently attached to said silane or silane derivative, wherein the functionalized molecule comprises a hydrogel or a polymer that comprises a recurring unit of Formula (I) and a recurring unit of Formula (II):
wherein
R 1 is H or alkyl;
R A is selected from the group consisting of azido, optionally substituted amino, optionally substituted alkenyl, optionally substituted hydrazone, optionally substituted hydrazine, carboxyl, hydroxy, optionally substituted tetrazole, optionally substituted tetrazine, nitrile oxide, nitrone and thiol;
X is an optionally substituted alkylene linker or an optionally substituted heteroalkylene linker;
R 4 , R 4′ , R 5 and R 5′ are each independently selected from the group consisting of H, R 6 , OR 6 , —C(O)OR 6 , —C(O)R 6 , —OC(O)R 6 , —C(O)NR 7 R 8 , and —NR 7 R 8 ;
R 6 is independently selected from the group consisting of H, OH, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl, heterocyclyl, and optionally substituted variants thereof; and
R 7 and R 8 are each independently H or alkyl, or R 7 and R 8 are joined together with the atom or atoms to which they are attached to form a heterocycle.
2. The substrate of claim 1 , wherein the first plurality of unsaturated moieties is selected from the group consisting of norbornene, heteronorbornenes, norbornene derivatives, trans-cyclooctene, trans-cyclooctene derivatives, and optionally substituted variants and combinations thereof.
3. The substrate of claim 1 , wherein the first plurality of unsaturated moieties is optionally substituted norbornene.
4. The substrate of claim 1 , wherein the linker is present and is covalently attached between silicon atoms of the silane or silane derivative and the first plurality of unsaturated moieties.
5. The substrate of claim 4 , wherein the linker is selected from the group consisting of optionally substituted alkylenes, optionally substituted heteroalkylenes, optionally substituted cycloalkylenes, optionally substituted heterocyclylenes, optionally substituted arylenes, optionally substituted heteroarylenes, optionally substituted polyethylene glycols, cleavable linkers, and combinations thereof.
6. The substrate of claim 5 , wherein the linker is selected from the group consisting of optionally substituted alkylenes and optionally substituted heteroalkylenes.
7. The substrate of claim 1 , wherein R A is selected from the group consisting of azido, optionally substituted alkenyl, optionally substituted hydrazone, optionally substituted hydrazine, and optionally substituted tetrazine.
8. The substrate of claim 1 , wherein:
R 4 is —C(O)NH 2 ;
R 5 is H or optionally substituted alkyl;
each R 4′ and R 5′ is H;
X is optionally substituted —(CH 2 ) p —; and
p is an integer in the range of 1 to 50.
9. The substrate of claim 8 , wherein p is 5 and R A is azido.
10. The substrate of claim 1 , further comprising oligonucleotides covalently attached to the functionalized molecule through a second plurality of unsaturated moieties on the oligonucleotides that are selected from the group consisting of cycloalkenes, cycloalkynes, heterocycloalkenes, heterocycloalkynes, alkynes, and optionally substituted variants and combinations thereof.
11. The substrate of claim 10 , wherein the second plurality of unsaturated moieties is selected from the group consisting of optionally substituted cyclooctyne, optionally substituted cyclooctyne, optionally substituted bicyclononyne, and optionally substituted bicyclo[6.1.0] non-4-yne.
12. The substrate of claim 10 , wherein the second plurality of unsaturated moieties on the oligonucleotides are alkynes or optionally substituted variants thereof.
13. The substrate of claim 1 , wherein the substrate is selected from the group consisting of a glass substrate, a silica substrate, a plastic substrate, a quartz substrate, a metal substrate, a metal oxide substrate, and combinations thereof.
14. The substrate of claim 1 , wherein the first surface is patterned with nanowells silanized with the silane or silane derivative, wherein the silane or silane derivative is linked to the hydrogel or polymer.
15. A method of preparing the first surface of the substrate of claim 1 , said method comprising:
applying silane or the silane derivative comprising the first plurality of unsaturated moieties covalently attached thereto onto the first surface of the substrate; and
covalently attaching the functionalized molecule to said silane or silane derivative by reacting the functionalized molecule with the first plurality of unsaturated moieties to form a coating layer.
16. The method of claim 15 , wherein the first plurality of unsaturated moieties of the silane or silane derivative is optionally substituted norbornene.
17. The method of claim 15 , wherein the silane or silane derivative are applied onto the first surface by chemical vapor deposition.
18. The method of claim 15 , further comprising
providing oligonucleotides comprising a second plurality of unsaturated moieties selected from the group consisting of cycloalkenes, cycloalkynes, heterocycloalkenes, heterocycloalkynes, alkynes, and optionally substituted variants and combinations thereof;
reacting the second plurality of unsaturated moieties of the oligonucleotides with the functionalized molecule to form covalent bonding.
19. The method of claim 18 , wherein the first plurality of unsaturated moieties is selected from the group consisting of norbornene, heteronorbornenes, norbornene derivatives, trans-cyclooctene, and trans-cyclooctene derivatives, and optionally substituted variants and combinations thereof; and wherein the second plurality of unsaturated moieties is selected from the group consisting of cyclooctyne and bicycloalkynes, and optionally substituted variants and combinations thereof.
20. The method of claim 18 , wherein the second plurality of unsaturated moieties of the oligonucleotides is selected from the group consisting of optionally substituted cyclooctyne, optionally substituted bicyclononyne, and optionally substituted bicyclo[6.1.0]non-4-yne.
21. The method of claim 18 , wherein the second plurality of unsaturated moieties of said oligonucleotides are alkynes or optionally substituted variants thereof.
22. The method of claim 15 , wherein the silane or silane derivative comprise linkers covalently attached between silicon atoms and the first plurality of unsaturated moieties.
23. The method of claim 22 , wherein the linkers are selected from the group consisting of optionally substituted alkylenes, optionally substituted heteroalkylenes, optionally substituted cycloalkylenes, optionally substituted heterocyclylenes, optionally substituted arylenes, optionally substituted heteroarylenes, optionally substituted polyethylene glycols, cleavable linkers, and combinations thereof.
24. The method of claim 22 , wherein the linkers are selected from the group consisting of optionally substituted alkylenes and optionally substituted heteroalkylenes.
25. The method of claim 15 , wherein:
R 4 is —C(O)NH 2 ;
R 5 is H or optionally substituted alkyl;
each R 4′ and R 5′ is H;
X is optionally substituted —(CH 2 ) p —; and
p is an integer in the range of 1 to 50.
26. The method of claim 25 , wherein p is 5 and R A is azido.
27. The method of claim 15 , if further comprising a washing step to remove excess unbounded functionalized molecules.
28. The method of claim 15 , further comprising a drying step.