Hydrogel-free surface functionalization for sequencing
Embodiments of the present application relate to substrate comprising a surface-bound azido functionalized organosilane wherein the substrate is free or substantially free of a hydrogel or a hydrophilic polymer. Methods of preparing such substrate surface for sequencing applications are also disclosed.
1 . A substrate comprising a surface-bound organosilane covalently attached to a plurality of biological molecules, wherein the surface-bound organosilane comprises azido groups that are covalent bonded with the plurality of biological molecules, each azido group is attached to a silicon atom of the organosilane through a linker moiety, and wherein the substrate is free or substantially free of a hydrogel or a hydrophilic polymer.
2 . The substrate of claim 1 , wherein the linker moiety comprises a hydrophobic region and a hydrophilic region.
3 . The substrate of claim 1 , the surface-bound organosilane comprises a plurality of the structure:
wherein linker comprises optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted heteroalkylene, optionally substituted arylene, optionally substituted cycloalkylene, optionally substituted heteroarylene, or optionally substituted heterocyclylene, or combinations thereof.
4 . The substrate of claim 1 , wherein the linker moiety comprises alkylene or —(CH 2 CH 2 O) m —, or a combination thereof, wherein m is an integer of 1 to 10.
5 . The substrate of claim 1 , each of the plurality of biological molecules comprises an alkynyl group, a DBCO moiety or a BCN moiety, and the azido group of the surface-bound organosilane forms a triazole moiety with the biological molecule.
6 . The substrate of claim 5 , wherein the plurality of biological molecules comprise nucleosides, nucleotides, oligonucleotides, polynucleotides, peptides, amino acids, or proteins, or combinations thereof.
7 . The substrate of claim 6 , wherein the plurality of biological molecules are oligonucleotides, each comprising a spacer and an adaptor sequence for hybridization with a template polynucleotide.
8 . The substrate of claim 7 , wherein the spacer comprises 10 to 50 T nucleotides, C 1-10 alkylene, PEG, or a combination thereof.
9 . The substrate of claim 8 , wherein the spacer comprises 30 T nucleotide and C 3 alkylene.
10 . The substrate of claim 1 , wherein the substrate is a flow cell, a nanoparticle, or a bead.
11 . The substrate of claim 1 , wherein the substrate comprises patterned nanowells separated by interstitial regions, and wherein the plurality of biological molecules reside inside the patterned nanowells.
12 . A method of functionalizing a surface of a substrate, comprising:
depositing an organosilane to the surface of the substrate to form a surface-bound organosilane layer, wherein the surface-bound organosilane layer comprises azido groups, each azido group is attached to a silicon atom of the organosilane through a linker moiety, and wherein the method does not comprise depositing a hydrogel or a hydrophilic polymer to the surface of the substrate prior to or after the deposition of the organosilane.
13 . A method of immobilizing oligonucleotides to a substrate, comprising:
contacting a plurality of oligonucleotides with an organosilane bound surface of the substrate, wherein the organosilane comprises azido groups, each azido group is attached to a silicon atom of the organosilane through a linker moiety, and wherein the substrate is free or substantially free of a hydrogel or a hydrophilic polymer; and
reacting the plurality of the oligonucleotides with the azido groups of the organosilane to covalently attach the oligonucleotides to the substrate.
14 . The method of claim 13 , wherein the substrate comprising a plurality of nanowells separated by interstitial regions.
15 . The method of claim 14 , further comprising removing the oligonucleotides from the interstitial regions of the surface such that the oligonucleotides are only present inside the nanowells.
16 . The method of claim 13 , wherein each of the plurality of oligonucleotides comprises an alkynyl group, a DBCO moiety, or a BCN moiety, and the azido group of the organosilane forms a triazole moiety with the oligonucleotide.
17 . The method of claim 13 , wherein each of the plurality of oligonucleotides comprises a spacer and an adaptor sequence complementary to at least a portion of a template polynucleotide sequence.
18 . The method of claim 17 , wherein the spacer comprises 10 to 50 T nucleotides, C 1-10 alkylene, or a combination thereof.
19 . The method of claim 18 , wherein the spacer comprises 30 T nucleotide and C 3 alkylene.
20 . The method of claim 13 , further comprising contacting the immobilized oligonucleotides with a plurality of template polynucleotides; and amplifying the template polynucleotides.
21 . The method of claim 13 , wherein the linker moiety comprises a hydrophobic region and a hydrophilic region.
22 . The method of claim 13 , the linker moiety comprises optionally substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted heteroalkylene, optionally substituted arylene, optionally substituted cycloalkylene, optionally substituted heteroarylene, or optionally substituted heterocyclylene, or combinations thereof.
23 . The method of claim 22 , wherein the linker moiety comprises alkylene or —(CH 2 CH 2 O) m —, or a combination thereof, wherein m is an integer of 1 to 10.