Substrate with orthogonally functional nanodomains
Embodiments of the present disclosure also relate to methods of fabricating flow cell substrates. Some exemplary workflows exploit orthogonal chemistries of substrate layers such that the process does not include polishing steps. Substrates prepared by the method described herein can include a first primer set and a second primer set compatible with simultaneous paired-end sequencing methods.
1 . A patterned substrate, comprising:
a base support;
a multi-layer stack positioned over the base support; the multi-layer stack comprising:
a first layer positioned over the base support;
a second layer positioned over the first layer, wherein the second layer comprises a plurality of depressions through the second layer and expose a surface of the first layer; and
a functionalized hydrogel within at least a portion of the plurality of depressions, wherein the functionalized hydrogel is covalently attached to the first layer;
wherein the first layer comprises poly(allylamine) (PAAm) and the second layer comprises poly (propargyl methacrylate) (PPMA), or the first layer comprises PPMA and the second layer comprises PAAm.
2 . The patterned substrate of claim 1 , wherein the depression comprises a swath, a fiducial, a lane, or a well.
3 . The patterned substrate of claim 1 , wherein the functionalized hydrogel comprises a polyacrylamide copolymer having functional groups that form covalent bonding with the PAAm or PPMA of the first layer of the multi-layer stack.
4 . The patterned substrate of claim 1 , wherein the first layer of the multi-layer stack comprises PAAm and the second layer of the multi-layer stack comprises PPMA.
5 . The patterned substrate of claim 4 , wherein the functionalized hydrogel comprises N-hydroxysuccinimide (NHS) ester groups that form amide bonds with the first layer.
6 . The patterned substrate of claim 1 , wherein the first layer of the multi-layer stack comprises PPMA and the second layer of the multi-layer stack comprises PAAm.
7 . The patterned substrate of claim 6 , wherein the functionalized hydrogel comprises azido groups that form triazoline bonds with the first layer.
8 . The patterned substrate of claim 7 , wherein the functionalized hydrogel comprises poly (N-(5-azidoacetamidylpentyl) acrylamide-co-acrylamide) (PAZAM).
9 . The patterned substrate of claim 1 , further comprising oligonucleotide or polynucleotide primers attached to the functionalized hydrogel.
10 . A patterned substrate, comprising:
a base support;
a multi-layer stack positioned over the base support; the multi-layer stack comprising:
a first layer positioned over the base support;
a second layer positioned over the first layer, wherein the second layer comprises a first plurality of depressions through the second layer which expose a surface of the first layer; and
a third layer positioned over the second layer, wherein the third layer comprises a second plurality of depressions through the third layer which expose a surface of the second layer, wherein the second plurality of depressions are spatially separated from the first plurality of depressions;
a first functionalized hydrogel within at least a portion of the first plurality of depressions, wherein the first functionalized hydrogel is covalently attached to the first layer; and
a second functionalized hydrogel within at least a portion of the second plurality of depressions, wherein the second functionalized hydrogel is covalently attached to the second layer;
wherein the first layer of the multi-layer stack comprises a first polymer having a plurality of first functional groups selected from the group consisting of an amino, an alkynyl, a cycloalkenyl, a cycloalkynyl, a carboxyl, a vinyl, and a thiol;
wherein the second layer of the multi-layer stack comprises a second polymer having a plurality of second functional groups selected from the group consisting of an amino, an alkynyl, a cycloalkenyl, a cycloalkynyl, a carboxyl, a vinyl, and a thiol, and the second functional groups are orthogonal to the first functional groups; and
wherein the third layer does not comprise either the first or the second functional groups, and wherein the third layer is inert to both the first functionalized hydrogel and the second functionalized hydrogel.
11 . The patterned substrate of claim 10 , wherein the first functionalized hydrogel comprises a first polyacrylamide copolymer having functional groups that form covalent bonding with the plurality of first functional groups.
12 . The patterned substrate of claim 10 , wherein the second functionalized hydrogel comprises a second polyacrylamide copolymer having functional groups that form covalent bonding with the plurality of second functional groups.
13 . The patterned substrate of claim 10 , wherein the first layer of the multi-layer stack comprises amino groups and the second layer of the multi-layer stack comprises alkynyl groups.
14 . The patterned substrate of claim 13 , wherein the first layer of the multi-layer stack comprises poly (allylamine) (PAAm) and the second layer of the multi-layer stack comprises poly (propargyl methacrylate) (PPMA).
15 . The patterned substrate of claim 13 , wherein the first functionalized hydrogel comprises N-hydroxysuccinimide (NHS) ester groups that form amide bonds with the first layer.
16 . The patterned substrate of claim 15 , wherein the second functionalized hydrogel comprises azido groups that form triazoline bonds with the second layer.
17 . The patterned substrate of claim 10 , wherein the first layer of the multi-layer stack comprises alkynyl groups and the second layer of the multi-layer stack comprises amino groups.
18 . The patterned substrate of claim 17 , wherein the first layer of the multi-layer stack comprises PPMA and the second layer of the multi-layer stack comprises PAAm.
19 . The patterned substrate of claim 17 , wherein the first functionalized hydrogel comprises azido groups that form triazoline bonds with the first layer.
20 . The patterned substrate of claim 19 , wherein the second functionalized hydrogel comprises N-hydroxysuccinimide (NHS) ester groups that formamide bonds with the second layer.
21 . The patterned substrate of claim 10 , wherein the first or second functionalized hydrogel comprises (N-( 5 -azidoacetamidylpentyl) acrylamide-co-acrylamide) (PAZAM).
22 . The patterned substrate of claim 10 , wherein the first plurality of depressions comprise or define a first region on the substrate, and the second plurality of depressions comprise or define a second region on the substrate, wherein the first region is either spatially separate from the second region, or the first region is overlapping with the second region.