REVERSIBLY CROSS-LINKED HYDROGELS, AND METHODS OF USING THE SAME FOR CLUSTER AMPLIFICATION

1 . A hydrogel on a substrate, comprising:

a three-dimensional network of polymer chains;

first functional groups coupled to the polymer chains;

amplification primers coupled to the polymer chains via the first functional groups; and

second functional groups coupled to the polymer chains and reversibly cross-linking the polymer chains to one another.

2 . The hydrogel of claim 1 , wherein the first and second functional groups are of different types than one another.

3 . The hydrogel of claim 1 , wherein the first and second functional groups are of the same type as one another.

4 . The hydrogel of claim 1 , wherein the first and second functional groups independently are selected from the group consisting of: azide, amine, thiol, diol, aldehyde, alkyne, strained cyclooctyne, and an inverse electron-demand (IED) Diels-Alder group.

5 . The hydrogel of claim 1 , wherein the second functional groups reversibly cross-link the polymer chains via cleavable molecules.

6 . The hydrogel of claim 5 , wherein the cleavable molecules are cleavable using a chemical agent, an enzyme, light, or heat.

7 . The hydrogel of claim 6 , wherein the chemical agent comprises an acid.

8 . The hydrogel of claim 7 , wherein the cleavable molecules comprise an acetal, ketal, imine, hydrazone, or t-butyl ester that is cleavable by the acid.

9 . The hydrogel of claim 6 , wherein the chemical agent comprises a reducing agent.

10 . The hydrogel of claim 8 , wherein the cleavable molecules comprise a disulfide bond or azidoalkyl ether that is cleavable using the reducing agent, or allyl ether that is cleavable using a palladium complex of the reducing agent.

11 . The hydrogel of claim 6 , wherein the enzyme comprises a DNAase, RNAase, protease, or restriction enzyme, and wherein the cleavable molecules comprise an oligonucleotide that is cleavable using the DNAase, RNAase, protease, or restriction enzyme.

12 . The hydrogel of claim 6 , wherein the enzyme comprises a protease enzyme or lysosomal enzyme, and wherein the cleavable molecules comprise a peptide that is cleavable using the protease enzyme or lysosomal enzyme.

13 . The hydrogel of claim 6 , wherein the cleavable molecules comprise a Diels-Alder conjugation that is cleavable using heat.

14 . The hydrogel of claim 6 , wherein the cleavable molecules comprise a coumarin or nitrobenzene group that is cleavable using light.

15 . The hydrogel of claim 1 , wherein the second functional groups comprise host molecules that reversibly cross-link the backbone via guest molecules.

16 . The hydrogel of claim 15 , wherein the guest molecules are removable via salt, heat, or pH.

17 . The hydrogel of claim 15 , wherein the guest molecules are removable via displacement with a binding partner to the guest molecules.

18 . The hydrogel of claim 15 , wherein the host molecules comprise crown ethers and the guest molecules comprise ammonium moieties.

19 . The hydrogel of claim 15 , wherein the host molecules comprise beta-cyclodextrins and the guest molecules comprise adamantanes, ferrocenes, or bipyridines.

20 . The hydrogel of claim 1 , wherein the second functional groups comprise ligand molecules that reversibly cross-link the backbone via multivalent binding proteins.

21 . The hydrogel of claim 20 , wherein the multivalent binding proteins are removable using a denaturing agent.

22 . A method of using a hydrogel, the method comprising:

depositing a hydrogel on a substrate, the hydrogel comprising three-dimensional network of polymer chains and at least first and second types of functional groups coupled to the polymer chains;

coupling amplification primers to the first functional groups of the deposited hydrogel; and

reversibly stabilizing the deposited hydrogel by reversibly cross-linking the second functional groups of the deposited hydrogel to which the amplification primers are coupled.

23 . A method of using a hydrogel, the method comprising:

depositing a hydrogel on a substrate, the hydrogel comprising a three-dimensional network of polymer chains, amplification primers coupled to the polymer chains, and functional groups coupled to the polymer chains; and

reversibly stabilizing the hydrogel by reversibly cross-linking the functional groups of the deposited hydrogel to which the amplification primers are coupled.

24 . A method of using a hydrogel, the method comprising:

depositing a hydrogel on a substrate, the hydrogel comprising three-dimensional network of polymer chains and first functional groups coupled to the polymer chains;

coupling amplification primers to a first subset of the first functional groups of the deposited hydrogel;

converting a second subset of the first functional groups to second functional groups; and

reversibly stabilizing the hydrogel by reversibly cross-linking the second functional groups.

25 . A method of using a hydrogel, the method comprising:

hybridizing a target polynucleotide to an amplification primer coupled to a hydrogel;

cleaving cross-linkages within the hydrogel within which the target polynucleotide is hybridized to the amplification primer; and

amplifying the target polynucleotide using additional amplification primers within the hydrogel within which the cross-linkages have been cleaved.

26 . The method of claim 25 , further comprising swelling the hydrogel after the cleaving and before the amplifying.