Stabilization of biomolecules by attachment of responsive polymers and sensors thereof
The present invention provides a biomolecule conjugate having one or more functionalized biomolecules wherein the biomolecule is functionalized with one or more reactive sites, and at least one polymer capable of undergoing a polymer growth reaction, wherein the polymer is attached to at least one of the reactive sites of the functionalized biomolecule and wherein the polymer envelopes the functionalized biomolecule to form a reversible nanoparticle structure which protects the biomolecule by dynamically collapsing to preserve the biomolecule when an adverse environmental stimulus is present. A method of protecting a biomolecule from environmental conditions is also provided.
1. A biomolecule conjugate comprising:
one functionalized biomolecule, said biomolecule is functionalized with one or more sites;
at least one polymer chain having a first end and a second end;
said first end of said chain is attached to said site on said functionalized biomolecule;
said second end of said chain is free moving;
said chain comprises a block copolymer having at least a first polymer block and a second polymer block, wherein said first polymer block is stimuli responsive, wherein said chain collapses in response to said stimuli and envelopes said functionalized biomolecule to form a reversible nanoparticle structure; and
wherein said chain in not cross-linked.
2. The biomolecule conjugate of claim 1 wherein said functionalized biomolecule is one or more enzymes, proteins, antibodies, or biological catalysts.
3. The biomolecule conjugate of claim 2 wherein said enzyme, protein, or biological catalyst remains in a folded position and retains active conformation within said nanoparticle structure.
4. The biomolecule conjugate of claim 1 wherein said biomolecule is functionalized with a site that is at least one of the moieties selected from a group consisting of an amine, a carboxylate, a vincinal diol, a lysine residue, and a thiol group.
5. The biomolecule conjugate of claim 1 wherein said first polymer block is thermally responsive and selected from the group consisting of a poly(N-isopropylacrylamide), a poly(isopropyl-N-vinylpyrrolidone), a polymer which undergoes a conformational change when heated, and combinations thereof.
6. The biomolecule conjugate of claim 1 that has improved stability over a native biomolecule at temperatures greater than 30 degrees Centigrade in both aqueous solutions and dry powders.
7. The biomolecule conjugate of claim 2 wherein said enzymes are selected from the group consisting of organophosphorous hydrolase (OPH), organophosphorous acid anhydrolase (OPAA), butyrylcholinesterase, glucose oxidase (GOX), acetylcholinesterase (AChE), dehalogenase (DHG), diisopropylfluorophosphatase (DFPase), chloroperoxidase, lipase and urease.
8. The biomolecule conjugate of claim 1 wherein said second polymer block provides an additional functionality than said chain collapsing response to a stimuli.
9. The biomolecule conjugate of claim 8 wherein said second polymer block includes one or more primary amines.
10. The biomolecule conjugate of claim 9 wherein said second polymer block is N-(3-aminopropyl)methacrylamide hydrochloride (APMA).
11. The biomolecule conjugate of claim 1 wherein said second polymer block is poly(ethylene glycol) methyl ether methacrylate.
12. The biomolecule conjugate of claim 1 wherein said chain is attached to said functionalized biomolecule using a polymerization initiator that has been covalently attached to the biomolecule prior to attaching said chain.
13. The biomolecule conjugate of claim 12 wherein said polymerization initiator is succinimidyl-tetraethylene glycol (TEG)-bromide.
14. The biomolecule conjugate of claim 1 wherein said chain is not cross-linked to a second chain on said biomolecule, or to another biomolecule or a chain on another biomolecule.
15. The biomolecule conjugate of claim 1 wherein said biomolecule conjugate is from 5 nm to 200 nm in size.
16. The biomolecule conjugate of claim 1 wherein said block copolymer further comprises a third polymer block.
17. A sensor comprising the biomolecule conjugate of claim 1 .
18. The sensor of claim 17 wherein said functionalized biomolecule is one or more enzymes, proteins, antibodies, or biological catalysts.
19. The sensor of claim 18 wherein said enzymes are selected from the group consisting of organophosphorous hydrolase (OPH), organophosphorous acid anhydrolase (OPAA), butyrylcholinesterase, glucose oxidase (GOX), acetylcholinesterase (AChE), dehalogenase (DHG), diisopropylfluorophosphatase (DFPase), urease, chloroperoxidase and Factor VIIa.
20. The sensor of claim 19 including a glucose sensing element with enhanced thermal stability.
21. The sensor of claim 20 wherein said glucose sensing element comprises a GOX containing hydrogel.
22. The sensor of claim 17 wherein said first polymer block is thermally responsive and selected from the group consisting of a poly(N-isopropylacrylamide), a poly(isopropyl-N-vinylpyrrolidone), a polymer which undergoes a conformational change when heated, and combinations thereof.
23. The sensor of claim 17 that has improved stability over a native biomolecule at temperatures greater than 30 degrees Centigrade in both aqueous solutions and dry powders.