Enzyme immobilization by crosslinking
View Patent ↗An analyte sensor for the continuous or semi-continuous monitoring of physiological parameters and a method for making the analyte sensor are disclosed. In one aspect, the analyte sensor includes an electrode, a sensing layer in contact with a surface of the electrode, and a protective membrane. The sensing layer is a crosslinked, hydrophilic copolymer including poly(alkylene oxide) and poly(vinyl pyridine), and an analyte sensing component is immobilized within the crosslinked, hydrophilic copolymer. The protective membrane is a crosslinked, hydrophilic copolymer including alkylene oxide, vinyl pyridine and styrene units. The method involves the formation of a sensing layer on a surface of an electrode, followed by the formation of a protective membrane on a surface of the sensing layer.
1. An analyte sensor comprising:
a sensing layer in contact with a surface of an electrode, wherein the sensing layer comprises a first crosslinked, hydrophilic, diblock copolymer having an immobilized analyte sensing component, wherein the first diblock copolymer comprises a first block of poly(alkylene oxide) and a second block of poly(vinyl pyridine); and
a protective membrane comprising a second crosslinked, hydrophilic, diblock copolymer, wherein the second diblock copolymer comprises a poly(alkylene oxide) capped random copolymer of vinyl pyridine and styrene.
2. The sensor according to claim 1 , wherein the first crosslinked, hydrophilic diblock copolymer having the formula:
wherein n and m are such that the poly(vinyl pyridine) block and the poly(ethylene glycol) block each have a number average molecular weight (M n ) of about 500 to about 10,000.
3. The sensor according to claim 1 , wherein the second crosslinked, hydrophilic diblock copolymer has the formula:
wherein x, y, v and s are such that the poly(ethylene glycol) block and the vinyl pyridine/styrene copolymer block each have a number average molecular weight (M n ) of about 1,000 to about 100,000.
4. The sensor according to claim 1 , wherein the sensing layer or the protective membrane has crosslinks of the formula:
wherein z is 0-10.
5. The sensor according to claim 1 , wherein the analyte sensing component is glucose oxidase.
6. The sensor according to claim 1 , wherein the sensing layer or protective membrane is about 1 μm to about 5 μm in thickness.
7. The sensor according to claim 1 , wherein the immobilized analyte sensing component is covalently bound, optionally through a linker, to the first crosslinked, hydrophilic, diblock copolymer.
8. The sensor according to claim 1 , wherein the first crosslinked, hydrophilic diblock copolymer has the formula:
wherein n and m are such that the poly(vinyl pyridine) block has a number average molecular weight (M n ) of about 5,000 and the poly(ethylene glycol) block has a number average molecular weight (M n ) of about 8,500;
the second crosslinked, hydrophilic diblock copolymer has the formula:
wherein x, y, v and s are such that the poly(ethylene glycol) block has a number average molecular weight (M n ) of about 5,000 and the vinyl pyridine/styrene copolymer block has a number average molecular weight (M n ) of about 40,000;
the sensing layer and the protective membrane have crosslinks of the formula:
wherein z is 10; and
the analyte sensing component is glucose oxidase.
9. A method for manufacturing an analyte sensor, the method comprising:
forming a first solution of a first hydrophilic, diblock copolymer, a first crosslinking agent and an analyte sensing component, wherein the first hydrophilic, diblock copolymer comprises a first block of poly(alkylene oxide) and second block of poly(vinyl pyridine);
depositing the first solution onto a surface of an electrode;
curing the first solution to provide a sensing layer;
forming a second solution of a second hydrophilic, diblock copolymer and a second crosslinking agent, wherein the second hydrophilic, diblock copolymer comprises a poly(alkylene oxide) capped random copolymer of vinyl pyridine and styrene;
depositing the second solution onto a surface of the sensing layer; and
curing the second solution to provide a protective membrane.
10. The method of claim 9 , wherein the first hydrophilic, diblock copolymer has the formula:
wherein n′ and m′ are selected such that the poly(vinyl pyridine) block and the poly(ethylene glycol) block each have a number average molecular weight (M n ) of about 500 to about 10,000.
11. The method of claim 9 , wherein the second hydrophilic, diblock copolymer has the formula:
wherein x′, y′, v′ and s′ are selected such that the poly(ethylene glycol) block and the vinyl pyridine/styrene copolymer block each have a number average molecular weight (M n ) of about 1,000 to about 100,000.
12. The method of claim 9 , wherein the first or second crosslinking agent has the formula:
wherein z′ is 0-10.
13. The method of claim 9 , wherein the sensing layer or the protective membrane is about 1 μm to about 5 μm in thickness.
14. The method of claim 9 , wherein the analyte sensing component is glucose oxidase.
15. The method of claim 9 , wherein the second solution comprises about 90% by weight second hydrophilic, diblock copolymer and about 10% by weight second crosslinking agent.
16. The method of claim 9 , wherein the forming, depositing, and curing of the second solution is repeated to increase the thickness of the protective membrane.
17. The method of claim 9 , wherein the amount of analyte sensing component is about 10% by weight to about 40% by weight of the first solution.
18. The method of claim 9 , wherein the amount of first crosslinking agent is about 20% by weight to about 30% by weight of the first solution.
19. The method of claim 9 , wherein the amount of first hydrophilic, diblock copolymer is about 20% by weight to about 70% by weight of the first solution.