Enzyme immobilization by crosslinking

Patent metadata

Field	Value
Publication number	US-9441258-B2
Application number	US-201313930890-A
Country	US
Kind code	B2
Filing date	Jun 28, 2013
Priority date	Jun 28, 2013
Publication date	Sep 13, 2016
Grant date	Sep 13, 2016

Abstract

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.

First claim

The invention claimed is:

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.

Assignees

• Verily Life Sciences Llc
• Verily Life Sciences LLP

Inventors

• Liu Zenghe

Classifications

• C12Q1/003

  Functionalisation · CPC title

• C12Q1/006

  for glucose · CPC title

• G01N27/40

  Semi-permeable membranes or partitions · CPC title

• C12Q1/002 Primary

  Electrode membranes · CPC title