Biosensor incorporating protein-immobilized membrane and method of immobilizing protein in biosensor

• US 9,702,843 B2
• Filed: 09/10/2015
• Issued: 07/11/2017
• Est. Priority Date: 05/20/2005
• Status: Active Grant

• Current Assignee: ARKRAY, Inc
• Original Assignee: ARKRAY, Inc
• Inventors: Yamaoka, Hideaki, Katsuki, Koji
• Primary Examiner(s): Van, Luan
• Assistant Examiner(s): Kessel, Maris R
• Application Number:
  US14/850,576
• Publication Number:
  US 20150377818A1
• Time in Patent Office: 670 Days
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C07K 14/80 Cytochromes
• CPC Class Codes: C12N 11/06 attached to the carrier via a bridging agent
• CPC Class Codes: C12N 11/087 Acrylic polymers
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12Q 1/006 for glucose
• CPC Class Codes: G01N 2333/80 Cytochromes
• CPC Class Codes: G01N 27/3272 Test elements therefor, i.e. disposable laminated substrates with electrodes, reagent and channels optical biosensors G01N33/52

Abstract

The present invention relates to a protein-immobilized membrane (14) including a cell membrane homologous layer (14A) and a protein (14B) immobilized to the cell membrane homologous layer (14A), where the protein contains cytochrome or a cytochrome complex. The present invention also relates to a method for forming a protein-immobilized membrane (14), and an enzyme-immobilized electrode and a biosensor (X1) provided with a protein-immobilized membrane (14). Preferably, the cell membrane homologous layer (14A) may contain a phospholipid polymer, and the protein (14B) may be CyGDH including an α subunit having a glucose dehydrogenase activity and cytochrome C having a function of electron transfer.

Claims

1. A biosensor, comprising:

a substrate;

a capillary formed on the substrate for passage of a fluid to be analyzed;

an electrode formed on the substrate; and

a reagent portion formed on the substrate to cover the electrode;

wherein the reagent portion comprises;

an orientation-controlling layer formed on the electrode in contact therewith; and

a layer of protein that is immobilized on a side of the orientation-controlling layer opposite the electrode in direct contact with the orientation-controlling layer, the layer of protein containing a cytochrome or a cytochrome complex, and the cytochrome or cytochrome complex of the layer of protein being oriented toward the orientation-controlling layer; and

wherein the orientation-controlling layer contains a phospholipid polymer, the phospholipid polymer being 2-methacryloyloxyethyl phosphorylcholine polymer.

2. The biosensor according to claim 1, wherein the orientation-controlling layer contains a silane coupling agent for bonding the orientation-controlling layer to the electrode.

3. The biosensor according to claim 2, wherein the silane coupling agent is tetraethoxysilane.

4. The biosensor according to claim 1, wherein the layer of protein comprises cytochrome glucose dehydrogenase containing an α

subunit having a glucose dehydrogenase activity and cytochrome C having a function of electron transfer, the cytochrome C of the layer of protein being oriented toward the orientation-controlling layer, and the α

subunit of the layer of protein being oriented away from the orientation-controlling layer.

5. A biosensor, comprising:

a substrate;

a capillary formed on the substrate for passage of a fluid to be analyzed;

an electrode formed on the substrate; and

a reagent portion formed on the substrate to cover the electrode;

wherein the reagent portion comprises;

an orientation-controlling layer formed on the electrode in contact therewith; and

a layer of protein that is immobilized on a side of the orientation-controlling layer opposite the electrode in direct contact with the orientation-controlling layer, the layer of protein containing a cytochrome or a cytochrome complex, and the cytochrome or cytochrome complex of the layer of protein being oriented toward the orientation-controlling layer; and

wherein the orientation-controlling layer contains a silane coupling agent for bonding the orientation-controlling layer to the electrode.

6. The biosensor according to claim 5, wherein the orientation-controlling layer contains a phospholipid polymer.

7. The biosensor according to claim 5, wherein the silane coupling agent is tetraethoxysilane.

8. The biosensor according to claim 5, wherein the layer of protein comprises cytochrome glucose dehydrogenase containing an α

subunit having a glucose dehydrogenase activity and cytochrome C having a function of electron transfer, the cytochrome C of the layer of protein being oriented toward the orientation-controlling layer, and the α

subunit of the layer of protein being oriented away from the orientation-controlling layer.

9. A method for immobilizing a protein in a biosensor, the biosensor comprising:

a substrate;

a capillary formed on the substrate for passage of a fluid to be analyzed; and

an electrode formed on the substrate;

the method comprising;

forming an orientation-controlling layer on a portion of the electrode in contact therewith; and

causing a layer of protein to immobilize on a side of the orientation-controlling layer opposite the electrode in direct contact with the orientation-controlling layer, the layer of protein containing cytochrome or a cytochrome complex, the cytochrome or cytochrome complex of the layer of protein being oriented toward the orientation-controlling layer; and

wherein the orientation-controlling layer contains a phospholipid polymer, the phospholipid polymer is 2-methacryloyloxyethyl phosphorylcholine polymer.

10. The protein immobilization method according to claim 9, further comprising subjecting said portion of the electrode to hydrophilic treatment before forming the orientation-controlling layer.

11. The protein immobilization method according to claim 10, wherein the orientation-controlling layer is formed to contain a silane coupling agent for bonding the orientation-controlling layer to the electrode.

12. The protein immobilization method according to claim 11, wherein the silane coupling agent is tetraethoxysilane.

13. The protein immobilization method according to claim 9, wherein the layer of protein comprises cytochrome glucose dehydrogenase containing an α

subunit having a glucose dehydrogenase activity and cytochrome C having a function of electron transfer, the protein being immobilized on said portion of the electrode in a manner such that the cytochrome C of the layer of protein is oriented toward the orientation-controlling layer while the α

subunit of the layer of protein is oriented away from the orientation-controlling layer.

14. The protein immobilization method according to claim 9, wherein the layer of protein is immobilized on the orientation-controlling layer by self-assembly.

15. A method for immobilizing a protein in a biosensor, the biosensor comprising:

a substrate;

a capillary formed on the substrate for passage of a fluid to be analyzed; and

an electrode formed on the substrate;

the method comprising;

forming an orientation-controlling layer on a portion of the electrode in contact therewith; and

causing a layer of protein to immobilize on a side of the orientation-controlling layer opposite the electrode in direct contact with the orientation-controlling layer, the layer of protein containing cytochrome or a cytochrome complex, the cytochrome or cytochrome complex of the layer of protein being oriented toward the orientation-controlling layer; and

wherein the orientation-controlling layer is formed to contain a silane coupling agent for bonding the orientation-controlling layer to the electrode.

16. The protein immobilization method according to claim 15, wherein the orientation-controlling layer contains a phospholipid polymer.

17. The protein immobilization method according to claim 16, wherein the phospholipid polymer is 2-methacryloyloxyethyl phosphorylcholine polymer.

18. The protein immobilization method according to claim 15, further comprising subjecting said portion of the electrode to hydrophilic treatment before forming the orientation-controlling layer.

19. The protein immobilization method according to claim 15, wherein the silane coupling agent is tetraethoxysilane.

20. The protein immobilization method according to claim 15, wherein the layer of protein comprises cytochrome glucose dehydrogenase containing an α

subunit having a glucose dehydrogenase activity and cytochrome C having a function of electron transfer, the protein being immobilized on said portion of the electrode in a manner such that the cytochrome C of the layer of protein is oriented toward the orientation-controlling layer while the α

subunit of the layer of protein is oriented away from the orientation-controlling layer.

21. The protein immobilization method according to claim 15, wherein the layer of protein is immobilized on the orientation-controlling layer by self-assembly.