Protein-Immobilized membrane, method for immobilization of protein, enzyme-immobilized electrode, and biosensor

• US 20090101499A1
• Filed: 05/18/2006
• Published: 04/23/2009
• Est. Priority Date: 05/20/2005
• Status: Abandoned Application

• Current Assignee: ARKRAY, Inc
• Original Assignee: ARKRAY, Inc
• Inventors: Yamaoka, Hideaki, Katsuki, Koji
• Application Number:
  US11/920,782
• Publication Number:
  US 20090101499A1
• Time in Patent Office: Days
• US Class Current: 204/403.140
• 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 protein-immobilized membrane comprising:

a cell membrane homologous layer; and

a protein immobilized to the cell membrane homologous layer, the protein containing cytochrome or a cytochrome complex.

2. The protein-immobilized membrane according to claim 1, wherein the cell membrane homologous layer contains a phospholipid polymer.

3. The protein-immobilized membrane according to claim 2, wherein the phospholipid polymer is 2-methacryloyloxyethyl phosphorylcholine polymer.

4. The protein-immobilized membrane according to claim 1, wherein the cell membrane homologous layer contains a silane coupling agent.

5. The protein-immobilized membrane according to claim 4, wherein the silane coupling agent is tetraethoxysilane.

6. The protein-immobilized membrane according to claim 1, wherein the protein is CyGDH containing an α

subunit having a glucose dehydrogenase activity and cytochrome C having a function of electron transfer.

7. A method for immobilizing a protein, the method comprising:

a first step of forming a cell membrane homologous layer at an intended portion of an immobilization target member; and

a second step of causing a protein to self-organize with respect to the cell membrane homologous layer, the protein containing cytochrome or a cytochrome complex.

8. The protein immobilization method according to claim 7, wherein the cell membrane homologous layer contains a phospholipid polymer.

9. The protein immobilization method according to claim 8, wherein the phospholipid polymer is 2-methacryloyloxyethyl phosphorylcholine polymer.

10. The protein immobilization method according to claim 7, further comprising a third step of subjecting the intended portion to hydrophilic treatment before the first step.

11. The protein immobilization method according to claim 10, wherein, in the second step, the cell membrane homologous layer is formed to contain a silane coupling agent.

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

13. The protein immobilization method according to claim 7, wherein the protein is CyGDH containing an α

subunit having a glucose dehydrogenase activity and cytochrome C having a function of electron transfer.

14. An enzyme-immobilized electrode comprising:

a substrate; and

an enzyme-containing layer immobilized to the substrate;

wherein the enzyme-containing layer includes a cell membrane homologous layer and an enzyme, the enzyme containing, as a subunit, cytochrome C immobilized to the cell membrane homologous layer by self organization.

15. The enzyme-immobilized electrode according to claim 14, wherein the cell membrane homologous layer contains a phospholipid polymer.

16. The enzyme-immobilized electrode according to claim 15, wherein the phospholipid polymer is 2-methacryloyloxyethyl phosphorylcholine polymer.

17. The enzyme-immobilized electrode according to claim 14, wherein the cell membrane homologous layer contains a silane coupling agent.

18. The enzyme-immobilized electrode according to claim 17, wherein the silane coupling agent is tetraethoxysilane.

19. The enzyme-immobilized electrode according to claim 14, wherein the enzyme is CyGDH containing an α

subunit having a glucose dehydrogenase activity and cytochrome C having a function of electron transfer.

20. A biosensor comprising:

a substrate; and

an enzyme-containing layer immobilized to the substrate;

wherein the enzyme-containing layer includes a cell membrane homologous layer and an enzyme, the enzyme containing, as a subunit, cytochrome C immobilized to the cell membrane homologous layer by self organization.

21. The biosensor according to claim 20, wherein the cell membrane homologous layer contains a phospholipid polymer.

22. The biosensor according to claim 21, wherein the phospholipid polymer is 2-methacryloyloxyethyl phosphorylcholine polymer.

23. The biosensor according to claim 20, wherein the cell membrane homologous layer contains a silane coupling agent.

24. The biosensor according to claim 23, wherein the silane coupling agent is tetraethoxysilane.

25. The biosensor according to claim 20, wherein the enzyme is CyGDH containing an α

subunit having a glucose dehydrogenase activity and cytochrome C having a function of electron transfer.

26. The biosensor according to claim 20, further comprising:

a flow path for moving a sample; and

a reagent portion-provided in the flow path.

27. The biosensor according to claim 26, further comprising a working electrode and a counter electrode which are partially exposed at the flow path and utilized for applying a voltage to a sample.

28. The biosensor according to claim 27, at least part of the cell membrane homologous layer is formed on the working electrode.

29. The biosensor according to claim 26, wherein the reagent portion contains a color former.

30. The biosensor according to claim 29, wherein the reagent portion includes a chromogenic layer containing the color former, the cell membrane homologous layer, and an enzyme-containing layer containing the enzyme.