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

Abstract

The present invention relates to a protein-immobilized membrane ( 14 ) including a cell membrane homologous layer ( 14 A) and a protein ( 14 B) immobilized to the cell membrane homologous layer ( 14 A), 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 (X 1 ) provided with a protein-immobilized membrane ( 14 ). Preferably, the cell membrane homologous layer ( 14 A) may contain a phospholipid polymer, and the protein ( 14 B) may be CyGDH including an α subunit having a glucose dehydrogenase activity and cytochrome C having a function of electron transfer.

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.