Biosensor and method for producing the same

Abstract

A biosensor for rapid quantification of a specific component contained in various biological samples with high accuracy has an electrically insulating base, an electrode system including a working electrode and a counter electrode formed on one face of the insulating base, and a reaction layer formed on the insulating base in close contact with the electrode system. The reaction layer contains at least a hydrophilic polymer, a buffer and an enzyme which is separated from the buffer.

What is claimed is: 
     
       1. A method for producing a biosensor comprising the steps of: forming a first layer containing an enzyme and a hydrophilic polymer by using water as the medium on a face of an insulating base in close contact with an electrode system including a working electrode and a counter electrode which are provided on said insulating base; and   forming a second layer containing a buffer on said first layer by using an organic solvent solution of a lipid which does not dissolve said hydrophilic polymer.   
     
     
       2. The method for producing a biosensor in accordance with claim 1, wherein said step of forming the first layer comprises spreading an aqueous solution which dissolves the enzyme and the hydrophilic polymer on the insulating base and drying the spread solution, and wherein said step of forming the second layer comprises spreading a solution obtained by dispersing the buffer in an organic solvent solution and drying the spread solution. 
     
     
       3. The method for producing a biosensor in accordance with claim 2, wherein said aqueous solution which dissolves the enzyme and the hydrophilic polymer further dissolves an electron acceptor. 
     
     
       4. A method for producing a biosensor comprising the steps of: forming a first layer containing a buffer and a hydrophilic polymer by using water as the medium on a face of an insulating base in close contact with an electrode system including a working electrode and a counter electrode provided on said insulating base; and   forming a second layer containing a hydrophilic polymer and an enzyme on said first layer by using an organic solvent as the medium that does not dissolve said hydrophilic polymer contained in the first layer.   
     
     
       5. The method for producing a biosensor in accordance with claim 4, wherein said step of forming the first layer comprises spreading an aqueous solution which dissolves the buffer and the hydrophilic polymer on the insulating base and drying the spread solution, and wherein said step of forming the second layer comprises spreading an organic solvent solution of the hydrophilic polymer on the first layer and drying the spread solution, and further dropping an aqueous solution of the enzyme on the second layer and drying the dropped solution. 
     
     
       6. The method for producing a biosensor in accordance with claim 5, wherein said aqueous solution of the enzyme further dissolves an electron acceptor. 
     
     
       7. The method for producing a biosensor in accordance with claim 5, further comprising a step of forming a third layer by spreading an organic solvent solution of a lipid over the second layer and drying the spread solution. 
     
     
       8. A method for producing a biosensor comprising: a first step of spreading an aqueous solution containing a hydrophilic polymer and a buffer on an insulating base in close contact with an electrode system including a working electrode and a counter electrode provided on a face of said insulating base and drying the spread solution, and   a second step of spreading an organic solvent solution containing at least an enzyme and a hydrophilic polymer over the layer and drying the spread solution.   
     
     
       9. The method for producing a biosensor in accordance with claim 8, wherein either of said aqueous solution employed in said first step on the organic solvent solution employed in said second step further contains an electron acceptor. 
     
     
       10. The method for producing a biosensor in accordance with claim 9, wherein the organic solvent solution employed in said second step further contains a hydrophilic polymer. 
     
     
       11. A method for producing a biosensor comprising the steps of: forming a first layer containing an enzyme and a hydrophilic polymer by using water as the medium on a face of an insulating base in close contact with an electrode system including a working electrode and a counter electrode which are provided on said insulating base; and   forming a second layer containing a buffer on said first layer by using an organic solvent solution of a hydrophilic polymer, which solvent does not dissolve said hydrophilic polymer contained in the first layer.   
     
     
       12. The method for producing a biosensor in accordance with claim 11, further comprising a step of forming a third layer by spreading an organic solvent solution of a lipid over the second layer and drying the spread solution. 
     
     
       13. The method for producing a biosensor in accordance with claim 11, wherein said step of forming the first layer comprises spreading an aqueous solution which dissolves the enzyme and the hydrophilic polymer on the insulating base and drying the spread solution, and wherein said step of forming the second layer comprises spreading a solution obtained by dispersing the buffer in the organic solvent solution and drying the spread solution. 
     
     
       14. The method for producing a biosensor in accordance with claim 13, wherein said aqueous solution which dissolves the enzyme and the hydrophilic polymer further dissolves an electron acceptor. .[.15. The method for producing a biosensor in accordance with claim 13, wherein said aqueous solution which dissolves the enzyme and the hydrophilic polymer 
     
     
        further dissolves an electron acceptor..].16. A biosensor comprising: an electrical insulating base,   an electrode system including a working electrode and a counter electrode which are provided on a face of said insulating base, and   a reaction layer formed on said insulating base in close contact with said electrode system; wherein   said reaction layer is a laminate of at least two layers, and wherein a first reaction layer is in contact with said electrode system and contains an enzyme, an electron acceptor and a hydrophilic polymer, and a second reaction layer is on top of said first reaction layer and contains a buffer and a lipid,   
     
     
       said enzyme being separated from said buffer. 17. A biosensor comprising, an electrical insulating base,   an electrode system including a working electrode and a counter electrode which are provided on a face of said insulating base, and   a reaction layer formed on said insulating base in close contact with said electrode system; wherein   said reaction layer is a laminate of at least two layers, and wherein a first reaction layer is in contact with said electrode system and contains an enzyme and an electron acceptor and a second reaction layer is on top of said first reaction layer and contains a buffer and a hydrophilic   
     
     
        polymer, said enzyme being separated from said buffer. 18. A biosensor comprising, an electrical insulating base,   an electrode system including a working electrode and a counter electrode which are provided on a face of said insulating base and   a reaction layer formed on said insulating base in close contact with said electrode system; wherein   said reaction layer is a laminate of at least three layers, and wherein a first reaction layer is in contact with said electrode system and contains an enzyme and an electron acceptor, a second reaction layer is on top of said first reaction layer and contains a buffer and a hydrophilic polymer and a third reaction layer is on top of said second reaction layer and contains a lipid,   
     
     
       said enzyme being separated from said buffer. .Iadd.19.  A method for producing a biosensor comprising the steps of: forming a first layer containing a buffer and a hydrophilic polymer by using water as the medium on a face of an insulating base in close contact with an electrode system including a working electrode and counter electrode provided on said insulating base; and forming a second layer containing a lipid-modified enzyme on said first layer by using an organic solvent as the medium that does not dissolve said hydrophilic polymer contained in the first layer..Iaddend..Iadd.20. The method for producing a biosensor in accordance with claim 19, wherein said step of forming the first layer comprises spreading an aqueous solution which dissolves the buffer and the hydrophilic polymer on the insulating base and drying the spread solution, and wherein said step of forming the second layer comprises spreading an organic solvent solution of the lipid-modified enzyme on the first layer and drying the spread solution..Iaddend..Iadd.21. The method for producing a biosensor in accordance with claim 20, wherein said aqueous solution further dissolves an electron acceptor..Iaddend..Iadd.22. The method for producing a biosensor in accordance with claim 20, wherein said organic solvent solution of the lipid-modified enzyme is dispersed with an electron acceptor..Iaddend..Iadd.23. The method for producing a biosensor in accordance with claim 19, wherein said step of forming the first layer comprises spreading an aqueous solution which dissolves the buffer and the hydrophilic polymer on the insulating base and drying the spread solution, and wherein said step of forming the second layer comprises spreading an organic solvent dispersed with the lipid-modified enzyme on the first layer and drying the spread solution..Iaddend..Iadd.24. The method for producing a biosensor in accordance with claim 23, wherein said aqueous   
     
     
        solution further dissolves an electron acceptor..Iaddend..Iadd.25.  The method for producing a biosensor in accordance with claim 23, wherein said organic solvent solution is further dispersed with an electron acceptor..Iaddend..Iadd.26. A biosensor comprising: an electrical insulating base,   an electrode system including a working electrode and a counter electrode which are provided on a face of said insulating base, and   a reaction layer formed on said insulating base in close contact with said electrode system;   wherein said reaction layer is a laminate of at least two layers, and   wherein a first layer is in contact with said electrode system and contains a buffer and a hydrophilic polymer, and a second layer is on top of said first layer and contains a lipid-modified enzyme, said enzyme being separated from said buffer..Iaddend..Iadd.27. The biosensor in accordance with claim 26, wherein said first layer contains an electron acceptor..Iaddend..Iadd.28. The biosensor in accordance with claim 26, wherein said second layer contains an electron acceptor..Iaddend.