Biosensor and method of manufacture

• US 20050150763A1
• Filed: 01/04/2005
• Published: 07/14/2005
• Est. Priority Date: 01/09/2004
• Status: Abandoned Application

• Current Assignee: Hypoguard USA Inc.
• Original Assignee: Hypoguard USA Inc.
• Inventors: Mayne, Christopher J., Butters, Colin W., Ho, Wah On, Rippeth, John J.
• Application Number:
  US11/028,942
• Publication Number:
  US 20050150763A1
• Time in Patent Office: Days
• US Class Current: 204/403.10
• CPC Class Codes: C12Q 1/001 Enzyme electrodes
• CPC Class Codes: G01N 27/3272 Test elements therefor, i.e. disposable laminated substrates with electrodes, reagent and channels optical biosensors G01N33/52

Abstract

A non-mediated biosensor for indicating amperometrically the catalytic activity of an oxidoreductase enzyme in the presence of a fluid containing a substance acted upon by said enzyme, the biosensor comprising: (a) a first substrate; (b) a working electrode and a counter electrode on the first substrate; (c) conductive tracks connected to said electrodes for making electrical connections with a test meter apparatus; (d) a second substrate overlying at least a part of the first substrate; and (e) a spacer layer having a channel therein and disposed between the first substrate and the second substrate, the spacer layer channel co-operating with adjacent surfaces to define a capillary flow path which does not contain a mesh and which extends from an edge of at least one of said substrates to said electrodes; wherein the working electrode includes: (f) an electrically-conductive base layer comprising particles of finely divided platinum-group metal or platinum-group metal oxide bonded together by a resin; (g) a top layer on the base layer, said top layer comprising a buffer; and (h) a catalytically-active quantity of said oxidoreductase enzyme in at least one of said base layer and said top layer.

Claims

1. A non-mediated biosensor for indicating amperometrically the catalytic activity of an oxidoreductase enzyme in the presence of a fluid containing a substance acted upon by said enzyme, the biosensor comprising:

(a) a first substrate;

(b) a working electrode and a counter electrode on the first substrate;

(c) conductive tracks connected to said electrodes for making electrical connections with a test meter apparatus;

(d) a second substrate overlying at least a part of the first substrate; and

(e) a spacer layer having a channel therein and disposed between the first substrate and the second substrate, the spacer layer channel co-operating with adjacent surfaces to define a capillary flow path which does not contain a mesh and which extends from an edge of at least one of said substrates to said electrodes;

wherein the working electrode includes;

(f) an electrically-conductive base layer comprising particles of finely divided platinum-group metal or platinum-group metal oxide bonded together by a resin;

(g) a top layer on the base layer, said top layer comprising a buffer; and

(h) a catalytically-active quantity of said oxidoreductase enzyme in at least one of said base layer and said top layer.

2. A biosensor according to claim 1, wherein the buffer is selected from a group comprising:

phosphate, ADA, MOPS, MES, HEPES, ACA, and ACES, or buffers with a pKa 7.4±

1.

3. A biosensor according to claim 1, wherein the buffer has a pH in the range 7 to 10.

4. A biosensor according to claim 3, wherein the buffer has a pH in the range 7 to 8.5.

5. A biosensor according to claim 1, further including a system stabiliser in the top layer, comprising a polyol which is not acted upon by the enzyme.

6. A biosensor according to claim 5, wherein the system stabiliser is trehalose.

7. A biosensor according to claim 1, wherein the oxidoreductase enzyme is glucose oxidase.

8. A biosensor according to claim 1, wherein the base layer also contains particles of finely-divided carbon or graphite.

9. A biosensor according to claim 8, wherein said finely divided particles of platinum group metal or oxide are carried on the surface of the finely-divided carbon or graphite.

10. A biosensor according to claim 8, wherein the finely divided particles comprise carbon, and wherein the base layer further includes a blocking agent for blocking active sites of the carbon.

11. A biosensor according to claim 10, wherein said blocking agent comprises a protein or a polyol.

12. A biosensor according to claim 11, wherein the blocking agent is bovine serum albumin (BSA) or trehalose.

13. A biosensor according to claim 1, wherein said oxidoreductase enzyme is located substantially in said top layer.

14. A biosensor according to claim 1, wherein the ratio of buffer to enzyme is in the range 10-70 mol/kg.

15. A biosensor according to claim 14, wherein the ratio of buffer to enzyme is in the range 20-40 mol/kg.

16. A biosensor according to claim 1, wherein the capillary flow path extends from parallel edges of both the first and second substrate to the electrodes.

17. A biosensor according to claim 1, wherein the counter electrode also functions as a reference electrode.

18. A non-mediated biosensor for indicating amperometrically the catalytic activity of an oxidoreductase enzyme in the presence of a fluid containing a substance acted upon by said enzyme, the biosensor comprising:

(a) a first substrate;

(b) a working electrode and a combined reference and counter electrode on the first substrate;

(c) conductive tracks connected to said electrodes for making electrical connections with a test meter apparatus;

(d) a second substrate overlying the first substrate; and

(e) a spacer layer having a channel therein and disposed between the first substrate and the second substrate, the spacer layer channel co-operating with adjacent surfaces to define a capillary flow path which does not contain a mesh and which extends from an edge of at least one of said substrates to said electrodes;

wherein the working electrode includes;

(f) an electrically-conductive base layer comprising particles of finely divided platinum-group metal or platinum-group metal oxide bonded together by a resin;

(g) a top layer on the base layer, said top layer comprising a buffer; and

(h) a catalytically-active quantity of said oxidoreductase enzyme in at least one of said base layer and said top layer.

19. A method of manufacturing a non-mediated biosensor for indicating amperometrically the catalytic activity of an oxidoreductase enzyme in the presence of a fluid containing a substance acted upon by said enzyme, the method comprising the steps of:

(a) taking a first substrate having a working electrode and a counter electrode thereon, and conductive tracks connected to said working and reference electrodes for making electrical connections with a test meter apparatus;

(b) wherein said working electrode is formed by printing on one of said conductive tracks an ink containing finely divided platinum-group metal or platinum-group metal oxide and a resin binder;

(c) causing or permitting said printed ink to dry to form an electrically conductive base layer comprising said platinum-group metal or platinum-group metal oxide bonded together by the resin;

(d) forming a top layer on the base layer by coating the base layer with a coating medium comprising or containing a buffer;

wherein

(e) a catalytically active quantity of said oxidoreductase enzyme is provided in at least one of the printed ink and the coating medium;

(f) providing a second substrate overlying part of the first substrate; and

(g) providing a spacer layer having a channel therein and disposed between the first substrate and the second substrate, whereby the spacer layer channel and adjacent surfaces together define a capillary flow path which does not contain a mesh and which extends from an edge of at least one of said substrates to said electrodes.

20. A method according to claim 19, wherein the coating medium is a coating fluid containing the buffer and wherein the method further comprises causing or permitting said coating fluid to dry to form a top layer on the base layer.

21. A method according to claim 20, wherein the coating fluid is applied by spray coating.

22. A method according to claim 20, wherein the coating fluid has a pH in the range 7 to 8.5.

23. A method according to claim 20, wherein the concentration of buffer in the coating fluid is in the range 300 mmol/l to 1 mol/l.

24. A method according to claim 20, wherein the coating fluid is applied by drop coating.

25. A method according to claim 24, wherein the volume of coating fluid applied to the base layer is in the range 90-160 nl.

26. A method according to claim 19, wherein said enzyme is provided in the coating medium.

27. A method according to claim 19, wherein the ratio of buffer to enzyme is in the range 10-70 mmol/g.

28. A method according to claim 27, wherein the ratio of buffer to enzyme is in the range 20-40 mmol/g.

29. A method according to claim 19, wherein the buffer comprises phosphate or ADA.

30. A method according to claim 19, wherein said finely divided platinum group metal or platinum group metal oxide in said ink is carried on the surface of particles of finely divided carbon or graphite.

31. A method according to claim 19, wherein the spacer layer is formed by printing a UV-curable composition on the first substrate and then curing the composition.

32. A method according to claim 19, wherein the spacer layer comprises double-sided adhesive tape.

33. A method according to claim 19, wherein the second substrate comprises a plastics material having a hydrophilic inner surface.

34. A method according to claim 33, wherein said hydrophilic inner surface comprises a heat-sealable adhesive whereby the second substrate may be adhered to the spacer layer by the action of heat and pressure.