Lactate biosensing strip and method for manufacture thereof

• US 20040134797A1
• Filed: 01/15/2003
• Published: 07/15/2004
• Est. Priority Date: 12/31/2002
• Status: Active Grant

• Current Assignee: Asha Chaubey, Bansi Dhar Malhotra, Krishan Kant Pande, Krishan Kumar Saini, Manoj Kumar Pandey, Rajendra Kumar Sharma, Rajesh
• Original Assignee: Asha Chaubey, Bansi Dhar Malhotra, Krishan Kant Pande, Krishan Kumar Saini, Manoj Kumar Pandey, Rajendra Kumar Sharma, Rajesh
• Inventors: Sharma, Rajendra Kumar, Chaubey, Asha, Malhotra, Bansi Dhar, Pandey, Manoj Kumar, Pande, Krishan Kant, Rajesh, Saini, Krishan Kumar
• Granted Patent: US 7,319,018 B2
• Time in Patent Office: Days
• US Class Current: 205/777.5
• CPC Class Codes: C12Q 1/001 Enzyme electrodes
• CPC Class Codes: C12Q 1/003 Functionalisation
• CPC Class Codes: C12Q 1/005 involving specific analytes or enzymes including groups of enzymes, e.g. oxydases; C12Q1/004 takes precedence
• CPC Class Codes: Y10S 435/817 Enzyme or microbe electrode

Abstract

The present invention provides a lactate biosensing strip comprising a working electrode and a reference electrode, the said two electrodes being deposited on an electrically insulated base support, the working electrode being formed by immobilizing an enzyme lactate oxidase and an electro mediator on an inorganic graphite matrix and the graphite layer being deposited on a silver layer and the reference electrode being formed by depositing silver chloride on an another silver layer.

Claims

1. A lactate biosensing strip comprising a working electrode and a reference electrode, the said two electrodes being deposited on an electrically insulated base support, the working electrode being formed by immobilizing an enzyme lactate oxidase and an electro mediator on an inorganic graphite matrix and the graphite layer being deposited on a silver layer and the reference electrode being formed by depositing silver chloride on an another silver layer.

2. A lactate biosensing strip as claimed in claim 1, further comprising

an electrically insulated base support (1),

a pair of first and second silver layers deposited thereon (2) separated by an appropriate space between the two said layers,

a pair of graphite layers, each one of said pair of graphite layers being deposited on one respective silver layer and being electrically connected to said respective silver layer (2),

the first silver layer being covered fully by the respective graphite layer,

the second silver layer being covered partly in the middle thereof with the respective graphite layer after leaving the connecting terminal and working zone area uncovered,

the uncovered working zone of said second silver electrode layer being deposited with silver chloride (4),

lactate oxidase being deposited with a mediator on the working zone of graphite layer covering the first silver layer (5),

the said silver/silver chloride layer forming reference electrode (4) and enzyme with mediator layer forming working electrode (5) being supported on said support (1),

the working zone of reference electrode (4) and working electrode (5) being covered with a hydrophilic membrane.

3. A biosensing strip as claimed in claim 1 wherein the electrically insulated base support used is made of polyvinyl chloride.

4. A biosensing Strip as claimed in claim 1 wherein the distance between the silver layers is in the range of 0.5 to 1 mm

5. A biosensing strip as claimed in claim 1 wherein the thickness of each silver layer is in the range of 15 to 25 microns.

6. A biosensing strip as claimed in claim 1 wherein the electron mediator used is selected from potassium ferricyanide or ferrocene.

7. A biosensing strip as claimed in claim 1 wherein the hydrophilic membrane used is made of nylon or polyester.

8. A biosensing strip as claimed in claim 1 wherein the working zone of an electrode is a target area used for dispensing the analyte sample.

9. A biosensing strip as claimed in claim 1 wherein the enzyme lactate oxidase activity in the strip is in the range of 70-80%

10. A biosensing strip as claimed in claim 1 wherein the response time for lactate detection is in the range of 30 to 40 seconds.

11. A biosensing strip as claimed in claim 1 wherein the amperometric linear response to lactate concentration is in the range of 2-8 mM.

12. A biosensing strip as claimed in claim 1 wherein the shelf life of the biosensing strip is about 4 months under refrigerated conditions.

13. A biosensing strip as claimed in claim 1 wherein the shelf life of the biosensing strip is about 2 months at a temperature in the range of 20-30°

C.

14. A biosensing strip as claimed in claim 1 wherein the strip is disposable.

15. A biosensing strip as claimed in claim 1 wherein the connecting terminal of electrode is an area used for connecting an electrode to an electrometer.

18. A method as claimed in claim 1, wherein the said silver layer is applied by the step of screen-printing.

16. A method for the preparation of lactate biosensing strip comprising an electrically insulated base support (1), a pair of isolated first and second silver layers deposited thereon (2), a pair of graphite layers, each one of said pair of graphite layers being deposited on one respective silver layer and electrically connected to said respective silver layer (2), the first silver layer being covered fully by the respective graphite layer, the second silver layer being covered partly in the middle thereof with the respective graphite layer after leaving the terminal and working zone area uncovered, a Ag/AgCl electrode (4) provided on top of the working zone area of said second silver electrode layer, lactate oxidase deposited with a mediator on the working zone area of graphite layer covering the first silver layer (5), the said silver/silver chloride electrode (4) and enzyme with mediator layer working electrode (5) being supported on said support (1), the working zone of silver/silver chloride reference electrode (4) and working electrode (5) being covered with a hydrophilic membrane, said lactate biosensing strip of the invention showing an activity of 75% and a response time for lactate detection in the range of 30 to 40 seconds;

shelf life of about 4 months under refrigerated conditions and about 2 months under ambient conditions (25 to 30°

C.), the strip of the invention being disposable, said process comprising

(a) depositing a pair of silver layers on an electrically insulated base support;

(b) depositing a pair of graphite layers on said silver layers, each of said silver layers being deposited with one graphite layer, a first graphite layer completely covering the first silver layer, and the second graphite layer covering the second silver layer only in part of the surface thereof that is away from the surface acing the base support;

(c) depositing a silver chloride layer on the second silver layer on the part thereof that is not deposited with a graphite layer to obtain a silver/silver chloride electrode;

(d) adsorbing physically lactate oxidase enzyme with an electron mediator on the first graphite deposited silver layer to obtain a working electrode; and

(e) applying an outer hydrophilic membrane on the above said first reference electrode and second working electrode to obtain the desired pair of electrodes on an electrically insulated base support in a single assembly.

17. A method as claimed in claim 16, wherein the electrically insulated base support used is polyvinyl chloride.

19. A method as claimed in claim 16, wherein the said graphite layer is applied by the step of screen-printing.

20. A method as claimed in claim 16, wherein the sample being tested is an aqueous lactate solution or blood sample in an amount of 25 to 30 μ

L.

21. A method as claimed in claim 16, wherein the electron mediator used is selected from potassium ferricyanide and ferrocene.

22. A method as claimed in claim 16, wherein the working zone are of electrode is a target area used for dispensing the analyte sample.

23. A method as claimed in claim 16, wherein the connecting terminal zone area of electrode is an area used for the connectivity of electrode to an electrometer.

24. A method as claimed in claim 16, wherein the hydrophilic membrane is made of nylon or polyester.