Enzyme electrode and process for preparation thereof

• US 20040149577A1
• Filed: 01/16/2003
• Published: 08/05/2004
• Est. Priority Date: 01/16/2003
• Status: Active Grant

• Current Assignee: Arun Kumar, Bansi Dhar Malhotra, Rajesh
• Original Assignee: Arun Kumar, Bansi Dhar Malhotra, Rajesh
• Inventors: Kumar, Arun, Malhotra, Bansi Dhar, Rajesh
• Granted Patent: US 7,267,837 B2
• Time in Patent Office: Days
• US Class Current: 204/403.01
• 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: C12Q 1/60 involving cholesterol

Abstract

The present invention relates to an enzyme electrode useful for estimation of cholesterol in aqueous medium, said electrode comprising:

i. an electrically conductive base plate,

ii. a film of sol gel derived material deposited thereon,

said sol gel derived material of step b) being microencapsulated cholesterol oxidase with an electron mediator. The present invention also relates to a process for the preparation of an enzyme electrode by coating an immobilized cholesterol oxidase (ChOx) and mediator on a silicate sol gel by microencapsulation.

Claims

1. An enzyme electrode useful for estimation of cholesterol in aqueous medium, said electrode comprising:

i. an electrically conductive base plate,

ii. a film of sol gel derived material deposited thereon,

iii. said sol gel derived material of step b) being microencapsulated Cholesterol oxidase with an electron mediator,

said enzyme electrode showing zero leaching of the encapsulated enzyme and of the electron mediator, a response time of 30 seconds, an amperometric linear response to cholesterol in the range of 1-8 mm, being reusable at least five times and a shelf life of six months.

2. An enzyme electrode as claimed in claim 1, wherein the electrically conductive base plate used is selected from indium tin oxide coated glass plate and a silver coated non-conducting polymer surface.

3. An enzyme electrode as claimed in claim 1, wherein the non-conducting polymer surface used is selected from a film and a sheet.

4. An enzyme electrode as claimed in claim 1, wherein the non-conducting polymer surface used is selected from the group consisting of polyacrylamide, polyvinyl chloride and polyethylene.

5. An enzyme electrode as claimed in claim 1 wherein the sol material used is silica sol.

6. An enzyme electrode as claimed in claim 1 wherein the silica sol used is selected from tetraethyl orthosilicate and tetramethyl orthosilicate.

7. An enzyme electrode as claimed in claim 1 wherein the electron mediator used is selected from potassium ferricyanide, ferrocene and Prussian blue.

8. An enzyme electrode as claimed in claim 1 wherein the enzyme electrode has a sensitivity of 0.4 volt.

9. An enzyme electrode as claimed in claim 1 wherein the strength of cholesterol oxidase used is in the range of 3-5 IU per 1×

1 cm² of sol-gel surface area.

10. A process for the preparation of enzyme electrode useful for estimation of cholesterol in aqueous medium, which comprises the steps of:

a. preparing a silicate solution,

b. immobilizing an enzyme cholesterol oxidase and an electron mediator by slowly adding 0.05-0.1 M phosphate buffer containing 3-5 IU of cholesterol oxidase arid about 0.01 M of electron mediator on to the above said silicate solution of step a),

c. allowing the resultant mixture to stand till the complete encapsulation of enzyme and mediator by observing turbidity,

d. spreading the resultant turbid mixture on a conductive base plate,

e. drying the conductive base plate with the spread mixture for at least one day at a temperature in a range of 25-30°

C. to obtain the enzyme electrode.

11. A process as claimed in claim 10 wherein the silicate sol used is selected from tetraethyl orthosilicate and tetramethyl orthosilicate

12. A process as claimed in claim 10 wherein the phosphate buffer used has a pH in a range of 6.5-7.2.

13. A process as claimed in claim 10 wherein the electrically conductive base plate is selected from indium tin oxide coated glass plate and silver-coated non-conducting polymer surface.

14. A process as claimed in claim 10 wherein the non-conducting polymer surface is selected from a film and a sheet.

15. A process as claimed in claim 14 wherein the non-conducting polymer surface is selected from the group consisting of polyacrylamide, polyvinyl chloride and polyethylene.

16. A process as claimed in claim 10 wherein the electron mediator is selected from potassium ferricyanide, ferrocene and Prussian blue.

17. A process as claimed in claim 10 wherein the strength of cholesterol oxidase is in the range of 3-5 IU per 1×

1 cm² of sol-gel surface area.

18. A process as claimed in claim 10 wherein the process of preparation of enzyme electrode is a single step process.