Polymer based enzyme electrode for estimation of cholesterol and process for preparation thereof

• US 7,175,746 B2
• Filed: 10/22/2002
• Issued: 02/13/2007
• Est. Priority Date: 10/22/2002
• Status: Active Grant

• Current Assignee: Council of Scientific & Industrial Research
• Original Assignee: Council of Scientific & Industrial Research
• Inventors: Kumar, Arun, Malhotra, Bansi Daar, Chaubey, Asha, Rajesh, , Grover, Surinder Krishan
• Primary Examiner(s): Noguerola; Alex
• Application Number:
  US10/277,089
• Publication Number:
  US 20040074772A1
• Time in Patent Office: 1,575 Days
• Field of Search: 204/415, 204/317, 20440301-40315, 435/11, 435/14, 435/25, 435/817, 205/317
• US Class Current: 204/403.14
• CPC Class Codes: C12Q 1/002 Electrode membranes
• CPC Class Codes: C12Q 1/005 involving specific analytes or enzymes including groups of enzymes, e.g. oxydases; C12Q1/004 takes precedence

Abstract

A polymer based enzyme electrode useful for estimation for cholesterol is disclosed. The electrode has an ion-doped layer to increase the conductivity of the poly pyrrole layer. The ion doping is achieved by incorporating dodecyl benzene sulphonate (DBS) in the poly pyrrole film deposited by electrochemical deposition The enhanced conductivity due to ion doping helps in faster estimation of cholesterol having a measured response time of 30 seconds. The shelf life of the electrode is at least eight weeks without a mediator at room temperature.

Claims

1. A polymer based enzyme electrode for estimation of cholesterol comprising:

i. an electrically conducting substrate comprising an indium tin oxide coated glass plate,

ii. an ion doped polypyrrole film provided on said electrically conducting substrate,

iii. physically immobilized cholesterol oxidase with a potassium ferricyanide mediator provided on the doped polypyrrole film coated conducting substrate wherein the ion doped polypyrrole film comprises dodecylbenzene sulphonate doped polypyrrole film, to provide an ITO/DBS-PPY/ChOx/Fe3+ electrode configuration.

2. An enzyme electrode as in claim 1 wherein the concentration of dodecylbenzene sulphonate in the polypyrrole film is 1:

1 v/v.

3. An enzyme electrode as in claim 1 wherein the strength of cholesterol oxidase is in the range of 3–

5 IU per square centimeter of polypyrrole film.

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

5. An enzyme electrode as in claim 1 wherein the enzyme electrode has a response time of about 30 seconds.

6. An enzyme electrode as in claim 1 wherein the enzyme electrode has a working range of 6.5–

7.2 pH.

7. An enzyme electrode as in claim 1 wherein the enzyme electrode has a shelf life of at least 8 weeks at a temperature in the range of 20–

25°

C.

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

a. cleaning an electrically conducting substrate of indium tin oxide coated on a glass plate by conventional methods;

b. mixing dodecylbenzene sulphonate (DBS) with pyrrole in a ratio of 1;

1 v/v,

c. electrochemically depositing DBS and pyrrole film on the clean electrically conducting substrate to obtain a DBS doped polypyrrole deposited electrically conducting substrate,

d. physically depositing cholesterol oxidase from a solution consisting of cholesterol oxidase and a potassium ferricynanide mediator in a buffer on the DBS doped polypyrrole deposited conducting substrate obtained in step c,

e. drying the coated conducting substrate of step d to provide an ITO/DBS-PPY/ChOx/Fe3+ electrode configuration.

9. A process as in claim 8 wherein the drying is done at a temperature in the range of 20–

25°

C. for a period of about 12 hrs.

10. A process as in claim 8 wherein the strength of DBS used is in the range of 0.05 to 0.15M.

11. A process as in claim 8 wherein the electrochemical deposition is carried out using a current of about 2 mA and a reference electrode.

12. A process as in claim 11 wherein the reference electrode is a platinum electrode.

13. A process as in claim 8 wherein the strength of cholesterol oxidase is in a range of 3–

5 IU.

14. A process as 8 wherein the buffer used is a phosphate buffer used with a pH in the range of 6.5–

7.2.

15. A polymer based enzyme electrode for estimation of cholesterol with a sensitivity of about 0.4 volt, a response time of about 30 seconds, a working range of 6.5–

7.2 pH and a shelf life of at least 8 weeks at a temperature in the range of 20–

25°

C., the polymer based enzyme electrode comprising;

i. an electrically conducting substrate comprising indium tin oxide coated on a glass plate,

ii. ion doped polypyrrole film provided on said electrically conducting substrate,

iii. physically immobilized cholesterol oxidase with a mediator of potassium ferricyanide provided on the doped polypyrrole film coated conducting substrate wherein the ion doped polypyrrole film comprises dodecylbenzene sulphonate doped polypyrrole film to provide an ITO/DBS-PPY/ChOx/Fe3+ electrode configuration.

16. An enzyme electrode as in claim 15 wherein the concentration of dodecylbenzene sulphonate in the polypyrrole film is 1:

1 v/v.

17. An enzyme electrode as in claim 15 wherein the strength of cholesterol oxidase is in the range of 3–

5 IU per square centimeter of polypyrrole film.