Electrochemical biosensors based on NAD(P)-dependent dehydrogenase enzymes

• US 8,500,990 B2
• Filed: 04/22/2009
• Issued: 08/06/2013
• Est. Priority Date: 04/22/2009
• Status: Active Grant

• Current Assignee: Nova Biomedical Corporation (Nova Chemicals)
• Original Assignee: Nova Biomedical Corporation (Nova Chemicals)
• Inventors: Pei, Jianhong, Young, Chung Chang
• Primary Examiner(s): Ball, J. Christopher
• Assistant Examiner(s): DIETERLE, JENNIFER M
• Application Number:
  US12/427,994
• Publication Number:
  US 20100270175A1
• Time in Patent Office: 1,567 Days
• Field of Search: 204400-40315, 205/787, 205/792, 205/777.5, 205/778, 435/287.1, 422/68.1
• US Class Current: 205/777.5
• CPC Class Codes: C12Q 1/006 for glucose
• 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 biosensor for measuring an analyte in a liquid sample includes a working electrode having a dispensed reagent thereon wherein the dispensed reagent contains an enzyme capable of catalyzing a reaction involving the analyte, a mediator that is considered an enzyme inhibitor, and an enzyme co-factor where the working electrode provides a stable and sensitive response even when the biosensor is stored at ambient conditions for a period of time selected from the group consisting of at least 3 months, 12 months and two years or more.

Claims

1. A method for determining information indicative of the level of an analyte in a liquid sample, the method comprising:

reacting the liquid sample with a working electrode having a reagent matrix disposed thereon, the reagent matrix formed from a dispensing reagent containing an enzyme capable of catalyzing a reaction involving the analyte, a mediator that is considered an enzyme inhibitor, and an enzyme co-factor wherein the mediator is selected from the group consisting of meldola'"'"'s blue, 4-methyl-1,2-benzoquinone, and 2,6-dichloroindophenol, the enzyme co-factor having a concentration in the dispensing reagent of about 0.01% to about 3.0% (w/w) and the mediator having a concentration in the dispensing reagent of about 0.05% to about 3.0% (w/w), wherein the working electrode provides a stable and sensitive response when the working electrode is stored at ambient conditions for a period of time of at least 3 months or longer;

generating an electrical output from the reagent matrix;

measuring the electrical output; and

determining the level of the analyte in the liquid sample using information comprising the measured electrical output.

2. The method of claim 1 wherein the reacting step further includes forming the reagent matrix by dissolving the enzyme, the mediator and the enzyme co-factor in an aqueous solvent forming a dispensing reagent wherein the amount of the enzyme co-factor to the mediator in the dispensing reagent is a ratio in the range of about 0.01:

1 to 15;

1.

3. The method of claim 2 wherein the dissolving step further includes dissolving an amount of the enzyme co-factor that is in the range of about 0.1% to about 0.6% (w/w) of the dispensing reagent.

4. The method of claim 3 wherein the dissolving step further includes selecting the enzyme co-factor from one of NAD⁺ and NADP⁺.

5. The method of claim 1 wherein the dissolving step further includes dissolving an amount of the mediator that is in the range of about 0.2% to about 1.0% (w/w) of the dispensing reagent.

8. The biosensor of claim 1 wherein the mediator has a concentration in the dispensing reagent of about 0.05% to about 2% (w/w).

9. The biosensor of claim 1 wherein the enzyme co-factor has a concentration in the dispensing reagent of about 0.1% to about 0.6% (w/w).

10. The biosensor of claim 9 wherein the enzyme co-factor is one of NAD⁺ and NADP⁺.

11. The biosensor of claim 1 wherein the mediator has a concentration in the dispensing reagent of about 0.2% to about 1.0% (w/w).

12. The biosensor of claim 1 wherein the enzyme is selected from one of glucose dehydrogenase, glucose 6-phosphate dehydrogenase, β

-hydroxybutyrate dehydrogenase, cholesterol dehydrogenase, lactate dehydrogenase, alcohol dehydrogenase, glycerol dehydrogenase, malate dehydrogenase, and fructose dehydrogenase.

13. The biosensor of claim 1 further comprising:

a laminated body having a sample inlet end and an electrical contact end;

a sample inlet;

a substantially flat sample chamber in communication with the sample inlet, the test chamber being adapted to collect a fluid sample through the sample inlet wherein the working electrode and the reference electrode are located within the sample chamber.

14. The biosensor of claim 13 further comprising a compensating electrode within the sample chamber and used in conjunction with the working electrode, the reference electrode or both to negate any interferent species effects from interferent species in the sample.

6. A biosensor for measuring an analyte in a liquid sample, the biosensor comprising:

a working electrode having a reagent matrix formed from a dispensing reagent containing an enzyme capable of catalyzing a reaction involving the analyte, a mediator that is considered as an enzyme inhibitor, and an enzyme co-factor wherein the mediator is selected from the group consisting of meldola'"'"'s blue, 4-methyl-1,2-benzoquinone, and 2,6-dichloroindophenol, the enzyme co-factor having a concentration in the dispensing reagent of about 0.01% to about 3.0% (w/w) and the mediator having a concentration in the dispensing reagent of about 0.05% to about 3.0% (w/w) or less, wherein the working electrode provides a stable and sensitive response even when the biosensor is stored at ambient conditions for a period of time selected from the group consisting of at least 3 months, 12 months and two years or more; and

a reference electrode.

7. The biosensor of claim 6 wherein a ratio of the enzyme co-factor to the mediator is in the range of about 0.01:

1 to 15;

1.

15. A biosensor for measuring an analyte in a liquid sample, the biosensor comprising:

a working electrode having a reagent matrix formed from a dispensing reagent containing a mediator that is considered an enzyme inhibitor, an enzyme and an enzyme co-factor wherein the mediator is selected from the group consisting of meldola'"'"'s blue, 4-methyl-1,2-benzoquinone, and 2,6-dichloroindophenol, the enzyme co-factor having a concentration in the dispensing reagent of about 0.01% to about 3.0% (w/w) and the mediator having a concentration in the dispensing reagent of about 0.05% to about 3.0% (w/w), wherein the working electrode is configured for producing a stable and sensitive response when the biosensor is stored at ambient conditions for 3 months or longer; and

a reference electrode.

16. The biosensor of claim 15 wherein the enzyme co-factor has a concentration in the dispensing reagent in a range of about 0.1% to about 0.6% (w/w) and the mediator has a concentration in the dispensing reagent in a range of about 0.05% to about 2% (w/w).

17. The biosensor of claim 15 wherein the enzyme co-factor has a concentration in the dispensing reagent in a range of about 0.1% to about 0.6% (w/w) and the mediator has a concentration in the dispensing reagent in a range of about 0.2% to about 1% (w/w).

18. A biosensor for measuring an analyte in a liquid sample, the biosensor comprising:

a working electrode having a reagent matrix formed from a dispensing reagent containing a mediator that is considered an enzyme inhibitor, an enzyme and an enzyme co-factor wherein the mediator is selected from the group consisting of meldola'"'"'s blue, 4-methyl-1,2-benzoquinone, and 2,6-dichloroindophenol, the enzyme co-factor having a concentration in the dispensing reagent of about 0.01% to about 3.0% (w/w) and the mediator having a concentration in the dispensing reagent of about 0.05% to about 3.0% (w/w), wherein the enzyme co-factor and the mediator are present in the dispensing reagent that is used to form the reagent matrix in a ratio that enables the working electrode to produce a stable and sensitive response when the biosensor is stored at ambient conditions for 3 months or longer; and

a reference electrode.

19. The biosensor of claim 18 wherein the enzyme co-factor has a concentration in the dispensing reagent in a range of about 0.1% to about 0.6% (w/w) and the mediator has a concentration in the dispensing reagent in a range of about 0.05% to about 2% (w/w).

20. The biosensor of claim 18 wherein the enzyme co-factor has a concentration in the dispensing reagent in a range of about 0.1% to about 0.6% (w/w) and the mediator has a concentration in the dispensing reagent in a range of about 0.2% to about 1% (w/w).

21. A biosensor based on NAD(P)-dependent dehydrogenase enzymes, the biosensor comprising:

a working electrode having a reagent matrix formed from a dispensing reagent containing a mediator that is considered an enzyme inhibitor, NAD(P)-dependent dehydrogenase enzymes and an enzyme co-factor wherein the mediator is selected from the group consisting of meldola'"'"'s blue, 4-methyl-1,2-benzoquinone, and 2,6-dichloroindophenol, the enzyme co-factor having a concentration in the dispensing reagent of about 0.01% to about 3.0% (w/w) and the mediator having a concentration in the dispensing reagent of about 0.05% to about 3.0% (w/w), wherein the working electrode is capable of measuring the concentration of an analyte in a liquid sample without calibrating the working electrode; and

a reference electrode.

22. The biosensor of claim 21 wherein the NAD(P)-dependent dehydrogenase enzymes have a concentration in the dispensing reagent in a range of about 0.1% to about 0.6% (w/w) and the mediator has a concentration in the dispensing reagent in a range of about 0.05% to about 2% (w/w).

23. The biosensor of claim 21 wherein the NAD(P)-dependent dehydrogenase enzymes and the mediator are in a ratio in the range of about 0.01:

1 to 15;

1.

24. The biosensor of claim 21 wherein the NAD(P)-dependent dehydrogenase enzymes have a concentration in the dispensing reagent in a range of about 0.1% to about 0.6% (w/w).

25. The biosensor of claim 21 wherein the mediator has a concentration in the dispensing reagent in a range of about 0.2% to about 1% (w/w).

26. A method of making a biosensor for measuring an analyte in a liquid sample where the biosensor has a mediator considered to be an enzyme inhibitor and is capable of long term storage, the method comprising:

forming a working electrode and a reference electrode on a substrate;

formulating a dispensing reagent containing an enzyme capable of catalyzing a reaction involving the analyte, a mediator that is considered an enzyme inhibitor, and an enzyme co-factor wherein the mediator is selected from the group consisting of meldola'"'"'s blue, 4-methyl-1,2-benzoquinone, and 2,6-dichloroindophenol, the enzyme co-factor having a concentration in the dispensing reagent of about 0.01% to about 3.0% (w/w) and the mediator having a concentration in the dispensing reagent of about 0.05% to about 3.0% (w/w), wherein the ratio of the enzyme co-factor and the mediator in the dispensing reagent enables the working electrode to have a stable response when the biosensor is stored at ambient conditions for 3 months or longer;

dispensing a quantity of the dispensing reagent onto the working electrode; and

evaporating the dispensing reagent forming a reagent matrix on the working electrode.

27. The method of claim 26 wherein the formulating step includes dissolving an amount of the enzyme co-factor and the mediator in the dispensing reagent, the amount being a ratio in the range of about 0.01:

1 to about 15;

1 of the enzyme co-factor to the mediator.

28. The method of claim 26 wherein the dissolving step further includes dissolving an amount of the enzyme co-factor that is in the range of about 0.1% to about 0.6% (w/w) of the dispensing reagent.

29. The method of claim 26 wherein the dissolving step further includes selecting the enzyme co-factor from one of NAD⁺ and NADP⁺.

30. The method of claim 26 wherein the dissolving step further includes dissolving an amount of the mediator that is in the range of about 0.2% to about 1.0% (w/w) of the dispensing reagent.