Covalently coupled cofactor modified electrodes and methods of synthesis and use

• US 4,704,193 A
• Filed: 05/14/1986
• Issued: 11/03/1987
• Est. Priority Date: 05/14/1986
• Status: Expired due to Fees

• Current Assignee: GTE Laboratories Incorporated (Lumen Technologies, Inc.)
• Original Assignee: GTE Laboratories Incorporated (Lumen Technologies, Inc.)
• Inventors: Tripathy, Sukant K., Druy, Mark A., Bowers, Mark L., Durfor, Charles N., Yenser, Barbara A., Smith, H. Gilbert
• Primary Examiner(s): Niebling, John F.
• Assistant Examiner(s): Rubino, Kathryn
• Application Number:
  US06/862,951
• Time in Patent Office: 538 Days
• Field of Search: 204/290 R, 204/403, 204/1 T, 204/418, 427/58, 427/77, 427/338, 427/414, 435/817, 435/291
• US Class Current: 205/777.5
• CPC Class Codes: C12Q 1/003 Functionalisation
• CPC Class Codes: Y10S 435/814 Enzyme separation or purification

Abstract

An electrically and covalently coupled cofactor modified electrode for mediating the heterogeneous or homogeneous catalysis of a biochemical reaction by an enzyme. The electrode coating comprises an imidazoleflavin derivative covalently bonded to the electrode surface. Methods for preparation and use of the modified electrode are also presented.

Claims

1. A cofactor modified electrode comprising:

an electrode substrate having a working surface of an electrically conducting or semiconducting material having an outer oxide or hydroxyl containing layer; and

a redox active material electrically and covalently bonded to the outer layer by means of a suitable linking group;

wherein the redox active material is derived from a redox active flavin derivative having a flavin cofactor moiety, and having the formula ##STR14## wherein R₁ is a ribose derivative, and at least one of R₂ and R₃ is an imidazole derivative, the other of R₂ and R₃ being the imidazole derivative or hydrogen.

2. An electrode according to claim 1 wherein the redox active material is oriented to permit coupling of the flavin cofactor moiety to a suitable apoenzyme in a solution to form a renatured enzyme and to permit electron transfer between the electrode surface and the renatured enzyme.

3. An electrode according to claim 1 wherein the redox active material is oriented to permit electron transfer between the flavin cofactor moiety and a holoenzyme in a solution to activate the holoenzyme.

4. An electrode according to claim 1 wherein R₁ is selected from the group consisting of --CH₂ (CHOH)₃ CH₂ OH, --CH₂ (CHOH)₃ CH₂ O--PO₃^2-, --CH₂ (CHOCOCH₃)₃ CH₂ OCOCH₃, and ##STR15##

5. An electrode according to claim 1 wherein the imidazole derivative is selected from the group consisting of ##STR16##

6. An electrode according to claim 1 wherein the working surface of the electrode is of a material selected from the group consisting of platinum, gold, tin dioxide, silicon, graphite, glassy carbon, and conducting polymers.

7. An electron according to claim 1 wherein the linking group is a derivative of a silane compound having the formula ##STR17## wherein R₄, R₅, and R₆ are independently selected from the group consisting of the Si ligands displaceable by a hydroxide anion, and n is a positive integer from 1 to 20.

8. A cofactor modified electrode comprising:

an electode substrate having a working surface of an electrically conducting or semiconducting material selected from the group consisting of platinum, gold, silicon, graphite, glassy carbon, or tin dioxide, and conducting polymers and having an outer oxide or hydroxyl layer; and

a redox active coating electrically and covalently bonded to the outer layer and comprising groups of the formula ##STR18## wherein R₁ is a ribose derivative selected from the group consisting of --CH₂ (CHOH)₃ --CH₂ OH, --CH₂ (CHOH)₃ CH₂ O--PO₃^2-, --CH₂ (CHOCOCH₃)₃ CH₂ OCOCH₃, and --CH₂ (CHOH)₃ CH₂ O--P--O--P--O--CH₂ ##STR19## the silane group is bonded to the imidazole moiety at the N-3, C-4 or C-5 position on the imidazole ring, and R₇ and R₈ are each an --O--C₂ H₅ group, a hydroxyl group, or an --O-- link to an adjacent silane group or to the outer layer of the electrode surface, and may be the same or different.

9. A method of effecting electron transfer between an electrode having a working surface of an electrically conducting or semiconducting material and an enzyme comprising the steps of:

modifying the working surface of the electrode by electrically and covalently bonding to an existing or derivatized outer oxide or hydroxyl layer on the working surface, by means of a suitable linking group, a redox active material derived from a redox active flavin derivative having a flavin cofactor moiety, and having the formula ##STR20## wherein at least one of R₂ and R₃ is an imidazole derivative, the other of R₂ and R₃ being the imidazole derivative or hydrogen; and

wherein the flavin cofactor moiety of the redox active flavin derivative is selected to activate a protein moiety of the enzyme, R₁ being a ribose derivative selected to provide the selected flavin cofactor moiety;

immersing the modified working surface of the electrode in an electrolyte solution containing the protein moiety of the enzyme; and

applying an electric potential to the electrode sufficient to effect the electron transfer between the electrode surface and the enzyme through the redox active material.

10. A method according to claim 9 wherein R₁ is selected from the group consisting of --CH₂ (CHOH)₃ CH₂ OH, --CH₂ (CHOH)₃ CH₂ O--PO₃^2-, --CH₂ (CHOCOCH₃)₃ CH₂ OCOCH₃, and ##STR21## the imidazole of derivative is selected from the group consisting of ##STR22## the working surface of the electrode is of a material selected from the group consisting of platinum, gold, tin dioxide, silicon, graphite, glassy carbon, and conducting polymers, and the linking group is a derivative of a silane compound having the formula ##STR23## wherein R₄, R₅ and R₆ are independently selected from the group consisting of Si ligands displaceable by a hydroxide anion, and n is a positive integer from 1 to 20.

11. A method of effecting electron transfer between an electrode having a working surface of an electrically conducting or semiconducting material and an enzyme comprising the steps of:

modifying the working surface of the electrode by electrically and covalently bonding to an existing or derivatized outer oxide or hydroxyl layer on the working surface, by means of a suitable linking group a redox active material derived from a redox active flavin derivative having a flavin cofactor moiety, and having the formula ##STR24## wherein at least one of R₂ and R₃ is an imidazole derivative, the other of R₂ and R₃ being the imidazole derivative or hydrogen; and

wherein the flavin cofactor moiety of the redox active flavin derivative is selected to activate the enzyme, R₁ being a ribose derivative selected to provide the selected flavin cofactor moiety;

immersing the modified working surface of the electrode in an electrolyte solution in an electrochemical cell;

introducing the enzyme to the electrochemical cell; and

applying an electric potential to the electrode sufficient to effect the electron transfer between the electrode surface and the enzyme through the redox active material.

12. A method according to claim 11 wherein the step of introducing the enzyme to the electrochemical cell comprises adding the enzyme to the electrolyte solution.

13. A method according to claim 11 wherein the step of introducing the enzyme to the electrochemical cell comprises immobilizing the enzyme on the working surface of the electrode.

14. A method according to claim 11 wherein R₁ is selected from the group consisting of --CH₂ (CHOH)₃ CH₂ OH, --CH₂ (CHOH)₃ CH₂ O--PO₃^2-, --CH₂ (CHOCOCH₃)₃ CH₂ OCOCH₃, and ##STR25## the imidazole derivative is selected from the group consisting of ##STR26## the working surface of the electrode is of a material selected from the group consisting of platinum, gold, tin dioxide, silicon, graphite, glassy carbon, and conducting polymers, and the linking group is a derivative of a silane compound having the formula ##STR27## wherein R₄, R₅ and R₆ are independently selected from the group consisting of Si ligands displaceable by a hydroxide anion, and n is a positive integer from 1 to 20.

15. A method of electrochemically mediating the catalysis of a reaction involving one or more biochemical substrates by an enzyme comprising the steps of:

modifying a working surface of a working electrode by electrically and covalently bonding to an existing or derivatized oxide or hydroxyl layer on the working surface, by means of a suitable linking group, a redox active material derived from a redox active flavin derivative having a flavin cofactor moiety, and having the formula ##STR28## wherein at least one of R₂ and R₃ is an imidazole derivative, the other of R₂ and R₃ being the imidazole derivative or hydrogen; and

wherein the flavin cofactor moiety of the redox active flavin derivative is selected to activate a protein moiety of the enzyme, R₁ being a ribose derivative selected to provide the selected flavin cofactor moiety;

establishing in an electrochemical cell having at least the modified electrode and an auxiliary electrode, an electrolyte solution containing the biochemical substrate(s) and the protein moiety of an enzyme suitable for catalyzing the reaction; and

applying an electric potential to the electrodes sufficient to effect electron transfer between the modified electrode and the biochemical substrate through the redox active material and the enzyme.

16. A method according to claim 15 wherein the enzyme is a metalloenzyme and the electrolyte solution further contains one or more metallic ions suitable for coupling with the protein moiety and the flavin cofactor moiety to complete the enzyme.

17. A method according to claim 15 wherein R₁ is selected from the group consisting of --CH₂ (CHOH)₃ CH₂ OH, --CH₂ (CHOH)₃ CH₂ O--PO₃^2-, --CH₂ (CHOCOCH₃)₃ CH₂ OCOCH₃, and ##STR29## the imidazole derivative is selected from the group consisting of ##STR30## the working surface of the electrode is of a material selected from the group consisting of platinum, gold, tin dioxide, silicon, graphite, glassy carbon, and conducting polymers, and the linking group is a derivative of a silane compound having the formula ##STR31## wherein R₄, R₅ and R₆ are independently selected from the group consisting of Si ligands displaceable by a hydroxide anion, and n is a positive integer from 1 to 20.

18. A method of electrochemically mediating the catalysis of a reaction involving one or more biochemical substrates by an enzyme comprising the steps of:

modifying a working surface of an electrode by electrically and covalently bonding to an existing or derivatized outer oxide or hydroxyl layer on the working surface by means of a suitable linking group, a redox active material derived from a redox active flavin derivative having a flavin cofactor moiety, and having formula ##STR32## wherein at least one of R₂ and R₃ is an imidazole derivative, the other of R₂ and R₃ being the imidazole derivative or hydrogen; and

wherein the flavin cofactor moiety of the redox active flavin derivative is selected to activate the enzyme, R₁ being a ribose derivative selected to provide the selected flavin cofactor moiety;

establishing in an electrochemical cell having at least the modified electrode and an auxiliary electrode, an electrolyte solution containing the biochemical substrate(s)

introducing to the electrochemical cell an enzyme suitable for catalyzing the reaction; and

applying an electric potential to the electrodes sufficient to effect electron transfer between the modified electrode and the biochemical substrate through the redox active material and the enzyme.

19. A method according to claim 18 wherein the step of introducing the enzyme to the electrochemical cell comprises adding the enzyme to the electrolyte solution.

20. A method according to claim 18 wherein the step of introducing the enzyme to the electrochemical cell comprises immobilizing the enzyme on the working surface of the electrode.

21. A method according to claim 18 wherein R₁ is selected from the group consisting of --CH₂ (CHOH)₃ CH₂ OH, --CH₂ (CHOH)₃ CH₂ O--PO₃^2-, --CH₂ (CHOCOCH₃)₃ CH₂ OCOCH₃, and ##STR33## the imidazole derivative is selected from the group consisting of ##STR34## the working surface of the electrode is of a material selected from the group consisting of platinum, gold, tin dioxide, silicon, graphite, glassy carbon, and conducting polymers, and the linking group is a derivative of a silane compound having the formula ##STR35## wherein R₄, R₅ and R₆ are independently selected from the group consisting of Si ligands displaceable by a hydroxide anion, and n is a positive integer from 1 to 20.

22. A method for coating an electrode substrate having a working surface of an electrically conductive or semiconductive material having an outer oxide or hydroxyl layer comprising:

electrically and covalently bonding a redox active material to the outer layer by means of a suitable linking group;

wherein the redox active material is derived from a redox active flavin derivative having the formula ##STR36## wherein R₁ is a ribose derivative and at least one of R₂ and R₃ is an imidazole derivative, the other of R₂ and R₃ being the imidazole derivative or hydrogen.

23. A method according to claim 22 wherein R₁ is selected from the group consisting of --CH₂ (CHOH)₃ CH₂ OH, --CH₂ (CHOH)₃ CH₂ O--PO₃^2-, --CH₂ (CHOCOCH₃)₃ CH₂ OCOCH₃, and ##STR37##

24. A method according to claim 22 wherein the imidazole derivative is selected from the group consisting of derivatives of ##STR38##

25. A method of preparing a working electrode for electrochemically mediating the catalysis of a biochemical reaction by an enzyme comprising the steps of:

placing within a vessel an electrode having a working surface of an electrically conducting or semiconducting material having an outer oxide layer on the working surface;

immersing the working surface in an electrolytic solution containing a redox active material derived from a redox active flavin derivative having the formula ##STR39## wherein R₁ is a ribose derivative, at least one of R₂ and R₃ is an imidazole derivative, the other of R₂ and R₃ being the imidazole derivative or hydrogen; and

a compound from which a suitable linking group may be derived, for a sufficient time to electrically and covalently bond sufficient redox active material to the outer layer by means of the linking group to provide a working electrode capable of electrochemically mediating the catalysis of the biochemical reaction.

26. A method according to claim 25 wherein the working surface of the electrode is of a material selected from the group consisting of platinum, gold, tin dioxide, silicon, graphite, glassy carbon and conducting polymers;

the ribose derivative is selected from the group consisting of --CH₂ (CHOH)₃ CH₂ OH, --CH₂ (CHOH)₃ CH₂ O--PO₃^2-, --CH₂ (CHOCOCH₃)₃ CH₂ OCOCH₃, and ##STR40## the imidazole derivative is selected from the group consisting of ##STR41## and the compound from which a suitable linking group may be derived is a silane compound having the formula ##STR42## wherein R₄, R₅, and R₆ are independently selected from the group consisting of Si ligands displaceable by a hydroxide anion, and n is a positive integer from 1 to 20.

27. A method according to claim 25 wherein the vessel is an electrochemical cell further containing an auxiliary electrode, and further comprising the step of establishing an electrical potential across the electrodes of sufficient density and for sufficient time to electrically and covalently bond sufficient redox active material to the outer layer by means of the linking group to provide a working electrode capable of electrochemically mediating the catalysis of the biochemical reaction.