Electrobiochemical analytical method and electrodes

• US 5,443,701 A
• Filed: 08/23/1993
• Issued: 08/22/1995
• Est. Priority Date: 08/25/1992
• Status: Expired due to Term

• Current Assignee: Yissum Research Development Company of the Hebrew University of Jerusalem Ltd. (Hebrew University of Jerusalem)
• Original Assignee: Yissum Research Development Company of the Hebrew University of Jerusalem Ltd. (Hebrew University of Jerusalem)
• Inventors: Shoham, Benjamin, Willner, Itamar, Katz, Evgeny, Kasher, Ron, Riklin, Azalia
• Primary Examiner(s): Niebling, John
• Assistant Examiner(s): Bell, Bruce F.
• Application Number:
  US08/109,922
• Time in Patent Office: 729 Days
• Field of Search: 204/403, 204/418, 204/153.21, 204/153.19, 204/153.12, 204/415, 204/290 R, 204/291, 435/817, 435/288
• US Class Current: 205/777.5
• CPC Class Codes: C12Q 1/003 Functionalisation
• CPC Class Codes: C12Q 1/004 mediator-assisted
• CPC Class Codes: Y10S 435/817 Enzyme or microbe electrode

Abstract

An analytical method for determining the presence or concentration of an analyte in a liquid medium is provided in which electrons are transferred from an electrode material of an electrode to the redox center of an enzyme, by the mediation of an electron mediator, whereby in the presence of an analyte the enzyme calalyzed a redox reaction in which the analyte is converted into a product. By measuring either the concentration or the product or the charge which flows in this process, the presence and/or concentration of the analyte in the medium is determined. Either or both of the enzyme and the electron mediator are immobilized on the electrode.

Claims

1. An analytical method for determining the presence or concentration of an analyte in a liquid medium comprising the steps of:

selecting an electrobiochemical system, the electrobiochemical system comprising a test electrode which has chemisorbed sulfur containing moieties;

selecting a redox enzyme, the redox enzyme selectively converts the analyte to a product;

binding the redox enzyme to the test electrode;

selecting an electron mediator material which transfers electrons to and from the redox enzyme and test electrode;

introducing a liquid medium with an analyte to the electrobiochemical system;

applying a constant or alternating electric potential to the test electrode so that electrons pass between the redox enzyme and the test electrode allowing the redox enzyme to catalyze the reaction of the analyte; and

measuring the concentration of the analyte.

2. A method according to claim 1 wherein the electron mediator material is immobilized on the test electrode.

3. The analytical method of claim 1 wherein the method further includes the step of selecting an electron mediator wherein the electron mediator is selected from the group consisting of viologen, pyridinium, acridine, ferrocene, phenothiazine, substituted pyridinium, substituted acridine, substituted phenothiazine and substituted ferrocene.

4. The analytical method of claim 3 wherein the method further includes the step of selecting an electron mediator from the group consisting of compounds having the following formulae (IV) to (VIII):

##STR14## wherein n is an integer between 1 and 16;

##STR15## wherein Y is CO₂ H or NH₂ ##STR16##

5. The analytical method of claim 1 wherein the method further includes the step of selecting an electron mediator having the general formulae (IX):

space="preserve" listing-type="equation">U--R.sup.4 --X.sup.3 (IX)

wherein U is a member selected from the group consisting of viologen, pyridinium, acridine, ferrocene, phenothiazine, substituted viologen, substituted pyridinium, substituted acridine, substituted ferrocene and substituted phenothiazine, R⁴ is a connecting group, and X³ is a functional group, which forms a covalent bond with a moiety of said at least one component and reacting the electron mediator with the moiety of at least one component to bond the election mediator in vicinity of the redox enzyme.

6. An analytical method according to claim 1 wherein the method further includes the steps of:

selecting an enzyme;

selecting a bridging group;

reacting the bridging group with the enzyme; and

reacting the enzyme with already bound enzyme through bridging groups to form a test electrode.

7. The method of claim 6 wherein the bridging group have the following formula (X):

space="preserve" listing-type="equation">W.sup.1 --R.sup.b --W.sup.2 (X)

wherein W¹ and W² may be the same or different from one another and form a covalent bond with a moiety of at least one component and R^b represents a connecting group.

8. The method of claim 6 wherein the bridging group is selected from the group consisting of proteins, polypeptides and polymers, the proteins, polypeptides and polymers having at least one functional group X² which forms a covalent bond with a moiety of at least one component.

9. The method of claim 6 wherein the enzyme molecules selected for the external layer are non-redox enzymes, the non-redox enzymes catalyzing the reaction of interfering material in the liquid medium.

29. The analytical method of claim 1 wherein the method further includes the steps of selecting a sulfur containing linking group;

binding the sulfur containing linking group to the redox enzyme to form a linking group-enzyme component; and

binding the linking group-enzyme component to the electrode material.

30. The analytical method of claim 29 wherein the method further includes the step of selecting a linking group with the following general formula (I):

space="preserve" listing-type="equation">Z--R.sup.1 --Q (I)

wherein;

Z represents a sulfur containing moiety,

Q represents a group selected from the group consisting of X¹ and P wherein X¹ is a functional group which forms a covalent bond with a moiety of at least one component and P is selected from the group consisting of proteins, polypeptides and polymers, each having at least one functional group X² which forms a covalent bond with a moiety of at least one component and R¹ represents a connecting group.

31. The analytical method of claim 30 wherein the method further includes the step of selecting the X¹ or X² linking group wherein X¹ and X² are independently selected from the group consisting of an amine group, a carboxyl group, --N=C=S,--N=C=O, aryl group and ##STR21##

32. The analytical method of claim 31, wherein the method further includes the step of selecting an acyl group which has the formula --R^a --CO--G,

wherein G is selected from the group consisting of OH, halogen and OR^b, and R^a or R^b being independently selected from the group consisting of C₁ -C₁₂ alkyl, C₁ -C₁₂ alkynyl, C_1- C₁₂ alkenyl and phenyl containing chains.

33. The analytical method of claim 30 wherein the method further includes the step of selecting a group P from the group consisting of polyamides and polypeptides with free amine or carboxyl groups.

34. The analytical method of claim 33 wherein the method further includes the step of selecting a group P from the group consisting of polylysine, polyglutamate and polyethyleneimine.

35. The analytical method claim 33 wherein P carries a plurality of functional groups X¹ wherein X¹ is a functional group which forms a covalent bond with a moiety of at least one component.

36. The analytical method of claim 30 wherein R¹ is selected from the group consisting of a covalent bond, alkylene chain, alkenyl chain, alkynyl chain and phenyl containing chain.

37. The analytical method of claim 30 wherein R¹ is selected from the group consisting of a chemical bond, and a group having the following formula (II a), (II b) and (III):

##STR22## wherein;

R² and R³ may be the same or different and are selected from the group consisting of a covalent bond and straight or branched alkylene, alkenylenes, alkynylene molecules having 1-12 carbon atoms;

A and B may be the same or different and are independently selected from the group consisting of oxygen and sulfur; and

Ph is a phenyl which is selected from the group consisting of phenyl, alkylphenyl, polyalkylphenyl, phenyl sulfate, alkylphenyl sulfate, polyalkylphenyl sulfate, polyalkylphenyl polysulfate.

10. An electrode comprising an electrode material has chemisorbed sulphur containing moieties, the electrode having immobilized thereon a plurality of complexes, each complex comprising a linking group having a sulphur containing moiety and at least one, of a redox enzyme component or a molecular electron mediator component, all components of the complex are covalently bound to one another, the electron mediator transfers electrons between the electrode material and the redox center of the redox enzyme whereby, in the presence of analyte, the enzyme catalyzes a reaction in which the analyte is convened to product.

11. The electrode of claim 10, wherein the linking group has the following general formula (I);

space="preserve" listing-type="equation">Z--R.sup.1 --Q

wherein;

Z represents a sulphur containing moiety,

Q represents a group selected from the group consisting of X¹ and P wherein X¹ is a functional group which forms a covalent bond with a moiety of at least one component and P is selected from the group consisting of proteins, polypeptides and polymers, each having at least one functional group X² which forms a covalent bond with a moiety of at least one component,

R¹ represents a connecting group.

12. An electrode according to claim 11, wherein X¹ or X² is selected from the group consisting of an amine group, a carboxyl group, -N=C=S,--N=C--O, an acyl group and ##STR17##

13. An electrode according to claim 12 wherein the acyl group has the formula --R^a --CO--G wherein G is selected from the group consisting of OH, halogen, and OR^b wherein R^a and R^b being independently selected from the group consisting of a C₁ -C₁₂ alkyl chain, C₁ -C₁₂ alkenyl chain, C₁ -C₁₂ alkynyl chain, a phenyl containing chain, a substituted C₁ -C₁₂ alkyl chain, a substituted C₁ -C₁₂ alkenyl chain, a substituted C₁ -C₁₂ alkynyl chain and a substituted phenyl containing chain.

14. An electrode according to any one of claims 11 to 13, comprising linking groups of the general formula (I), wherein P is polyamine or a polypeptide with free amine or carboxyl groups.

15. An electrode according to claim 14 wherein P is selected from the group consisting of polylysine, polyglutamate and polyethyleneimine.

16. An electrode according to claim 14, wherein P carries at least one functional group X¹ wherein X¹ is a functional group which forms a covalent bond with a moiety of at least one component.

17. An electrode according to claim 11, wherein R¹ is selected from the group consisting of a covalent bond, alkylene chains, alkenylene chains, alkynylene chains, and phenyl containing chains.

18. An electrode according to claim 17 wherein R¹ is selected from the group consisting of a chemical bond, and a group having the following formula (II a), (II b) and (III):

wherein;

R² and R³ may be the same or different and are selected from the group consisting of a covalent bond and straight or branched alkylene, alkenylenes, alkynylene molecules having 1-12 carbon atoms;

A and B may be the same or different and are selected from the group consisting of oxygen and sulfur;

Ph is a phenyl which is selected from the group consisting of phenyl, alkylphenyl, polyalkylphenyl, phenyl sulfate, alkylphenyl sulfate, polyalkylphenyl sulfate, polyalkylphenyl polysulfate.

19. An electrode according to anyone of claims 10-18, wherein the electron mediators have a mediator moiety U being a member selected from the group consisting of viologen, pyridinium, acridine, ferrocene, phenothiazine, substituted pyridinium, substituted acridine, substituted phenothiazine and substituted ferrocene.

20. An electrode according to claim 19, wherein U is a member selected from the group consisting of compounds having the following formulae (IV) to (VIII):

##STR18## wherein n is an integer between 1 and 16;

##STR19## wherein Y is CO₂ H or NH₂ ##STR20##

21. An electrode according to any one of claims 10-18 wherein the electron mediator group forms part of said complex and has the general formula (IX):

space="preserve" listing-type="equation">U--R.sup.4 --X.sup.3 (IX)

wherein U is a member selected from the group consisting of viologen, pyridinium, acridine, ferrocene, phenothiazine, substituted viologen, substituted pyridinium, substituted acridine, substituted ferrocene and substituted phenothiazine, R⁴ is a connecting group, and X³ is a functional group, which forms a covalent bond with a moiety of at least one component.

22. An electrode according to claim 10 comprising several layers of enzymes, enzymes of one layer being linked to enzymes of an adjacent layer by means of bridging groups.

23. An electrode according to claim 22, wherein the bridging groups have the following formula (X):

space="preserve" listing-type="equation">W.sup.1--R.sup.6 --W.sup.2 (X)

wherein W¹ and W² may be the same of different from one another and forms a covalent bond with a moiety of at least one component and R⁶ represents a connecting group.

24. An electrode according to claim 22, wherein the bridging group is selected from the group consisting of proteins, polypeptides and polymers, the proteins, polypeptides and polymers having at least one functional group X² which forms a covalent bond with a moiety of at least one component.

25. An electrode according to any one of claims 22 to 24 wherein the enzyme molecules in the external layer are non-redox enzymes which catalyst the reaction of agents other than the analyte.

26. A process for preparing an electrode comprising the steps:

selecting a sulfur chemisorption electrode material;

selecting a complex comprising a linking group having a sulphur containing moiety, chemisorption of the sulfur containing moiety onto the electrode material;

selecting at least one of a redox enzyme and an electron mediator and reacting at least one of the redox enzyme and an electron mediator with the chemisorbed linking group.

28. A process according to claims 26 or 27, further comprising:

(a) unfolding the enzyme molecule;

(b) binding electron mediator groups to the unfolded enzyme molecule;

(c) refolding the enzyme.

27. A process for preparing an electrode comprising the steps:

selecting a sulfur chemisorption electrode material,

selecting a sulfur containing linking group;

selecting at least one of a redox enzyme and an electron mediator;

reacting the selected linking group with at least one of the selected redox enzymes and electron mediators to form a complex; and

reacting the complex with the electrode material to form the electrode.

38. An analytical method for determining the presence or concentration of analyte in a liquid medium comprising the steps of;

providing an electrobiochemical system comprising a test electrode which has a sulfur containing moiety;

providing an electron mediator;

chemisorbing the election mediator onto the test electrode;

introducing a liquid medium with an analyte to the electrobiochemical system;

providing a redox enzyme for oxidizing or reducing the analyte and for interacting with the electron mediator;

introducing the redox enzyme into the liquid medium;

applying a constant or alternate electric potential to the test electrode so that electrons pass between the mediator group, test electrodes and redox enzyme;

allowing the enzyme to catalyze the reaction of the analyte; and

measuring the concentration of the analyte.

39. The method of claim 38 wherein the method further includes the steps of selecting an electron mediator group;

reacting said electron mediator group with the redox enzyme to form a redox enzyme-electron mediator group; and

introducing the redox enzyme-electron mediator complex into the liquid medium.

40. The method of claim 39 wherein the analyte concentration is measured by measuring the flow of electric charge.

41. The method of claim 39 wherein the analyte concentration is measured by measuring the concentration of the redox reaction product in the liquid medium.

42. An analytical method for determining the presence or concentration of analyte in a liquid medium comprising the steps of;

providing an electrobiochemical system comprising a test electrode which has a sulfur containing moiety;

providing an electron mediator;

chemisorbing the election mediator onto the test electrode;

introducing a liquid medium with an analyte to the electrobiochemical system;

providing a redox enzyme for oxidizing or reducing the analyte and for interacting with the electron mediator;

introducing the redox enzyme into the liquid medium;

applying a constant or alternate electric potential to the test electrode so that electrons pass between the mediator group, test electrodes and redox enzyme;

allowing the enzyme to catalyze the reaction of the analyte; and

measuring the concentration of the analyte by measuring the flow of electric charge.

43. An analytical method for determining the presence or concentration of analyte in a liquid medium comprising the steps of;

providing an electrobiochemical system comprising a test electrode which has a sulfur containing moiety;

providing an electron mediator;

chemisorbing the election mediator onto the test electrode;

introducing a liquid medium with an analyte to the electrobiochemical system;

providing a redox enzyme for oxidizing or reducing the analyte and for interacting with the electron mediator;

introducing the redox enzyme into the liquid medium;

applying a constant or alternate electric potential to the test electrode so that electrons pass between the mediator group, test electrodes and redox enzyme;

allowing the enzyme to catalyze the reaction of the analyte; and

measuring the concentration of the analyte by measuring the concentration of the redox reaction product in the liquid medium.

44. An analytical method for determining the presence or concentration of an analyte in a liquid medium comprising the steps of:

selecting an appropriate electrobiochemical system, the electrobiochemical system comprising selecting a test electrode which has chemisorption of sulfur containing moieties;

selecting a redox enzyme, the redox enzyme selectively converts the analyte to a product;

binding the redox enzyme to the test electrode;

selecting an electron mediator material which transfers electrons to and from the redox enzyme and test electrode;

introducing a liquid medium with an analyte to the electrobiochemical system;

applying a constant or alternating electric potential to the test electrode so that electrons pass between the redox enzyme and the test electrode allowing the redox enzyme to catalyze the reaction of the analyte; and

measuring the concentration of the analyte by measuring the flow of electron charge.

45. An analytical method for determining the presence or concentration of an analyte in a liquid medium comprising the steps of:

selecting an appropriate electrobiochemical system, the electrobiochemical system comprising selecting a test electrode which has chemisorption of sulfur containing moieties;

selecting a redox enzyme, the redox enzyme selectively converts the analyte to a product;

binding the redox enzyme to the test electrode;

selecting an electron mediator material which transfers electrons to and from the redox enzyme and test electrode;

introducing a liquid medium with an analyte to the electrobiochemical system;

applying a constant or alternating electric potential to the test electrode so that electrons pass between the redox enzyme and the test electrode allowing the redox enzyme to catalyze the reaction of the analyte; and

measuring the concentration of the analyte by measuring the concentration of the redox reaction product in the liquid medium.