Method and agent for the simultaneous colorimetric and electrochemical measurement of an analyte

• US 5,858,691 A
• Filed: 06/13/1996
• Issued: 01/12/1999
• Est. Priority Date: 06/13/1995
• Status: Expired due to Fees

• Current Assignee: Roche Diagnostics GmbH (Roche Holding AG)
• Original Assignee: Boehringer Mannheim GmbH (Roche Holding AG)
• Inventors: Marouant, Michael, Kotzan, Holger, Unkrig, Volker, Hoenes, Joachim
• Primary Examiner(s): Dees, Jose G.
• Assistant Examiner(s): JONES, DAMERON LEVEST
• Application Number:
  US08/663,349
• Time in Patent Office: 943 Days
• Field of Search: 435/4, 435/25, 435/26, 435/805, 422/50, 422/82.01
• US Class Current: 435/25
• CPC Class Codes: C12Q 1/26 involving oxidoreductase

Abstract

The invention concerns a method for the simultaneous colorimetric and electrochemical measurement of an analyte using an oxidizing enzyme and a chromogen A that accepts electrons from the enzyme which is characterized in that the chromogen A after reduction to A'"'"' reacts by coupling to a substance BX to form a colored substance A'"'"'B the concentratin of which is measured colorimetrically as a measure for the presence or amount of the analyte and in that an electrochemically measurable group X^- is cleaved off during the coupling reaction the concentration of which is measured electrochemically as a measure for the amount of the analyte.

Claims

1. A method for the simultaneous colorimetric and electrochemical measurement of an analyte, comprising:

(a) reducing a chromogen A to a compound A'"'"' by reacting the analyte with an oxidizing enzyme to oxidize the analyte and transferring electrons produced thereby onto the chromogen A,

(b) reacting the compound A'"'"' with a substance BX to form a colored substance A'"'"'B and an electrochemically measurable group X^-,

(c) colorimetrically measuring or detecting the colored substance A'"'"'B as an indication of amount or presence, respectively, of the analyte, and

(d) electrochemically measuring the electrochemically measurable group X^- as an indication of amount of the analyte.

2. The method of claim 1, wherein in the reducing step (a), the electrons are transferred onto the chromogen A via a mediator.

3. The method of claim 2, wherein the mediator comprises phenazine methosulfate or diaphorase.

4. The method of claim 1, wherein the oxidizing enzyme comprises a dehydrogenase.

5. The method of claim 1, wherein the chromogen A comprises an aromatic nitroso compound.

6. The method of claim 5, wherein the aromatic nitroso compound comprises a compound of formula I ##STR8## wherein R¹ is selected from the group consisting of hydrogen;

hydroxy;

alkyl which is unsubstituted or has a substituent selected from the group consisting of (a) hydroxy, (b) COOH, (c) PO₃ H₂ and (d) SO₃ H;

alkoxy;

alkylthio;

aryloxy;

arylthio;

halogen; and

amino which is unsubstituted or has at least one alkyl substituent, which at least one alkyl substituent is unsubstituted or has a substituent selected from the group consisting of (a) hydroxy, (b) PO₃ H₂, (c) dialkyl-phosphinyl, (d) SO₃ H and (e) CO₂ H;

R² is selected from the group consisting of hydroxy;

alkoxy;

aryloxy;

arylthio;

alkylthio; and

amino group NR³ R⁴,

wherein the alkyl residue of the alkoxy or alkylthio is unsubstituted or has a substituent selected from the group consisting of (a) hydroxy, (b) alkoxy and (c) amino which is unsubstituted or has at least one substituent selected from the group consisting of (1) alkyl, (2) PO₃ H₂, or a salt thereof, (3) SO₃ H, or a salt thereof, and (4) CO₂ H, or a salt thereof, and

wherein R³ and R⁴ are the same or different, and each is independently selected from the group consisting of (a) hydrogen, (b) aryl and (c) alkyl, wherein each of (b) aryl or (c) alkyl is unsubstituted or has a substituent selected from the group consisting of (1) hydroxy, (2) alkoxy, (3) hydroxyalkoxy, (4) polyalkoxy which is unsubstituted or substituted by hydroxy, (5) PO₃ H₂, or a salt thereof, (6) SO₃ H, or a salt thereof, (7) COOH, or a salt thereof, and (8) an amino group which is unsubstituted or substituted at least once by alkyl, or

wherein R³ and R⁴ together are an alkylene residue which is uninterrupted or interrupted by oxygen, sulphur or nitrogen, wherein the nitrogen is substituted by a substituent selected from the group consisting of alkyl, hydroxyalkyl, hydroxyalkoxyalkyl, alkoxyhydroxyalkyl, alkoxycarbonylalkyl, dioxanylylalkyl and polyalkoxyalkyl, wherein each substituent of the nitrogen is unsubstituted or substituted in the alkyl moiety by a hydroxy residue, or

if R¹ is in the ortho position relative to NR³ R⁴, one of R³ and R⁴ is additionally selected from an alkylene residue together with R¹.

7. The method of claim 6, wherein R¹ is hydrogen or C₁ -C₆ alkyl.

8. The method of claim 6, wherein R is C₁ -C₆ alkoxy or amino group NR³ R⁴.

9. The method of claim 6, wherein the compound is selected from the group consisting of

N,N'"'"'-bis-(2-hydroxyethyl)-p-nitrosoaniline

N,N'"'"'-dimethyl-p-nitrosoaniline

N,N'"'"'-diethyl-p-nitrosoaniline

N-methyl-N'"'"'-(4-nitrosophenyl)-piperazine

N-(2-hydroxyethyl)-5-nitrosoindoline

2,4-dimethoxy-nitrosobenzene

N,N'"'"'-bis-(2-methoxyethyl)-4-nitrosoaniline

N-(4-nitrosophenyl)-morpholine

N-(2,2-diethoxy-ethyl)N'"'"'-(4-nitrosophenyl)-piperazine

p-nitrosophenol

3-methoxy-4-nitrosophenol.

10. The method of claim 5, wherein the aromatic nitroso compound comprises a compound of formula II ##STR9## wherein X--Y is NR⁵ --CO or N═

CR⁶

R⁵ is selected from the group consisting of (a) hydrogen, (b) alkyl, which is unsubstituted or has a substituent selected from the group consisting of (1) hydroxy, (2) carboxy, (3) SO₃ H₂, (4) PO₃ H₂ and (5) dialkylphosphinyl,

R₆ is selected from the group consisting of (a) hydrogen, (b) alkyl, (c) alkenyl, (d) alkoxy, (e) alkylthio, (f) aryl, (g) arylthio, (h) aralkyl, wherein each of (b)-(h) is unsubstituted or has a substituent selected from the group consisting of (1) hydroxy, (2) carboxy, or a salt thereof, (3) SO₃ H, or a salt thereof, (4) PO₃ H₂, or a salt thereof, and (5) alkoxycarbonyl,

(i) carboxy, (j) alkoxycarbonyl, (k) carboxyamido, (1) halogen, (m) amino which is unsubstituted or substituted by one or two alkyl residues which are unsubstituted or substituted by at least one residue selected from the group consisting of hydroxy, carboxy, and alkoxycarbonyl, wherein when amino is substituted by two alkyl residues, the two alkyl residues are unclosed or closed to from a ring that, in addition to the N atom of the amino group, is uninterrupted or interrupted by oxygen, sulphur or a further nitrogen atom, and (n) amino which is unsubstituted or substituted by up to two substituents, each independently selected from the group consisting of acyl, alkoxy, aralkoxycarbonyl, H₂ N--CO, alkyl, aralkyl and arylcarbamoyl,

R⁷ is selected from the group consisting of (a) alkyl, (b) thioalkyl, (c) aralkyl, wherein each of (a)-(c) is unsubstituted or has a substituent selected from the group consisting of (1) hydroxy, (2) carboxy, (3) SO₃ H and (4) PO₃ H₂, and (d) amino which is unsubstituted or substituted by one or two alkyl groups, which alkyl groups are unsubstituted or have a substituent selected from the group consisting of (1) hydroxy, (2) carboxy, (3) SO₃ H, (4) dialkylphosphinyl and (5) PO₃ H₂,

wherein at least one of R⁶ and R⁷ are an amino group and

R⁸ is alkyl or aralkyl, wherein R⁸ is unsubstituted or has a substituent selected from the group consisting of hydroxy, carboxy, SO₃ H and PO₃ H₂, or

R⁷ and R⁸ together are a saturated or unsaturated chain having 3 or 4 members of nitrogen atoms or of carbon atoms, which saturated or unsaturated chain further contains zero or at least one nitrogen or sulphur atom, wherein the carbon atoms are unsubstituted or have a substituent selected from the group consisting of alkyl, alkoxy, alkylthio, hydroxy, aralkyl, aryl, carboxy, carboxamido, alkoxycarbonyl, cyano, halogen and amino, which amino is unsubstituted or substituted by one or two alkyl residues which are each unsubstituted or substituted by one or more substituents selected from the group consisting of (a) hydroxy, (b) carboxy, and (c) alkoxycarbonyl, wherein nitrogen atoms that are not bound via a double bond are substituted by alkyl or aralkyl, which alkyl or aralkyl are each independently unsubstituted or have a substituent selected from the group consisting of hydroxy, SO₃ H, PO₃ H₂, carboxy and dialkylphosphinyl, or two neighboring chain substituents form an alkylene group which is unsubstituted or substituted with aryl or is anellated,

a tautomeric form or a salt thereof.

11. The method of claim 10, wherein the aromatic nitroso compound comprises a compound selected from the group consisting of formulae III to XII ##STR10## wherein R⁹, R¹⁰, R¹¹ and R¹² are the same or different and each independently is selected from the group consisting of hydrogen, hydroxy, halogen, alkyl, akloxy, aklylthio, aralkyl, aryl, carboxy, alkoxycarbonyl, carboxamido, cyano, amino which is unsubstituted or substituted by one or two alkyl residues each of which is unsubstituted or substituted by at least one substituent selected from the group consisting of hydroxy, carboxy and alkoxycarbonyl,

wherein two neighboring residues form an alkylene group which is unsubstituted or substituted by aryl or is anellated, and

R¹³ is alkyl or aralkyl, each of which is unsubstituted or substituted by a substituent selected from the group consisting of hydroxy, carboxy, SO₃ H, PO₃ H₂ and dialkylphosphinyl.

12. The method of claim 11, wherein the aromatic nitroso compound comprises a compound selected from the group consisting of formulae III, IV, V, VII, VIII and IX.

13. The method of claim 11, wherein X--Y is N═

CR⁶.

14. The method of claim 5, wherein the aromatic nitroso compound is selected from the group consisting of

3-nitroso-2-methyl-pyrazolo- 1.5a!-pyridine,

3-nitroso-pyrazolo- 1.5a!-pyridine and

3-nitroso-pyrazolo 3.2-c!-s-triazole,

or a salt thereof.

15. The method of claim 1, wherein in the substance BX, B comprises an aromatic compound and X comprises a substituent which is substituted or unsubstituted.

16. The method of claim 15, wherein B comprises phenol or naphthol.

17. The method of claim 15, wherein X comprises halogen or SO₃ H.

18. The method of claim 1, wherein BX comprises aniline or naphthylamine.

19. The method of claim 1, wherein BX comprises 2,4,6-trihalogen-3-hydroxy-benzoic acid, 2,4-dihalogen-1-naphthol, 1-naphthol-4-sulfonic acid, 4-monohalogen-aniline, 4-monohalogen-phenol and 4-monohalogen-naphthol.

20. The method of claim 1, wherein the electrochemically measurable group X^- is measured potentiometrically.

21. The method of claim 1, wherein the electrochemically measurable group X^- is measured amperometrically.

22. The method of claim 1, wherein the analyte is present in a concentration of 10^-6 to 10^-2 mol/l.

23. The method of claim 1, wherein the chromogen A is present in a concentration of 10^-4 to 10^-1 mol/l.

24. The method of claim 1, wherein the oxidizing enzyme is present in a concentration of 1 mU/ml to 1 U/ml.

25. The method of claim 1, wherein the substance BX is present in from a stoichiometric ratio to a 2-fold excess, compared to the chromogen A.

26. A kit for the simultaneous colorimetric and electrochemical measurement of an analyte, comprising:

(a) a set of reagents, comprising

(1) an oxidizing enzyme which oxidizes the analyte,

(2) a chromogen A which accepts electrons produced in an oxidation reaction of the oxidizing enzyme and the analyte to produce a compound A'"'"', and

(3) a substance BX which reacts with the compound A'"'"' to form a colored substance A'"'"'B and an electrochemically measurable group X^- ; and

(b) a sensor electrode for electrochemically measuring the electrochemically measurable group X^-.

27. The kit of claim 26, wherein each of the set of reagents is separately packaged.

28. The kit of claim 26, wherein the set of reagents are packaged together.

29. The kit of claim 28, further comprising an aqueous solution, wherein the aqueous solution contains the set of reagents, the aqueous solution comprising a buffer which sets a pH value of between 4 and 9 in the aqueous solution.

30. The kit of claim 26, further comprising an optical measuring device for colorimetrically measuring or detecting the colored substance A'"'"'B.

31. The kit of claim 26, further comprising a mediator which transfers electrons produced in the oxidation reaction of the oxidizing enzyme and the analyte to the chromogen A.

32. The kit of claim 31, wherein the mediator comprises phenazine methosulfate or diaphorase.

33. The kit of claim 26, wherein the oxidizing enzyme comprises a dehydrogenase.

34. The kit of claim 26, wherein the chromogen A comprises an aromatic nitroso compound.

35. The kit of claim 34, wherein the aromatic nitroso compound comprises a compound of formula I ##STR11## wherein R¹ is selected from the group consisting of hydrogen;

hydroxy;

alkyl which is unsubstituted or has a substituent selected from the group consisting of (a) hydroxy, (b) COOH, (c) PO₃ H₂ and (d) SO₃ H;

alkoxy;

alkylthio;

aryloxy;

arylthio;

halogen; and

amino which is unsubstituted or has at least one alkyl substituent, which at least one alkyl substituent is unsubstituted or has a substituent selected from the group consisting of (a) hydroxy, (b) PO₃ H₂, (c) dialkyl-phosphinyl, (d) SO₃ H and (e) CO₂ H;

R² is selected from the group consisting of hydroxy;

alkoxy;

aryloxy;

arylthio;

alkylthio; and

amino group NR³ R⁴,

wherein the alkyl residue of the alkoxy or alkylthio is unsubstituted or has a substituent selected from the group consisting of (a) hydroxy, (b) alkoxy and (c) amino which is unsubstituted or has at least one substituent selected from the group consisting of (1) alkyl, (2) PO₃ H₂, or a salt thereof, (3) SO₃ H, or a salt thereof, and (4) CO₂ H, or a salt thereof, and

wherein R³ and R⁴ are the same or different, and each is independently selected from the group consisting of (a) hydrogen, (b) aryl and (c) alkyl, wherein each of (b) aryl or (c) alkyl is unsubstituted or has a substituent selected from a group consisting of (1) hydroxy, consisting of (1) hydroxy, (2) alkoxy, (3) hydroxyalkoxy, (4) polyalkoxy which is unsubstituted or substituted by hydroxy, (5) PO₃ H₂, or a salt thereof, (6) SO₃ H, or a salt thereof, (7) COOH, or a salt thereof, and (8) an amino group which is unsubstituted or substituted at least once by alkyl, or

wherein R³ and R⁴ together are an alkylene residue which is uninterrupted or interrupted by oxygen, sulphur or nitrogen, wherein the nitrogen is substituted by a substituent selected from the group consisting of alkyl, hydroxyalkyl, hydroxyalkoxyalkyl, alkoxyhydroxyalkyl, alkoxycarbonylalkyl, dioxanylylalkyl and polyalkoxyalkyl, wherein each substituent of the nitrogen is unsubstituted or substituted in the alkyl moiety by a hydroxy residue, or

if R¹ is in the ortho position relative to NR³ R⁴, one of R³ and R⁴ is additionally selected from an alkylene residue together with R¹.

36. The kit of claim 35, wherein R¹ is hydrogen or C₁ -C₆ alkyl.

37. The kit of claim 35, wherein R² is C₁ -C₆ alkoxy or amino group NR³ R⁴.

38. The kit of claim 35, wherein the compound is selected from the group consisting of

N,N'"'"'-bis-(2-hydroxyethyl)-p-nitrosoaniline

N,N'"'"'-dimethyl-p-nitrosoaniline

N,N'"'"'-diethyl-p-nitrosoaniline

N-methyl-N'"'"'-(4-nitrosophenyl)-piperazine

N-(2-hydroxyethyl)-5-nitrosoindoline

2,4-dimethoxy-nitrosobenzene

N,N'"'"'-bis-(2-methoxyethyl)-4-nitrosoaniline

N-(4-nitrosophenyl)-morpholine

N-(2,2-diethoxy-ethyl)N'"'"'-(4-nitrosophenyl)-piperazine

p-nitrosophenol

3-methoxy-4-nitrosophenol.

39. The kit of claim 34, wherein the aromatic nitroso compound comprises a compound of formula II ##STR12## wherein X--Y is NR⁵ --CO or N═

CR⁶

R⁵ is selected from the group consisting of (a) hydrogen, (b) alkyl, which is unsubstituted or has a substituent selected from the group consisting of (1) hydroxy, (2) carboxy, (3) SO₃ H, (4) PO₃ H₂ and (5) dialkylphosphinyl,

R₆ is selected from the group consisting of (a) hydrogen, (b) alkyl, (c) alkenyl, (d) alkoxy, (e) alkylthio, (f) aryl, (g) arylthio, (h) aralkyl, wherein each of (b)-(h) is unsubstituted or has a substituent selected from the group consisting of (1) hydroxy, (2) carboxy, or a salt thereof, (3) SO₃ H or a salt thereof, (4) PO₃ H₂, or a salt thereof, and (5) alkoxycarbonyl,

(i) carboxy, (j) alkoxycarbonyl, (k) carboxyamido, (l) halogen, (m) amino which is unsubstituted or substituted by one or two alkyl residues which are unsubstituted or substituted by at least one residue selected from the group consisting of hydroxy, carboxy, and alkoxycarbonyl, wherein when amino is substituted by two alkyl residues, the two alkyl residues may additionally be closed to from a ring that, in addition to the N atom of the amino group, is uninterrupted or interrupted by oxygen, sulphur or a further nitrogen atom, and (n) amino which is unsubstituted or substituted by up to two substituents, each independently selected from the group consisting of acyl, alkoxy, aralkoxycarbonyl, H₂ N--CO, alkyl, aralkyl and arylcarbamoyl,

R⁷ is selected from the group consisting of (a) alkyl, (b) thioalkyl, (c) aralkyl, wherein each of (a)-(c) is unsubstituted or has a substituent selected from the group consisting of (1) hydroxy, (2) carboxy, (3) SO₃ H and (4) PO₃ H₂, and (d) amino which is unsubstituted or substituted by one or two alkyl groups, which alkyl groups are unsubstituted or have a substituent selected from the group consisting of (1) hydroxy, (2) carboxy, (3) SO₃ H, (4) dialkylphosphinyl and (5) PO₃ H₂,

wherein at least one of R⁶ and R⁷ are an amino group and

R⁸ is alkyl or aralkyl, wherein R⁸ is unsubstituted or has a substituent selected from the group consisting of hydroxy, carboxy, SO₃ H and PO₃ H₂,

R⁷ and R⁸ together are a saturated or unsaturated chain having 3 or 4 members of nitrogen atoms or of carbon atoms, which saturated or unsaturated chain further contains zero or at least one nitrogen or sulphur atom, wherein the carbon atoms are unsubstituted or have a substituent selected from the group consisting of alkyl, alkoxy, alkylthio, hydroxy, aralkyl, aryl, carboxy, carboxamido, alkoxycarbonyl, cyano, halogen and amino, which amino is unsubstituted or substituted by one or two alkyl residues which are each unsubstituted or substituted by one or more substituents selected from the group consisting of (a) hydroxy, (b) carboxy, and (c) alkoxycarbonyl, wherein nitrogen atoms that are not bound via a double bond are substituted by alkyl or aralkyl, which alkyl or aralkyl are each independently unsubstituted or have a substituent selected from the group consisting of hydroxy, SO₃ H, PO₃ H₂, carboxy and dialkylphosphinyl, or two neighboring chain substituents may additionally form an alkylene group which is unsubstituted or substituted with aryl or is anellated,

a tautomeric form or a salt thereof.

40. The kit of claim 39, wherein the aromatic nitroso compound comprises a compound selected from the group consisting of formulae III to XII ##STR13## wherein R⁹, R¹⁰, R¹¹ and R¹² are the same or different and each independently is selected from the group consisting of hydrogen, hydroxy, halogen, alkyl, akloxy, aklylthio, aralkyl, aryl, carboxy, alkoxycarbonyl, carboxamido, cyano, amino which is unsubstituted or substituted by one or two alkyl residues each of which is unsubstituted or substituted by at least one substituent selected from the group consisting of hydroxy, carboxy and alkoxycarbonyl,

wherein two neighboring residues can additionally form an alkylene group which is unsubstituted or substituted by aryl or is anellated, and

R¹³ is alkyl or aralkyl, each of which is unsubstituted or substituted by a substituent selected from the group consisting of hydroxy, carboxy, SO₃ H, PO₃ H₂ and dialkylphosphinyl.

41. The kit of claim 40, wherein the aromatic nitroso compound comprises a compound selected from the group consisting of formulae III, IV, V, VII, VIII and IX.

42. The kit of claim 40, wherein X--Y is N═

CR⁶.

43. The kit of claim 35, wherein the aromatic nitroso compound is selected from the group consisting of

3-nitroso-2-methyl-pyrazolo- 1.5a!-pyridine,

3-nitroso-pyrazolo- 1.5a!-pyridine and

3-nitroso-pyrazolo 3.2-c!-s-triazole,

or a salt thereof.

44. The kit of claim 26, wherein in the substance BX, B comprises an aromatic compound and X comprises a substituent which is substituted or unsubstituted.

45. The kit of claim 44, wherein B comprises phenol or naphthol.

46. The kit of claim 44, wherein X comprises halogen or SO₃ H.

47. The kit of claim 26, wherein BX comprises aniline or naphthylamine.

48. The kit of claim 26, wherein BX comprises 2,4,6-trihalogen-3-hydroxy-benzoic acid, 2,4-dihalogen-1-naphthol, 1-naphthol-4-sulfonic acid, 4-monohalogen-aniline, 4-monohalogen-phenol and 4-monohalogen-naphthol.

49. The kit of claim 26, wherein the chromogen A is present in a concentration of 10^-4 to 10^-1 mol/l.

50. The kit of claim 26, wherein the oxidizing enzyme is present in a concentration of 1 mU/ml to 1 U/ml.

51. The kit of claim 26, wherein the substance BX is present in from a stoichiometric ratio to a 2-fold excess, compared to the chromogen A.

52. A test element for the simultaneous colorimetric and electrochemical measurement of an analyte, comprising a set of reagents, present in or on at least one solid carrier, the set of reagents comprising:

(a) an oxidizing enzyme which oxidizes the analyte;

(b) a chromogen A which accepts electrons produced in an oxidation reaction of the oxidizing enzyme and the analyte to produce a compound A'"'"'; and

(c) a substance BX which reacts with the compound A'"'"' to form a colored substance A'"'"'B and an electrochemically measurable group X^-.

53. The test element of claim 52, further comprising a mediator which transfers electrons produced in the oxidation reaction of the oxidizing enzyme and the analyte to the chromogen A.

54. The test element of claim 53, wherein the mediator comprises phenazine methosulfate or diaphorase.

55. The test element of claim 52, wherein the oxidizing enzyme comprises a dehydrogenase.

56. The test element of claim 52, wherein the chromogen A comprises an aromatic nitroso compound.

57. The test element of claim 56, wherein the aromatic nitroso compound comprises a compound of formula I ##STR14## wherein R¹ is selected from the group consisting of hydrogen;

hydroxy;

alkyl which is unsubstituted or has a substituent selected from the group consisting of (a) hydroxy, (b) COOH, (c) PO₃ H₂ and (d) SO₃ H;

alkoxy;

alkylthio;

aryloxy;

arylthio;

halogen; and

amino which is unsubstituted or has at least one alkyl substituent, which at least one alkyl substituent is unsubstituted or has a substituent selected from the group consisting of (a) hydroxy, (b) PO₃ H₂, (c) dialkyl-phosphinyl, (d) SO₃ H and (e) CO₂ H;

R² is selected from the group consisting of hydroxy;

alkoxy;

aryloxy;

arylthio;

alkylthio; and

amino group NR³ R⁴,

wherein the alkyl residue of the alkoxy or alkylthio is unsubstituted or has a substituent selected from the group consisting of (a) hydroxy, (b) alkoxy and (c) amino which is unsubstituted or has at least one substituent selected from the group consisting of (1) alkyl, (2) PO₃ H₂, or a salt thereof, (3) SO₃ H, or a salt thereof, and (4) CO₂ H, or a salt thereof, and

wherein R³ and R⁴ are the same or different, and each is independently selected from the group consisting of (a) hydrogen, (b) aryl and (c) alkyl, wherein each of (b) aryl or (c) alkyl is unsubstituted or has a substituent selected from the group consisting of (1) hydroxy, (2) alkoxy, (3) hydroxyalkoxy, (4) polyalkoxy which is unsubstituted or substituted by hydroxy, (5) PO₃ H₂, or a salt thereof, (6) SO₃ H, or a salt thereof, (7) COOH, or a salt thereof, and (8) an amino group which is unsubstituted or substituted at least once by alkyl, or

wherein R³ and R⁴ together are an alkylene residue which is uninterrupted or interrupted by oxygen, sulphur or nitrogen, wherein the nitrogen is substituted by a substituent selected from the group consisting of alkyl, hydroxyalkyl, hydroxyalkoxyalkyl, alkoxyhydroxyalkyl, alkoxycarbonylalkyl, dioxanylylalkyl and polyalkoxyalkyl, wherein each substituent of the nitrogen is unsubstituted or substituted in the alkyl moiety by a hydroxy residue, or

if R¹ is in the ortho position relative to NR³ R⁴, one of R³ and R⁴ is additionally selected from an alkylene residue together with R¹.

58. The test element of claim 57, wherein R¹ is hydrogen or C₁ -C₆ alkyl.

59. The test element of claim 57, wherein R² is C₁ -C₆ alkoxy or amino group NR³ R⁴.

60. The test element of claim 57, wherein the compound is selected from the group consisting of

N,N'"'"'-bis-(2-hydroxyethyl)-p-nitrosoaniline

N,N'"'"'-dimethyl-p-nitrosoaniline

N,N'"'"'-diethyl-p-nitrosoaniline

N-methyl-N'"'"'-(4-nitrosophenyl)-piperazine

N-(2-hydroxyethyl)-5-nitrosoindoline

2,4-dimethoxy-nitrosobenzene

N,N'"'"'-bis-(2-methoxyethyl)-4-nitrosoaniline

N-(4-nitrosophenyl)-morpholine

N-(2,2-diethoxy-ethyl)N'"'"'-(4-nitrosophenyl)-piperazine

p-nitrosophenol

3-methoxy-4-nitrosophenol.

61. The test element of claim 56, wherein the aromatic nitroso compound comprises a compound of formula II ##STR15## wherein X--Y is NR⁵ --CO or N═

CR⁶

R⁵ is selected from the group consisting of (a) hydrogen, (b) alkyl, which is unsubstituted or has a substituent selected from the group consisting of (1) hydroxy, (2) carboxy, (3) SO₃ H, (4) PO₃ H₂ and (5) dialkylphosphinyl,

R₆ is selected from the group consisting of (a) hydrogen, (b) alkyl, (c) alkenyl, (d) alkoxy, (e) alkylthio, (f) aryl, (g) arylthio, (h) aralkyl, wherein each of (b)-(h) is unsubstituted or has a substituent selected from the group consisting of hydroxy, carboxy or a salt thereof, SO₃ H or a salt thereof, PO₃ H₂ or a salt thereof, and alkoxycarbonyl,

(i) carboxy, (j) alkoxycarbonyl, (k) carboxyamido, (1) halogen, (m) amino which is unsubstituted or substituted by one or two alkyl residues which are unsubstituted or substituted by at least one residue selected from the group consisting of hydroxy, carboxy, and alkoxycarbonyl, wherein when amino is substituted by two alkyl residues, the two alkyl residues are unclosed or closed to from a ring that, in addition to the N atom of the amino group, is uninterrupted or interrupted by oxygen, sulphur or a further nitrogen atom, and (n) amino which is unsubstituted or substituted by up to two substituents, each independently selected from the group consisting of acyl, alkoxy, aralkoxycarbonyl, H₂ N--CO, alkyl, aralkyl and arylcarbamoyl,

R⁷ is selected from the group consisting of (a) alkyl, (b) thioalkyl, (c) aralkyl, wherein each of (a)-(c) is unsubstituted or has a substituent selected from the group consisting of (1) hydroxy, (2) carboxy, (3) SO₃ H and (4) PO₃ H₂, and (d) amino which is unsubstituted or substituted by one or two alkyl groups, which alkyl groups are unsubstituted or have a substituent selected from the group consisting of (1) hydroxy, (2) carboxy, (3) SO₃ H, (4) dialkylphosphinyl and (5) PO₃ H₂,

wherein at least one of R⁶ and R⁷ are an amino group and

R⁸ is alkyl or aralkyl, wherein R⁸ is unsubstituted or has a substituent selected from the group consisting of hydroxy, carboxy, SO₃ H and PO₃ H₂,

or wherein

R⁷ and R⁸ together are a saturated or unsaturated chain having 3 or 4 members of nitrogen atoms or of carbon atoms, which saturated or unsaturated chain further contains zero or at least one nitrogen or sulphur atom, wherein the carbon atoms are unsubstituted or have a substituent selected from the group consisting of alkyl, alkoxy, alkylthio, hydroxy, aralkyl, aryl, carboxy, carboxamido, alkoxycarbonyl, cyano, halogen and amino, which amino is unsubstituted or substituted by one or two alkyl residues which are each unsubstituted or substituted by one or more substituents selected from the group consisting of (a) hydroxy, (b) carboxy, and (c) alkoxycarbonyl, wherein nitrogen atoms that are not bound via a double bond are substituted by alkyl or aralkyl, which alkyl or aralkyl are each independently unsubstituted or have a substituent selected from the group consisting of hydroxy, SO₃ H, PO₃ H₂, carboxy and dialkylphosphinyl, or two neighboring chain substituents form an alkylene group which is unsubstituted or substituted with aryl or is anellated,

a tautomeric form or a salt thereof.

62. The test element of claim 61, wherein the aromatic nitroso compound comprises a compound selected from the group consisting of formulae III to XII ##STR16## wherein R⁹, R¹⁰, R¹¹ and R¹² are the same or different and each independently is selected from the group consisting of hydrogen, hydroxy, halogen, alkyl, akloxy, aklylthio, aralkyl, aryl, carboxy, alkoxycarbonyl, carboxamido, cyano, amino which is unsubstituted or substituted by one or two alkyl residues each of which is unsubstituted or substituted by at least one substituent selected from the group consisting of hydroxy, carboxy and alkoxycarbonyl,

wherein two neighboring residues form an alkylene group which is unsubstituted or substituted by aryl or is anellated, and

R¹³ is alkyl or aralkyl, each of which is unsubstituted or substituted by a substituent selected from the group consisting of hydroxy, carboxy, SO₃ H, PO₃ H₂ and dialkylphosphinyl.

63. The test element of claim 62, wherein the aromatic nitroso compound comprises a compound selected from the group consisting of formulae III, IV, V, VII, VIII and IX.

64. The test element of claim 62, wherein X--Y is N═

CR⁶.

65. The test element of claim 57, wherein the aromatic nitroso compound is selected from the group consisting of

3-nitroso-2-methyl-pyrazolo- 1.5a!-pyridine,

3-nitroso-pyrazolo- 1.5a!-pyridine and

3-nitroso-pyrazolo 3.2-c!-s-triazole,

or a salt thereof.

66. The test element of claim 52, wherein in the substance BX, B comprises an aromatic compound and X comprises a substituent which is substituted or unsubstituted.

67. The test element of claim 66, wherein B comprises phenol or naphthol.

68. The test element of claim 66, wherein X comprises halogen or SO₃ H.

69. The test element of claim 52, wherein BX comprises aniline or naphthylamine.

70. The test element of claim 52, wherein BX comprises 2,4,6-trihalogen-3-hydroxy-benzoic acid, 2,4-dihalogen-1-naphthol, 1-naphthol-4-sulfonic acid, 4-monohalogen-aniline, 4-monohalogen-phenol and 4-monohalogen-naphthol.

71. The test element of claim 52, wherein the chromogen A is present in a concentration of 10^-3 to 1 mol/l.

72. The test element of claim 52, wherein the oxidizing enzyme is present in an amount of 0.1 to 100 U/test zone.

73. The test element of claim 52, wherein the substance BX is present in from a stoichiometric ratio to a 2-fold excess, compared to the chromogen A.

74. A test system for the simultaneous colorimetric and electrochemical measurement of an analyte, comprising

the test element of claim 52, in liquid contact with a sample liquid containing the analyte to form a liquid environment system;

and

an electrode for electrochemically measuring the electrochemically measurable group X^-, in liquid conductive contact with the liquid environment system.