Electrochemical enzyme assay

• US 6,110,696 A
• Filed: 06/26/1995
• Issued: 08/29/2000
• Est. Priority Date: 08/27/1993
• Status: Expired due to Fees

• Current Assignee: Microgenics Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Boehringer Mannheim GmbH (Roche Holding AG), Roche Diagnostics Corporation (Roche Holding AG)
• Inventors: Kuhn, Lance S., McEnroe, Robert J., Muddiman, Rebecca W., Brown, Mary E., Ochs, Mary Luann, Hurrell, John G. R., Guder, Hans-Joachim
• Primary Examiner(s): SAUNDERS, DAVID A
• Application Number:
  US08/494,668
• Time in Patent Office: 1,891 Days
• Field of Search: 435/7.6, 435/18, 435/23, 435/24, 435/817, 435/287.2, 435/287.7, 435/287.9, 204/403
• US Class Current: 435/7.6
• CPC Class Codes: C12Q 1/001 Enzyme electrodes
• CPC Class Codes: G01N 33/535 with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/5438 Electrodes
• CPC Class Codes: G01N 33/581 with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
• CPC Class Codes: Y10S 435/817 Enzyme or microbe electrode
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive

Abstract

A diagnostic kit, method, and apparatus for electrochemically determining the presence or concentration of an analyte in a sample. A mixture is formed which includes the sample, an enzyme acceptor polypeptide, an enzyme donor polypeptide, and a labeled substrate. The enzyme donor polypeptide is capable of combining with the enzyme acceptor polypeptide to form an active enzyme complex. The formation of such the active enzyme complex is responsive to the presence or concentration of the analyte in the fluid sample. The active enzyme hydrolyzes the labeled substrate, resulting in the generation of an electroactive label, which can then be oxidized at the surface of an electrode. A current resulting from the oxidation of the electroactive compound can be measured and correlated to the concentration of the analyte in the sample.

Claims

1. A diagnostic kit for determining the presence or concentration of an analyte in a fluid sample, comprising:

(a) an enzyme donor reagent which comprises

1) an enzyme donor polypeptide conjugate; and

2) a labeled substrate, comprising an enzyme substrate cleavably linked to an electroactive label;

(b) an enzyme acceptor reagent which comprises

1) an enzyme acceptor polypeptide capable of combining with the enzyme donor polypeptide conjugate to form an active enzyme complex capable of catalyzing the cleavage of the electroactive label from the enzyme substrate; and

2) a first antibody capable of immunologically, competitively binding to the analyte and the enzyme donor polypeptide conjugate and hindering formation of the active enzyme complex when bound to the enzyme donor polypeptide conjugate; and

(c) an electrochemical immunosensor.

2. The diagnostic kit of claim 1, wherein the enzyme donor polypeptide conjugate is a conjugate of an enzyme donor polypeptide and at least one of an analyte or an analyte analog.

3. The diagnostic kit of claim 2, wherein the enzyme acceptor reagent further comprises an amount of the analyte sufficient to ensure that changes in concentration of the analyte in the fluid sample are substantially linearly related to changes in the current measured by an electrochemical measurement.

4. The diagnostic kit of claim 3, wherein the enzyme donor reagent further comprises an amount of added peptides, protein fragments, or proteins sufficient to reduce hydrolysis of the enzyme donor polypeptide conjugate from proteases in the fluid sample.

5. The diagnostic kit of claim 2, wherein the enzyme acceptor reagent further comprises a second antibody capable of immunologically binding to the first antibody, thereby further hindering formation of the active enzyme complex when the first antibody is bound to the enzyme donor polypeptide conjugate.

6. The diagnostic kit of claim 1, wherein the electrochemical immunosensor includes a first insulating substrate, first and second electrodes affixed to the first insulating substrate, and a second insulating substrate, which overlays the first and second electrodes has a window for exposing at least a portion of the first and second electrodes, and has a cutout portion at one end to allow contact between the electrodes and a meter and a power source.

7. The diagnostic kit of claim 6, wherein the first electrode is a working electrode and the second electrode is a counter electrode.

8. The diagnostic kit of claim 7, wherein the working and counter electrodes are palladium, platinum, gold, silver, titanium, copper, or carbon.

9. The diagnostic kit of claim 6, wherein the first electrode is a working electrode and the second electrode is a reference electrode.

10. The diagnostic kit of claim 1, wherein the enzyme substrate comprises μ

-D-galactopyranoside and the electroactive label comprises 4-(1,4,7,10-tetraoxadecyl)-1-naphthyl, 4-methoxy-1-naphthyl, p-aminophenyl, p-nitrophenyl, chlorophenol red, o-nitrophenyl, umbelliferyl, o-methoxy-p-nitrophenyl, 3,4 dinitrophenyl, m-cyano-p-nitrophenyl, 4-nitrosalicylaldehyde, or 4-methyl-umelliferyl.

11. An electrochemical immunoassay method for determining the presence or concentration of an analyte in a fluid sample, comprising:

(a) preparing a mixture which includes;

(1) the fluid sample;

(2) an enzyme donor reagent, which comprises;

(i) an enzyme donor polypeptide conjugate; and

(ii) a labeled substrate, comprising an enzyme substrate cleavably linked to an electroactive label; and

(3) an enzyme acceptor reagent, which comprises (i) an enzyme acceptor polypeptide capable of combining with the enzyme donor polypeptide conjugate to form an active enzyme complex capable of catalyzing the cleavage of the electroactive label from the enzyme substrate; and

(ii) a first antibody capable of immunologically, competitively binding to the analyte and the enzyme donor polypeptide conjugate and hindering formation of the active enzyme complex when bound to the enzyme donor polypeptide conjugate;

(b) applying the mixture to an electrochemical cell having first and second electrodes;

(c) applying, after incubation of the mixture, a potential difference between the first and second electrodes sufficient to oxidize the electroactive label that has been cleaved from the enzyme substrate, thereby generating a current; and

(d) measuring the current and correlating the current to the presence or concentration of the analyte.

12. The electrochemical immunoassay method of claim 11, wherein the enzyme substrate comprises β

-D-galactopyranoside and the electroactive label comprises 4-(1,4,7,10-tetraoxadecyl)-1-naphthyl, 4-methoxy-1-naphthyl, p-aminophenyl, p-nitrophenyl, chlorophenol red, o-nitrophenyl, umbelliferyl, o-methoxy-p-nitrophenyl, 3,4 dinitrophenyl, m-cyano-p-nitrophenyl, 4-nitrosalicylaldehyde, or 4-methyl-umelliferyl.

13. The electrochemical immunoassay method of claim 11, wherein the enzyme donor polypeptide conjugate is a conjugate of an enzyme donor polypeptide and at least one of an analyte or an analyte analogy.

14. The electrochemical immunoassay method of claim 13, wherein the enzyme acceptor reagent further comprises a second antibody capable of immunologically binding to the first antibody, thereby further hindering formation of the active enzyme complex when the first antibody is bound to the enzyme donor polypeptide conjugate.

15. The electrochemical immunoassay method of claim 13, wherein the enzyme acceptor reagent further comprises an amount of the analyte sufficient to ensure that changes in concentration of the analyte in the fluid sample are substantially linearly related to changes in the current measured by an electrochemical measurement.

16. The electrochemical immunoassay method of claim 15, wherein the enzyme donor reagent further comprises an amount of added peptides, protein fragments, or proteins sufficient to reduce hydrolysis of the enzyme donor polypeptide conjugate from proteases in the fluid sample.

17. The electrochemical immunoassay method of claim 16, wherein the electrochemical cell comprises a first insulating substrate, first and second electrodes affixed to the first insulating substrate, and a second insulating substrate, which overlays the first and second electrodes, has a window for exposing at least a portion of the first and second electrodes, and has a cutout portion at one end to allow contact between the electrodes and a meter and a power source.

18. The electrochemical immunoassay method of claim 17, wherein the first electrode is a working electrode and the second electrode is a reference electrode.

19. The electrochemical immunoassay method of claim 17, wherein the first electrode is a working electrode and the second electrode is a counter electrode.

20. The electrochemical immunoassay method of claim 19, wherein the working and counter electrodes are palladium, platinum, gold, silver, titanium, copper, or carbon.

21. An immunosensor useful for an electrochemical immunoassay of an analyte in a fluid sample, comprising:

(a) a first insulating substrate;

(b) first and second electrodes affixed to the first insulating substrate;

(c) a second insulating substrate, which overlays the first and second electrodes, has a window for exposing at least a portion of the first and second electrodes, and has a cutout portion at one end to allow contact between the electrodes and a meter and a power source;

(d) a porous substrate, which is impregnated with an enzyme donor reagent, overlays the window, and is spatially displaced from the working and counter electrodes, the enzyme donor reagent including;

(1) an enzyme donor polypeptide conjugate; and

(2) a labeled substrate, comprising an enzyme substrate cleavably linked to an electroactive label;

(e) an enzyme acceptor reagent, which is placed on the second insulating substrate, the enzyme acceptor reagent including;

(1) an enzyme acceptor polypeptide capable of combining with the enzyme donor polypeptide conjugate to form an active enzyme complex capable of catalyzing the cleavage of the electroactive label from the enzyme substrate; and

(2) an antibody capable of immunologically, competitively binding to the analyte and the enzyme donor polypeptide conjugate and hindering formation of the active enzyme complex when bound to the enzyme donor polypeptide conjugate;

(f) a third insulating substrate, which overlays the second insulating substrate and has a cutout portion for exposing the enzyme donor reagent, the enzyme acceptor reagent, and the window in the second insulating substrate; and

(g) a fourth insulating substrate, which overlays the third insulating substrate such that a capillary space is formed within the cutout portion of the third insulating substrate, has a window for exposing a portion of the enzyme acceptor reagent, and has a vent hole.

22. The immunosensor of claim 21, wherein the first electrode is a working electrode and the second electrode is a reference electrode.

23. Thee immunosensor of claim 21, wherein the enzyme substrate comprises β

-D-galactopyranoside and the electroactive label comprises 4-(1,4,7,10-tetraoxadecyl)-1-naphthyl, 4-methoxy-1-naphthyl, p-aminophenyl, p-nitrophenyl, chlorophenol red, o-nitrophenyl, umbelliferyl, o-methoxy-p-nitrophenyl, 3,4 dinitrophenyl, m-cyano-p-nitrophenyl, 4-nitrosalicylaldehyde, or 4-methyl-umelliferyl.

24. The immunosensor of claim 21, wherein the first electrode is a working electrode and the second electrode is a counter electrode.

25. The immunosensor of claim 24, wherein the working and counter electrodes are palladium, platinum, gold, silver, titanium, copper, or carbon.

26. The immunosensor of claim 25, wherein the working and counter electrodes are made of the same material and are substantially the same size.

27. The immunosensor of claim 26, further comprising the power source in electrical connection with the working and counter electrodes and capable of supplying an electrical potential difference between the working and counter electrodes sufficient to cause electrooxidation of the reduced form of the label at the surface of the working electrode, and the meter in electrical connection with the working and counter electrodes and capable of measuring the current produced by the oxidation of the reduced form of the label at the surface of the working electrode.

28. An immunosensor useful for an electrochemical immunoassay of an analyte in a fluid sample, comprising:

(a) a first insulating substrate;

(b) first and second electrodes affixed to the first insulating substrate;

(c) a second insulating substrate, which overlays the first and second electrodes, has a window for exposing at least a portion of the first and second electrodes, and has a cutout portion at one end to allow contact between the electrodes and a meter and a power source;

(d) an enzyme donor reagent which is placed on the second insulating substrate, the enzyme donor reagent including

(1) an enzyme donor polypeptide conjugate; and

(2) a labeled substrate, comprising an enzyme substrate cleavably linked to an electroactive label;

(e) an enzyme acceptor reagent, which is placed on the second insulating substrate, the enzyme acceptor reagent including (1) an enzyme acceptor polypeptide capable of combining with the enzyme donor polypeptide conjugate to form an active enzyme complex capable of catalyzing the cleavage of the electroactive label from the enzyme substrate; and

(f) an antibody capable of immunologically, competitively binding to the analyte and the enzyme donor polypeptide conjugate and hindering formation of the active enzyme complex when bound to the enzyme donor polypeptide conjugate;

(g) a polymer, which is placed on the second insulating substrate between the enzyme acceptor reagent and the enzyme donor reagent;

(h) a third insulating substrate, which overlays the second insulating substrate and has a cutout portion for exposing the enzyme donor reagent, the enzyme acceptor reagent, the polymer, and the window in the second insulating substrate; and

(i) a fourth insulating substrate, which overlays the third insulating substrate such that a capillary space is formed within the cutout portion of the third insulating substrate, has a window for exposing a portion of the enzyme acceptor reagent, and has a vent hole.

29. The immunosensor of claim 28, wherein the first electrode is a working electrode and the second electrode is a counter electrode.

30. The immunosensor of claim 28, wherein the first electrode is a working electrode and the second electrode is a reference electrode.

31. The immunosensor of claim 28, wherein the enzyme substrate comprises β

-D-galactopyranoside and the electroactive label comprises 4-(1,4,7,10-tetraoxadecyl)-1-naphthyl, 4-methoxy-1-naphthyl, p-aminophenyl, p-nitrophenyl, chlorophenol red, o-nitrophenyl, umbelliferyl, o-methoxy-p-nitrophenyl, 3,4 dinitrophenyl, m-cyano-p-nitrophenyl, 4-nitrosalicylaldehyde, or 4-methyl-umbelliferyl.