Electrochemical enzyme assay

• US 20030017519A1
• Filed: 02/27/2002
• Published: 01/23/2003
• Est. Priority Date: 08/27/1993
• Status: Abandoned Application

• Current Assignee: Hans-Joachim Guder, Harvev B. Buck Jr, John G.R. Hurrell, Lance S. Kuhn, Mary E. Brown, Mary Luann Ochs, Rebecca W. Muddiman, Robert J. Mcenroe
• Original Assignee: Hans-Joachim Guder, Harvev B. Buck Jr, John G.R. Hurrell, Lance S. Kuhn, Mary E. Brown, Mary Luann Ochs, Rebecca W. Muddiman, Robert J. Mcenroe
• Inventors: McEnroe, Robert J., Buck, Harvev B. Jr., Muddiman, Rebecca W., Brown, Mary E., Guder, Hans-Joachim, Hurrell, John G.R., Ochs, Mary Luann, Kuhn, Lance S.
• Application Number:
  US10/090,141
• Publication Number:
  US 20030017519A1
• Time in Patent Office: Days
• US Class Current: 435/7.92
• 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, in a direct manner, the presence or concentration of an analyte in a fluid sample, wherein the diagnostic kit utilizes an electrochemical measurement, comprising:

(a) an enzyme donor reagent which comprises

1) an enzyme donor polypeptide conjugate comprising an enzyme donor polypeptide conjugated to an antibody; and

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

(b) an enzyme acceptor reagent which comprises 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 substrate, wherein the amount of active enzyme complex formed is directly related to the amount of analyte in the fluid sample.

2. The diagnostic kit of claim 1, wherein the enzyme substrate comprises 13-D-galactopyranoside.

3. The diagnostic kit of claim 1, wherein the electroactive label comprises at least one of 4-(1,4,7,10-tetraoxadecyl)-1-naphthyl, 4-methoxy-1-naphthyl, 4-chloro-1-naphthyl, 4-propoxy-1-naphthyl, 4-isopropoxy-1-naphthyl, 4-benzyloxy-1-naphthyl, 4-trifluoroethoxy-1-naphthyl, p-nitrophenyl, chlorophenol red, o-nitrophenyl, umbelliferyl, o-methoxy-p-nitrophenyl, 3,4 dinitrophenyl, m-cyano-p-nitrophenyl, 4-nitrosalicylaldehyde, or 4-methyl-umelliferyl.

4. The diagnostic kit of claim 1, wherein the enzyme acceptor reagent further comprises a first antibody capable of immunologically binding to the analyte.

5. 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 comprising an enzyme donor polypeptide conjugated to an antibody; and

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

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

(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 substrate, thereby generating a current; and

(d) measuring the current and correlating the current to the presence or concentration of the analyte, wherein the amount of active enzyme complex formed is directly related to the amount of analyte in the fluid sample.

6. The electrochemical immunoassay method of claim 5, wherein the enzyme substrate comprises β

-D-galactopyranoside.

7. The electrochemical immunoassay method of claim 5, wherein the electroactive label comprises at least one of 4-(1,4,7,10-tetraoxadecyl)-1-naphthyl, 4-methoxy-1-naphthyl, 4-chloro-1-naphthyl, 4-propoxy-1-naphthyl, 4-isopropoxy-1-naphthyl, 4-benzyloxy-1-naphthyl, 4-trifluoroethoxy-1-naphthyl, p-nitrophenyl, chlorophenol red, o-nitrophenyl, umbelliferyl, o-methoxy-p-nitrophenyl, 3,4 dinitrophenyl, m-cyano-p-nitrophenyl, 4-nitrosalicylaldehyde, or 4-methyl-umelliferyl.

8. The electrochemical immunoassay method of claim 5, wherein the enzyme acceptor reagent further comprises a first antibody capable of immunologically binding to the analyte.

9. An immunosensor useful in a direct 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 comprising

(1) an enzyme donor polypeptide conjugate comprising an enzyme donor polypeptide conjugated to an antibody; 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 comprising 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 from the substrate wherein the amount of active enzyme complex formed is directly related to the amount of analyte in the fluid sample; and

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

(g) 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

(h) 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.

10. The immunosensor of claim 9, wherein the enzyme substrate comprises β

-D-galactopyranoside.

11. The immunosensor of claim 9, wherein the electroactive label comprises at least one of 4-(1,4,7,10-tetraoxadecyl)-1-naphthyl, 4-methoxy-1-naphthyl, 4-chloro-1-naphthyl, 4-propoxy-1-naphthyl, 4-isopropoxy-1-naphthyl, 4-benzyloxy-1-naphthyl, 4-trifluoroethoxy-1-naphthyl, p-nitrophenyl, chlorophenol red, o-nitrophenyl, umbelliferyl, o-methoxy-p-nitrophenyl, 3,4 dinitrophenyl, m-cyano-p-nitrophenyl, 4-nitrosalicylaldehyde, or 4-methyl-umelliferyl.

12. The immunosensor of claim 9, wherein the enzyme acceptor reagent further comprises a first antibody capable of immunologically binding to the analyte.