Conductometric detection process

• US 20030003523A1
• Filed: 06/18/2002
• Published: 01/02/2003
• Est. Priority Date: 06/19/2001
• Status: Abandoned Application

• Current Assignee: Mark Jensen
• Original Assignee: Mark Jensen
• Inventors: Jensen, Mark
• Application Number:
  US10/174,869
• Publication Number:
  US 20030003523A1
• Time in Patent Office: Days
• US Class Current: 435/25
• CPC Class Codes: C12Q 1/6832 Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 2537/125 Sandwich assay format
• CPC Class Codes: C12Q 2563/137 Metal/ion, e.g. metal label
• CPC Class Codes: G01N 27/041 of a solid body
• 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)

Abstract

The present invention provides a method for determining in a sample the presence and the level of a substance to be detected that can specifically bind to a capture substance. The method involves providing complexes comprising the substance to be detected, the capture substance and an oxidative enzyme linked to the complexes on a base along a path defined by a pair of electrical conductors. The oxidative enzyme promotes amplifying deposition of a metal layer along the path such that the metal layer becomes more electrically conductive, thereby enhancing the detection sensitivity. One can determine the presence and the level of the detected substance by monitoring the current flow of an electric circuit that includes the path.

Claims

1. A method for detecting a detected substance in a sample, the method comprising the steps of:

providing, in a path between spaced-apart electrical conductors on a substantially non-conductive base, aggregates of a complex between the detected substance, a capture substance specifically bound thereto and an oxidative enzyme;

exposing the aggregates in the path to an substrate capable of being oxidized by the oxidative enzyme and to transition metal ions so that the substrate is oxidized and the metal ions are reduced to zero-valence metal atoms that precipitate on the aggregates to form a transition metal layer; and

measuring electrical resistance of an electrical circuit comprising the conductors, the transition metal layer and an electrical energy source, whereby a change in resistance relative to a control lacking the detected substance indicates the detected substance in the sample.

2. The method of claim 1 further comprising the step of depositing a second metal layer on the transition metal precipitate layer, the second metal layer being more catalytic for reducing silver ions than the transition metal layer.

3. The method of claim 2, wherein the step of depositing the second metal layer comprises the step of exposing the transition metal precipitate layer to a metal salt under suitable metal depositing conditions, the metal being selected from the group consisting of gold, platinum and palladium.

4. The method of claim 2 further comprising the step of depositing silver metal on the second metal layer.

5. The method of claim 4, wherein the step of depositing the silver metal comprises the step of exposing the second metal layer to a silver salt and a reducing agent.

6. The method of claim 5, wherein the silver salt is selected from the group consisting of silver nitrate, silver lactate and silver acetate.

7. The method of claim 1, wherein the oxidative enzyme is selected from the group consisting of a peroxidase and an acetate.

8. The method of claim 1, wherein the enzyme is horseradish peroxidase.

9. The method of claim 1, wherein the substrate capable of being oxidized by the oxidative enzyme is selected from the group consisting of diaminobenzidine and 3,3′

,5,5′

-tetramethylbenzidine.

10. The method of claim 1, wherein the oxidative substrate is diaminobenzidine.

11. The method of claim 1, wherein the transition metal is selected from the group consisting of nickel, cobalt, copper, lead, and cadmium.

12. The method of claim 1, wherein the detected substance is selected from the group consisting of a nucleic acid and a polypeptide.

13. The method of claim 1 wherein the detected substance is a nucleic acid labeled with biotin and the capture substance is a nucleic acid anchored to the path that hybridizes at least in part to the detected biotin-labeled nucleic acid, wherein the oxidative enzyme is linked to the complex via a biotin-streptavidin linkage.

14. The method of claim 1 wherein the detected substance is a nucleic acid, the capture substance is a nucleic acid anchored to the path that hybridizes at least in part to the detected nucleic acid, the complex further comprising a biotin-labeled nucleic acid hybridized to the detected nucleic acid, wherein the oxidative enzyme is linked to the complex via a biotin-streptavidin linkage.

15. The method of claim 1 wherein the detected substance is an antigen, the capture substance is anchored to the path and is a first antibody capable of specific binding to the antigen, wherein the oxidative enzyme is linked to the complex via a second antibody capable of specific binding to the antigen.