Apoenzyme reactivation electrochemical detection method and assay

• US 20050196820A1
• Filed: 02/17/2005
• Published: 09/08/2005
• Est. Priority Date: 03/03/2004
• Status: Active Grant

• Current Assignee: Stephen Eliot Zweig
• Original Assignee: Stephen Eliot Zweig
• Inventors: Zweig, Stephen Eliot
• Granted Patent: US 7,166,208 B2
• Time in Patent Office: Days
• US Class Current: 435/14
• CPC Class Codes: C12Q 1/004 mediator-assisted
• CPC Class Codes: G01N 33/5438 Electrodes

Abstract

The invention discloses a methods in which dry reagent enzyme based electrochemical biosensors, which are in a relatively mature form due to the extensive amount of development pioneered by the blood glucose monitoring industry, may be simply adapted to perform tests for blood coagulation, enzymatic activity, or immunochemical assays for antigens present in a fluid sample. In particular, the utility of combining apoenzyme based dry reagent electrochemical biosensors with apoenzyme reactivation technology is taught. This combination creates a novel combination dry reagent test technology capable of detecting a wide range of different analytes.

Claims

1. :

An electrochemical detection device for detecting an analyte in a liquid sample, said device comprising;

an electrode containing an apoenzyme or otherwise inactive form of an enzyme that, in the active form, would produce an electrochemical change in said electrode in response to an enzyme substrate;

an apoenzyme cofactor, prosthetic group or other activation moiety that converts the inactive form of said enzyme to an active form;

said cofactor, prosthetic group or activation moiety being present in the form of a complex that changes its structure due to interactions with said analyte;

said complex being incapable of activating the apoenzyme or otherwise inactive form of the enzyme in the absence of said analyte;

wherein said analyte induces changes in said complex, enabling said prosthetic group or said activation moiety to activate said apoenzyme or said inactive form of an enzyme;

resulting in a detectable electrochemical change in said electrode.

2. :

The device of claim 1, in which the apoenzyme is glucose oxidase, and the complex contains FAD as the prosthetic group or activation moiety.

3. :

The device of claim 1, in which the analyte in a liquid sample comprises an active or inactive form of a protease, and said complex contains a protease substrate, wherein the protease activity of said sample liberates said cofactor, prosthetic group or said activation moiety from said complex, resulting in activation of said enzyme, and a change in the electrochemical status of said electrode.

4. :

The device of claim 1, in which the analyte is an antigen, and said complex contains an antibody to said antigen, wherein presence of excess antigen from said sample causes a liberation of said cofactor, prosthetic group or said activation moiety from said complex, resulting in activation of said enzyme, and a change in the electrochemical status of said electrode.

5. :

The device of claim 1, in which said complex is on a solid surface that is spatially separated from the region of the apparatus where the apoenzyme or inactive form of the enzyme is located.

6. :

The device of claim 1, in which the interaction between the analyte, the complex, and the apoenzyme or inactive form of the enzyme take place within a porous electrode.

7. The device of claim 1, in which said complex induced changes in the activity of said enzyme is selected from the group consisting of enzyme cofactor addition, prosthetic group addition, allosteric regulator binding, covalent enzyme modification, or proteolytic cleavage.

8. :

The device of claim 1, additionally containing integral means for producing heat to keep the device at a constant temperature during the reaction.

9. :

An electrochemical detection device for detecting an active or inactive form of an analyte enzyme in a liquid sample, said detection device comprising;

an electrode containing an apoenzyme or otherwise inactive form of an electrochemically active enzyme that, in the active form, would produce an electrochemical change in said electrode in response to an electrode enzyme substrate;

an apoenzyme cofactor, prosthetic group or other activation moiety that converts the inactive form of said electrochemically active enzyme to an active form;

said cofactor, prosthetic group or activation moiety being present in the form of a complex that contains an analyte enzyme substrate that changes its structure due to interactions with said analyte enzyme;

said complex being incapable of activating said apoenzyme or otherwise inactive form of the electrochemically active enzyme in the absence of said analyte enzyme;

wherein said analyte enzyme induces changes in said complex, enabling said cofactor, prosthetic group or said activation moiety to activate said apoenzyme or said inactive form of an electrochemically active enzyme;

resulting in a detectable electrochemical change in said electrode.

10. :

The device of claim 9, used to analyze the status of a coagulation pathway, in which the analyte enzyme is a coagulation pathway protease, the enzyme substrate is the peptide substrate to the coagulation pathway protease, and in which the device additionally contains chemical means to trigger the formation of one or more coagulation pathways in said sample.

11. :

The device of claim 9 used to perform a prothrombin time assay, in which the analyte enzyme is thrombin, the analyte enzyme substrate is a thrombin substrate, and the device additionally contains thromboplastin.

12. :

The device of claim 9, in which the apoenzyme is glucose oxidase, and the complex contains FAD as the prosthetic group or activation moiety.

13. :

The device of claim 9, in which said complex is on a solid surface that is spatially separated from the region of the device where the apoenzyme or inactive form of the electrically active enzyme is located.

14. :

The device of claim 9, in which the interaction between the complex and the apoenzyme or inactive form of the electrochemically active enzyme take place within a porous electrode.

15. The device of claim 9, in which said complex induced changes in the activity of said electrochemically active enzyme is selected from the group consisting of enzyme cofactor addition, prosthetic group addition, allosteric regulator binding, covalent enzyme modification, or proteolytic cleavage.

16. :

An electrochemical detection device for detecting an antigen in a liquid sample, said detection device comprising;

an electrode containing an apoenzyme or otherwise inactive form of an electrochemically active enzyme that, in the active form, would produce an electrochemical change in said electrode in response to a substrate to the electrically active enzyme;

an apoenzyme cofactor, prosthetic group, or other activation moiety that converts the inactive form of said electrochemically active enzyme to an active form;

said cofactor or activation moiety being present in the form of a complex that changes its structure due to interactions with said antigen;

said complex additionally containing an antibody capable of binding to said antigen;

said complex being incapable of activating the apoenzyme or otherwise inactive form of the electrochemically active enzyme in the absence of said antigen;

wherein said antigen induces changes in said complex, enabling said cofactor, prosthetic group or said activation moiety to activate said apoenzyme or said inactive form of an electrochemically active enzyme;

resulting in a detectable electrochemical change in said electrode.

17. :

The device of claim 16, in which the apoenzyme is glucose oxidase, and the complex contains FAD as the prosthetic group or activation moiety.

18. :

The device of claim 16, in which said complex is on a solid surface that is spatially separated from the region of the device where the apoenzyme or inactive form of the electrically active enzyme is located.

19. :

The device of claim 16, in which the interaction between the analyte, the complex, and the apoenzyme or inactive form of the enzyme take place within a porous electrode.

20. :

The device of claim 16, in which said complex induced changes in the activity of said enzyme is selected from the group consisting of enzyme cofactor addition, prosthetic group addition, allosteric regulator binding, covalent enzyme modification, or proteolytic cleavage.

21. :

A method for detecting an analyte in a liquid sample, said method comprising;

an electrode containing an apoenzyme or otherwise inactive form of an enzyme that, in the active form, would produce an electrochemical change in said electrode in response to an enzyme substrate;

an apoenzyme cofactor, prosthetic group or other activation moiety that converts the inactive form of said enzyme to an active form;

said cofactor, prosthetic group or activation moiety being present in the form of a complex that changes its structure due to interactions with said analyte;

said complex being incapable of activating the apoenzyme or otherwise inactive form of the enzyme in the absence of said analyte;

wherein said analyte induces changes in said complex, enabling said cofactor, prosthetic group or said activation moiety to activate said apoenzyme or said inactive form of an enzyme;

resulting in a detectable electrochemical change in said electrode;

in which analyte is added to the electrode, the electrochemical status of the electrode is assessed, and the relative amount of analyte present in the sample is detected.