Apoenzyme reactivation electrochemical detection method and assay
First Claim
1. An electrochemical detection device for detecting the activity of one or more hydrolase analyte enzymes in a liquid sample, said device comprising;
- at least one electrode containing an apoenzyme or otherwise inactive form of an electrochemical enzyme that, in the active form, would produce an electrochemical change in at least one of said electrodes in response to an electrochemical enzyme substrate;
an apoenzyme cofactor, prosthetic group or other activation moiety that converts the inactive form of said electrochemical enzyme to an active form;
said cofactor, prosthetic group or activation moiety being present in the form of at least one complex that contains at least one target substrate which is cleaved by at least one of said hydrolase analyte enzymes;
said complex being incapable of activating the apoenzyme or otherwise inactive form of said electrochemical enzyme when said target substrate is not cleaved;
wherein at least one of said hydrolase analyte enzymes cleaves at least one of said target substrates, enabling said cofactor, prosthetic group or said activation moiety to activate said apoenzyme or said inactive form of said electrochemical enzyme;
resulting in a detectable electrochemical change in at least one of said electrodes; and
wherein said complex is on a surface that is spatially separated from the region or regions of the apparatus where the apoenzyme or inactive form of said electrochemical enzyme are located.
0 Assignments
0 Petitions
Accused Products
Abstract
The invention discloses a device and method by 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 test technology capable of detecting a wide range of different analytes, and operating in a wide variety of wet or dry, in vivo or in vitro environments.
-
Citations
20 Claims
-
1. An electrochemical detection device for detecting the activity of one or more hydrolase analyte enzymes in a liquid sample, said device comprising;
-
at least one electrode containing an apoenzyme or otherwise inactive form of an electrochemical enzyme that, in the active form, would produce an electrochemical change in at least one of said electrodes in response to an electrochemical enzyme substrate; an apoenzyme cofactor, prosthetic group or other activation moiety that converts the inactive form of said electrochemical enzyme to an active form; said cofactor, prosthetic group or activation moiety being present in the form of at least one complex that contains at least one target substrate which is cleaved by at least one of said hydrolase analyte enzymes; said complex being incapable of activating the apoenzyme or otherwise inactive form of said electrochemical enzyme when said target substrate is not cleaved; wherein at least one of said hydrolase analyte enzymes cleaves at least one of said target substrates, enabling said cofactor, prosthetic group or said activation moiety to activate said apoenzyme or said inactive form of said electrochemical enzyme; resulting in a detectable electrochemical change in at least one of said electrodes; and wherein said complex is on a surface that is spatially separated from the region or regions of the apparatus where the apoenzyme or inactive form of said electrochemical enzyme are located. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
-
-
12. An immunochemical detector device for performing immunoassays for one or more test antigens, said detector comprising:
-
one or more electrodes; one or more hybrid antibodies formed from the protein produced by a recombinant fusion hybrid between an antibody immunoglobulin gene and the gene for an electrically active enzyme; the enzyme protein portion of said hybrid antibodies being present in an apoenzyme or otherwise enzymatically inactive form; in which said apoenzyme portion or said inactive form of said hybrid antibodies, in the active form, would produce an electrochemical change in said electrode in response to an amplification substrate to the electrically active enzyme portion of the hybrid antibody; said device additionally containing an apoenzyme cofactor, prosthetic group, or other activation moiety that converts the enzymatically inactive form of said hybrid antibody to an enzymatically active form; said hybrid antibody or said cofactor or activation moiety being present in the form of a complex that changes its structure due to interactions with a test antigen in a test sample; wherein said test antigen induces changes in said complex, enabling said cofactor, prosthetic group or said activation moiety to activate said enzymatically inactive hybrid antibody, resulting in a detectable electrochemical change in one or more of said electrodes. - View Dependent Claims (13, 14, 15, 16, 17, 18, 19)
-
-
20. A method for detecting the activity of one or more hydrolase analyte enzymes in a liquid sample, said method comprising;
-
obtaining a device comprising at least one electrode containing an apoenzyme or otherwise inactive form of an electrochemical enzyme that, in the active form, would produce an electrochemical change in at least one of said electrodes in response to an electrochemical enzyme substrate; an apoenzyme cofactor, prosthetic group or other activation moiety that converts the inactive form of said electrochemical enzyme to an active form; said cofactor, prosthetic group or activation moiety being present in the form of at least one complex that that contains at least one target substrate which is cleaved by at least one of said hydrolase analyte enzymes; said complex being incapable of activating the apoenzyme or otherwise inactive form of said electrochemical enzyme when said target substrate is not cleaved; wherein at least one of said hydrolase analyte enzyme cleaves at least one of said target substrates, enabling said cofactor, prosthetic group or said activation moiety to activate said apoenzyme or said inactive form of said electrochemical enzyme;
resulting in a detectable electrochemical change in at least one of said electrodes;in which one or more hydrolase analyte enzymes is added to the device, the electrochemical status of various device electrodes is assessed, and the relative activity of the various hydrolase analyte enzymes present in the sample is determined; and wherein said method is used to analyze the status of a coagulation pathway, in which the hydrolase analyte enzyme is a coagulation pathway protease, the target substrate which is cleaved by said hydrolase analyte enzyme 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 liquid sample.
-
Specification