Apoenzyme reactivation electrochemical detection method and assay

• US 20070289880A1
• Filed: 01/22/2007
• Published: 12/20/2007
• Est. Priority Date: 09/04/2001
• Status: Active Grant

• Current Assignee: Stephen Zweig
• Original Assignee: Stephen Zweig
• Inventors: Zweig, Stephen
• Granted Patent: US 8,118,991 B2
• Time in Patent Office: Days
• US Class Current: 205/777.500
• CPC Class Codes: C12Q 1/005 involving specific analytes or enzymes including groups of enzymes, e.g. oxydases; C12Q1/004 takes precedence
• CPC Class Codes: C12Q 1/34 involving hydrolase

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.

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 complexes 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 lest 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.

2. :

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

3. :

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

4. :

The device of claim 1, in which the hydrolase analyte enzyme is selected from the group consisting of proteases (EC-3.4), esterases (EC-3.1), and glycosylases (EC-3.2).

5. :

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

6. :

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

7. :

The device of claim 1, in which one or more of the hydrolase analyte enzyme target substrates are enzyme target substrates for enzymes selected from the group consisting of clinical markers for coagulation, angiogenesis, inflammation, sepsis, cardiovascular status, kidney disease, kidney injury, cancer and ischemia.

8. :

The device of claim 1, in which the device is in the form of a disposable test strip, and in which the test strip contains integral means for producing heat to keep the device at a constant temperature during the reaction.

9. :

The detection device of claim 1, mounted on an invasive device selected from the group consisting of surgical tools, catheters, implantable electrodes, implantable chips, and implantable biosensors, in which the detection device is initially protected from body fluids by a removable covering.

10. :

The device of claim 1, in which the protease analyte enzyme is a coagulation pathway protease, the target substrate that is cleaved by said protease 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.

11. :

The device of claim 10, in which the protease analyte enzyme is thrombin, the target substrate which is cleaved by thrombin is a thrombin substrate, and the chemical means to trigger the formation of one or more coagulation pathways in said liquid sample comprise thromboplastin or another coagulation factor VII activating substance.

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.

13. :

The immunochemical detector device of claim 12, in which said gene for said electrically active enzyme is a gene for a mutant form of said electrically active enzyme with an affinity for the enzyme'"'"'s cofactor or prosthetic group that is lower than that of the wild type gene'"'"'s affinity for the enzyme'"'"'s cofactor or prosthetic group.

14. :

The immunochemical detector device of claim 12, in which said one or more of said hybrid antibodies are produced by phage display technology using recombinant genes composed of fused antibody immunoglobulin genes and electrically active enzyme genes.

15. :

The immunochemical detector device of claim 12, in which the test antigen is selected from the group consisting of markers for sepsis, angiogenesis, pregnancy, ovulation, cardiovascular status, infectious disease, drugs of abuse, therapeutic drugs, kidney disease, ischemia, coagulation and cancer diagnostics.

16. :

The immunochemical detector device of claim 12, in which the test antigen is selected from the group of consisting of markers for calcitonin, procalcitonin, c-reactive protein, endogenous activated protein C, tumor necrosis factor, interleukin-6, interleukin 10, endotoxin, lipopolysacharide binding protein, pro-atrial natriuretic peptide, C difficile, Hepatitis, HIV, influenza, legionelly, pneumonia, RSV, strep, syphilis, early pregnancy factor, human chorionic gonadotropin, luteinising hormone, amphetamines, barbiturates, benzodiazepines, cocaine, methamphetamines, opiates, phencyclidine, THC, trycyclic antidepressants, acetaminophen, carbamazepine, phenyloin, theophylline, drotrecogin alfa, tissue plasminogen activator, cystantin C, neutrophil gelatinase-associated lipocalin, interleukin-18, kidney injury molecule-1, proatrialnatriuretic peptide, d-dimer, troponin I, CKMB, myoglobin, nt Pro BNP, ischemia modified albumin, myeloperoxidase, S-100 beta, and prostate specific antigen.

17. :

The immunochemical detector device of claim 12, mounted on a device selected from the group consisting of surgical tools, catheters, implantable electrodes, implantable chips, and implantable biosensors.

18. :

The immunochemical detector device of claim 12, mounted on an implantable biosensor, in which the biosensor contains electronic means to transmit signals from said detector outside the body, said means selected from the group consisting of radiofrequency signals, light signals, infrared signals, sonic signals, and vibration signals.

19. :

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 lease 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.

20. :

The method of claim 19, used to analyze the status of a coagulation pathway, in which the protease analyte enzyme is a coagulation pathway protease, the target substrate which is cleaved by said protease 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.