METHODS AND DEVICES FOR THE DETECTION AND MEASUREMENT OF FREE METALS IN FLUIDS AND METHODS FOR DIAGNOSING METAL-RELATED DISEASES AND FOR DETERMINING PHARMACOLOGIC DOSING REGIMENS

• US 20070209950A1
• Filed: 02/12/2007
• Published: 09/13/2007
• Est. Priority Date: 02/10/2006
• Status: Abandoned Application

• Current Assignee: Pipex Incorporated
• Original Assignee: Pipex Incorporated
• Inventors: KANZER, Steve, ALTHAUS, John
• Application Number:
  US11/674,036
• Publication Number:
  US 20070209950A1
• Time in Patent Office: Days
• US Class Current: 205/792
• CPC Class Codes: C12Q 1/001 Enzyme electrodes
• CPC Class Codes: G01N 33/5735 co-enzymes or co-factors, e.g. NAD, ATP
• CPC Class Codes: G01N 33/6896 Neurological disorders, e.g. Alzheimer's disease

Abstract

A detection method or a method for a point of care diagnostic assay which comprises detecting non-covalently bound copper (free copper) concentrations in a fluid.

Claims

1. A detection method or a method for a point of care diagnostic assay which comprises detecting non-covalently bound copper (free copper) concentrations in a fluid.

2. The method of claim 1, wherein the fluid comprises a bodily fluid selected from the group consisting of blood, plasma, serum, saliva, urine, tears, cerebrospinal fluid and sweat.

3. The method of claim 1, wherein the free copper is measured by electrochemical detection, preferably using a two or a three electrode electrochemical detection system.

4. A method of claim 3, wherein the electrode system comprises three electrodes, a working electrode, a reference electrode, and a counter or auxiliary electrode, or a working electrode, and a combined reference and counter or auxiliary electrode.

5. The method of claim 3, wherein at least one of the electrodes is formed of a material selected from the group consisting of carbon, gold, silver, silver chloride, platinum and palladium.

6. A method of diagnosing Alzheimer'"'"'s disease, Parkinson'"'"'s disease, schizophrenia, Wilson'"'"'s disease or other copper metabolic disorder in an individual, which comprises directly measuring a free copper pool in a fluid sample of that individual.

7. An assay device for detection of free or loosely bound copper comprising an enzyme capable of forming a holo-enzyme in the presence of copper.

8. An assay device as claimed in claim 7, wherein the holo-enzyme comprises an active enzyme, preferably an active apo-enzyme, or an inactive apo-enzyme.

11. The assay device as claimed in claim 8, wherein (a) apo-enzyme is prepared by removing copper, or (b) apo-enzyme is inactive in the absence of copper, or (c) when a sample containing copper is added to an apo-enzyme, activity is restored;

or (d) wherein when copper is added to an apo-enzyme to form a holo-enzyme, activity is restored, or (e) wherein activity of a holo-enzyme is measured.

9. An assay device for detection of free or loosely bound copper, which comprises an enzyme which uses copper as a co-factor.

10. The assay device as claimed in claim 9, wherein the enzyme comprises amine oxidase.

17. The assay device as claimed in claim 10, wherein the amine oxidase is oxidized whereby to generate a flow of electrons from the amine substrate;

the electrons are captured by a chemical mediator that exists in an oxidized state, whereby the mediator is chemically transformed into a reduced state; and

the chemical mediator is then oxidized at the surface of an electrode, wherein facilitation of the oxidation of the reduced chemical mediator occurs when a fixed potential is applied to the electrode.

12. An assay device for detection of copper, wherein enzyme activity is measured using a spectrophotometric or electrochemical system.

13. The assay device as claimed in claim 12, wherein, in the case of spectrophotometric system, changes in optical properties of a substrate or an electrochemical mediator are measured, and in the case of an electrochemical system, changes in electrochemical properties of a solution are measured.

14. The assay device as claimed in claim 12, wherein electrochemical changes in a solution are measured electrochemically in the presence of a chemical mediator.

15. The assay device as claimed in claim 14, wherein a chemical mediator is used to transduce electrochemical changes to an electrode.

16. The assay device as claimed in claim 15, wherein said electrochemical changes include an increase or a decrease in current flow when a fixed potential is applied.

18. The assay device as claimed in claim 12, wherein a two electrode system is used to detect enzyme activity, in which a working electrode acts as a catalyst for the oxidation of a chemical mediator, and an auxiliary electrode acts as a counter electrode and as a reference electrode for a flow of electrons, wherein in the case of oxidation of the chemical mediator the flow of electrons is inward.

19. The assay device as claimed in claim 12, wherein a three electrode system is used to detect enzyme activity, in which a working electrode acts a catalyst for the oxidation of the chemical mediator, an auxiliary electrode acts to complete a circuit and to provide a flow of electrons, and a reference electrode provides feedback and establishes an absolute potential depending on a chemical composition of the reference electrode.

20. The assay device as claimed in claim 12, wherein an electrode is connected to a potentiostat which provides for electrochemical measurements, the potentiostat is programmed to establish a fixed potential in a range for oxidizing an enzymatically reduced mediator, wherein at a fixed potential setting, a current that results from oxidation of the chemical mediator is recorded as a change in current above background.

21. The assay device as claimed in claim 12, wherein a change in current provides a measure of enzyme activity, in which enzyme activity is a direct measure of the free copper concentration which converts inactive apo-enzyme into active holo-enzyme.

22. The assay device as claimed in claim 19, wherein calibration of current vs. free copper in solution at various concentrations provides utility regarding a single-point measurement of the concentration of free copper in a test solution.

23. The assay device as claimed in claim 19, wherein the potentiostat is interfaced to a display for providing a reading indicating a concentration of free copper in a solution including blood.

24. The assay device as claimed in claim 19, wherein the electrodes are constructed so that measurement of free copper is made using solution phase or solid-state phase methods.

25. The assay device as claimed in claim 24, characterized by one or more of the following features:

(a) wherein in the case of a solution phase method, the working electrode is used in an unmodified state, and all of the reagents exit in a solution phase;

(b) wherein in the case of a solid phase method, the working electrode is modified so that one or more reagents exit with the working electrode in a solid state;

(c) wherein the enzyme and/or the chemical mediator are mixed with materials forming the working electrode;

(d) wherein the working electrode is screen printed with conductive ink that is doped with enzyme and/or chemical mediator;

(e) wherein a surface of the working electrode is screen printed with a conductive carbon ink doped with enzyme and/or chemical mediator and covered with a membrane that is selective for copper permeation;

(f) wherein the working electrode is screen printed with a conductive ink which also includes a conductive material, preferably carbon, gold or silver;

(g) wherein the working electrode is screen printed with conductive ink that includes a doping material selected from amine oxidase or other redox-type enzyme, or ferrocine, PMS, DCPIP or other type chemical mediator; and

(h) wherein a membrane formed of a polymerizing material that provides copper selectivity covers the working electrode at least in part, wherein the membrane preferably is formed of nafion.

26. The assay device as claimed in claim 12, wherein a two electrode system is used in which current is generated using a first working, electrode specific to copper detection and a second working electrode that is not specific to copper detection;

and specific copper detection is achieved by subtracting a current generated at the non-specific electrode from the current generated at the specific electrode.

27. A device that measures the state of copper in blood as a gradient of free to loosely to tightly bound copper is measured, wherein free copper in blood is based on a measurement of enzyme activity as an initial rate and not at equilibrium.

28. The device as claimed in claim 27, wherein enzyme activity as an initial rate provides for measurement of the free form of copper in blood.

29. The device as claimed in claim 27, using the assay as claimed in claim 50(e), wherein specific measurement of the free form of copper as an initial rate is achieved due to a limited number of enzymes doped into the conductive carbon ink as compared to the total number of free copper molecules in a drop of blood.

30. The device as claimed in claim 29, characterized by one or more of the following features:

(a) wherein at 1 uM of free copper in a 20 ul sample of blood, the total number of copper molecules is 1 E+13, the total number of copper binding sites on the electrode is E+9 or less, and even if all the free copper binding sites on the electrode were occupied, the free copper concentration of copper the would not be significantly altered even for normal patients;

(b) wherein, enzyme activity is based on the most accessible, free copper concentration; and

(c) wherein initial rates of enzyme activity is measured within 1 minute to avoid saturation of enzyme sites on the electrode, and further to avoid perturbation of copper homeostasis in the measured drop of blood.

31. An assay device for detection of free or loosely bound copper comprising a copper-dependent enzyme attached to a bead.

32. The assay device of claim 31, wherein the enzyme comprises an apo-enzyme attached to a bead by removing copper.

33. A method for measuring the concentration of copper in serum or blood by reconstitution of an enzyme attached to a bead from an apo-form to a holo-form.

34. The method of claim 33, wherein activity of a reconstituted apo-enzyme from copper in serum or blood is proportional to the concentration of copper in the serum of blood.