Electrochemical methods of assay

• US 4,945,045 A
• Filed: 06/23/1988
• Issued: 07/31/1990
• Est. Priority Date: 07/06/1984
• Status: Expired due to Term

• Current Assignee: Serono Diagnostics Ltd.
• Original Assignee: Serono Diagnostics Ltd.
• Inventors: Forrest, Gordon C., Hill, Hugh A. O., Robinson, Grenville A., Rattle, Simon J.
• Primary Examiner(s): Rosen, Sam
• Application Number:
  US07/212,043
• Time in Patent Office: 768 Days
• Field of Search: 435/7, 435/14, 435/25, 435/817, 436/806, 436/808, 204/403
• US Class Current: 435/25
• CPC Class Codes: G01N 27/3271 Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood amperometry per se G01N27/49; aspects concerning the enzyme reagent C12Q1/001
• CPC Class Codes: G01N 33/5438 Electrodes
• CPC Class Codes: Y10S 435/817 Enzyme or microbe electrode
• CPC Class Codes: Y10S 435/975 Kit
• CPC Class Codes: Y10S 436/806 Electrical property or magnetic property

Abstract

A ligand in a sample can be assayed using electrochemical apparatus containing a working electrode and components comprising:

(a) the sample,

(b) a specific binding partner to the ligand, or a specific binding partner to the ligand and selected from ligand analogues and specific binding partners, and

(c) an electron-source or electron-acceptor; at least one of components (b) being labelled with an electron-transfer mediator capable of aiding the transfer of electrons between the electron-source or electron-acceptor and the working electrode by accepting electrons from the electron-source and donating them to the working electrode, or accepting the electrons from the electrode and donating them to the electron-acceptor.

The method includes the step of determining whether, and, if desired, the extent to which, the said transfer of electrons is perturbed by at least one of (i) formation of a complex of said ligand with a specific binding partner and (ii) controlled external influence which produces a perturbation of said transfer of electrons as a function of said complex formation.

Claims

1. In a homogeneous method of assaying a ligand in a sample using electrochemical apparatus containing a working electrode and components comprising:

(a) the sample, and

(b) a specific binding partner to the ligand, or a specific binding partner to the ligand and at least one further reagent selected from ligand analogues and specific binding partners to the ligand, the improvement which comprises said components including

(c) an electron-source or electron-acceptor selected from oxidoreductase enzymes in cooperation with a substrate therefor and non-enzyme electron sources;

one of components (b) being labelled with an electron-transfer mediator capable of aiding the transfer of electrons between the electron-source or electron-acceptor and the working electrode by accepting electrons from the electron-source and donating them to the working electrode, or accepting electrons from said electrode and donating them to the electron-acceptor; and

which method includes the step of determining whether the said transfer of electrons is perturbed by formation of a complex of said ligand with a specific binding partner.

2. A method as claimed in claim 1 wherein component (c) is an oxidoreductase enzyme in co-operation with a substrate therefor.

3. A method as claimed in claim 1 wherein the electron-transfer media comprises ferrocene or a derivative thereof.

4. A method as claimed in claim 3 wherein the electron-transfer mediator comprises a derivative of ferrocene containing one or more side chains of the formula --CHO, --(CH₂)_n COOH or --(CH₂)_m NR¹ R² (where n and m are each from 0 to 6) and R¹ and R², which may be the same or different, each represents hydrogen or an alkyl group containing 1 to 4 carbon atoms).

5. A method as claimed in claim 1 wherein the perturbation in the transfer of electrons is determined from a perturbation in the peak current observed under the application of a preselected potential across the components.

6. A method as claimed in claim 1 wherein the ligand is an antigen or an antibody.

7. A method as claimed in claim 1, wherein the extent to which the said transfer of electrons is perturbed is determined, the extent of perturbation being an indication of the amount of ligand in the sample.

8. A method as claimed in claim 2, wherein the extent to which the said transfer of electrons is perturbed is determined, the extent of perturbation being an indication of the amount of ligand in the sample.

9. A method as claimed in claim 7, wherein said electron source is ascorbate or NADH.

10. A method as claimed in claim 1, wherein said electron source is ascorbate or NADH.

11. A kit for carrying out a method of assay as claimed in claim 1 comprising in at least one container:

(i) at least one specific binding partner to the ligand or a specific binding partner to the ligand and at least one further reagent selected from ligand analogues and specific binding partners to the ligand one of the components (i) being labelled with an electron transfer mediator, and (ii) a non-enzyme electron-source capable of donating electrons to said electron transfer mediator.

12. A kit as claimed in claim 11 which further comprises an electrochemical apparatus containing a working electrode.

13. A kit for carrying out a method of assay as claimed in claim 2 comprising in separate containers:

(i) at least one specific binding partner to the ligand or a specific binding partner to the ligand and at least one further reagent selected from ligand analogues and specific binding partners to the ligand, one of the components (i) being labelled with an electron transfer mediator; and

(ii) an oxidoreductase enzyme which in cooperation with a substrate therefor is capable of donating electrons to said electron transfer mediator or accepting electrons from said electron transfer mediator.

14. A kit as claimed in claim 13 which further comprises an electrochemical apparatus containing a working electrode.

15. A method as claimed in claim 1 wherein a specific binding partner to the ligand or a ligand analogue is immobilized on the working electrode.

16. In a heterogeneous method of assaying a ligand in a sample using electrochemical apparatus containing a working electrode and components comprising:

(a) the sample, and

(b) the specific binding partner to the ligand, or a specific binding partner to the ligand and at least one further reagent selected from ligand analogues and specific binding partners to the ligand, the improvement which comprises said components including

(c) an electron-source or electron-acceptor selected from oxidoreductase enzymes in cooperation with a substrate therefor and non-enzyme electron sources;

one of components (b) being labelled with an electron-transfer mediator capable of aiding the transfer of electrons between the electron-source or electron-acceptor

17. A method as claimed in claim 16 wherein component (c) is an oxidoreductase enzyme in cooperation with a substrate therefor.

18. A method as claimed in claim 16 wherein the electron-transfer mediator comprises ferrocene or a derivative thereof.

19. A method as claimed in claim 16 wherein the ligand is an antigen or an antibody.

20. A method as claimed in claim 16, wherein the extent to which the said transfer of electrons is perturbed is determined, the extent of perturbation being an indication of the amount of ligand in the sample.

21. A method as claimed in claim 16 wherein an unlabelled specific binding partner to the ligand under assay is immobilized on a solid support.

22. A kit for carrying out a method of assay as claimed in claim 16 comprising in at least one container:

(i) at least one specific binding partner to the ligand or a specific partner to the ligand and at least one further reagent selected from ligand analogs and specific binding partners to the ligand, one of the components (i) being labelled with an electron-transfer mediator, and (ii) a non-enzyme electron-source capable of donating electrons to said electron transfer mediator.

23. A kit for carrying out a method of assay as claimed in claim 16 comprising in separate containers:

(i) at least one specific binding partner to the ligand or a specific binding partner to the ligand and at least one further reagent selected from ligand analogs and specific binding partners to the ligand, one of the components (i) being labelled with an electron-transfer mediator; and

(ii) an oxidoreductase enzyme which in cooperation with a substrate therefor is capable of donating electrons to said electron transfer mediator or accepting electrons from said electron transfer mediator. and the working electrode by accepting electrons from the electron-source and donating them to the working electrode, or accepting electrons from said electrode and donating them to the electron-acceptor; and

which method includes the steps of (i) providing means for displacement of any mediator-labelled complex containing said ligand relative to any other mediator-labelled component, or where a mediator-labelled ligand analogue is present as component (b), providing means for displacement of any complex containing said ligand analog relative to non-complexed mediator-labelled ligand analog and (ii) determining whether the said transfer of electrons is thereby perturbed.