Protein detection method

• US 9,772,329 B2
• Filed: 01/11/2012
• Issued: 09/26/2017
• Est. Priority Date: 01/11/2011
• Status: Active Grant

• Current Assignee: The Governing Council of the University of Toronto (University of Toronto)
• Original Assignee: The Governing Council of the University of Toronto (University of Toronto)
• Inventors: Das, Jagotamoy, Kelley, Shana O.
• Primary Examiner(s): Brown, Melanie Y
• Application Number:
  US13/978,372
• Publication Number:
  US 20140005068A1
• Time in Patent Office: 2,085 Days
• Field of Search: None
• CPC Class Codes: G01N 27/3277 being a redox reaction, e.g. detection by cyclic voltammetry voltammetry per se G01N27/42, G01N27/48
• CPC Class Codes: G01N 33/54353 with ligand attached to the carrier via a chemical coupling agent coatings G01N33/54393
• CPC Class Codes: G01N 33/5438 Electrodes

Abstract

Described herein are systems and methods for detecting a target analyte in a sample with electrodes, comprising a linker and an antibody attached to the linker, and measuring an electrocatalytic signal changes generated by binding of an analyte in the sample to the antibody. Also disclosed herein are kits for electrochemical detection of protein analytes.

Claims

1. A method for electrochemical detection of cancer antigen 125 (CA-125), comprising:

providing a nanostructured microelectrode including a linker on its surface, the linker being attached to an antibody or fragment thereof capable of binding CA-125;

contacting the nanostructured microelectrode with a sample including CA-125 and with a redox reporter, wherein binding of CA-125 to the antibody or fragment thereof hinders electron transfer between the redox reporter and the nanostructured microelectrode, the nanostructured microelectrode having a diameter of about 100 microns or less, the nanostructured microelectrode having a detection limit for CA-125 from about 0.1 U/ml to about 10 U/ml;

applying a potential to the nanostructured microelectrode;

measuring, in response to the applied potential, an electrochemical signal generated by the nanostructured microelectrode; and

comparing the electrochemical signal to a control signal;

deeming a change of the electrochemical signal detected relative to the control signal as indicative of the presence of CA-125 in the sample.

2. The method of claim 1, wherein the linker includes a functional group capable of direct or indirect coupling to the antibody or fragment thereof.

3. The method of claim 1, wherein the nanostructured microelectrode is labeled with multiple copies of the antibody or fragment thereof.

4. The method of claim 1, wherein the nanostructured microelectrode is capable of interfacial electron transfer.

5. The method of claim 1, wherein the measuring step includes employing a voltammetry-based approach.

6. The method of claim 1, wherein the nanostructured microelectrode is a first nanostructured microelectrode, the linker is a first linker, the antibody or fragment thereof is a first antibody or fragment thereof, the electrochemical signal is a first electrochemical signal, and the control signal is a first control signal, further comprising:

providing a second nanostructured microelectrode including a second linker on its surface, the second linker being attached to a second antibody or fragment thereof;

contacting the second nanostructured microelectrode with the sample and with the redox reporter, the second linker being attached to the second antibody or fragment thereof and capable of binding a protein analyte of the sample, the protein analyte being different than CA-125;

applying the potential to the second nanostructured microelectrode;

measuring, in response to the applied potential, a second electrochemical signal generated by the second nanostructured microelectrode; and

comparing the second electrochemical signal to a second control signal;

deeming a change of the second electrochemical signal detected relative to the second control signal as indicative of the presence of the protein analyte in the sample.

7. The method of claim 6, wherein providing the first nanostructured electrode and providing the second nanostructured electrode includes providing a microfabricated chip having the first nanostructured electrode and the second nanostructured electrode fabricated thereon.

8. The method of claim 6, wherein the second nanostructured microelectrode is a reference control to the first nanostructured microelectrode.

10. The method of claim 1, wherein the deeming includes deeming a reduction in a signal magnitude of the electrochemical signal detected relative to the control signal as indicative of the presence of CA-125 in the sample.

12. The method of claim 6, wherein the first and second nanostructured microelectrode are on different microfabricated chips,

wherein providing the first nanostructured electrode includes providing a first microfabricated chip having the first nanostructured electrode fabricated thereon, and

wherein providing the second nanostructured electrode includes providing a second microfabricated chip having the second nanostructured electrode fabricated thereon.

13. The method of claim 6, wherein the second protein analyte includes abundant serum protein.

9. A method for monitoring progression or response in a subject having cancer by electrochemical detection of cancer antigen 125 (CA-125) in a biological sample of the subject, comprising:

providing a nanostructured microelectrode including a linker on its surface, the linker being attached to an antibody or fragment thereof;

contacting the nanostructured microelectrode with the biological sample and with a redox reporter, wherein binding of CA-125 to the antibody or fragment thereof hinders electron transfer between the redox reporter and the nanostructured microelectrode, the nanostructured microelectrode having a diameter of about 100 microns or less, the nanostructured microelectrode having a detection limit for CA-125 from about 0.1 U/ml to about 10 U/ml;

applying a potential to the nanostructured microelectrode;

measuring in response to the applied potential, an electrochemical signal generated by the nanostructured microelectrode; and

comparing the electrochemical signal to a control signal;

deeming a change of the electrochemical signal detected relative to the control signal as indicative of the presence of CA-125 in the biological sample.

11. The method of claim 9, wherein the deeming includes deeming a reduction in a signal magnitude of the electrochemical signal detected relative to the control signal as indicative of the presence of CA-125 in the sample.

14. The method of claim 9, wherein the nanostructured microelectrode is less than about 10 microns in diameter and has a detection limit for CA-125 from about 0.1 U/ml to about 1 U/ml.