Label-free electrochemical biosensor

A method of measuring concentration of an analyte in a fluid, the method comprising:

• a) providing an electrochemical biosensor comprising:
• A) a sensing electrode having deposited onto its surface a polyaniline (PANI)-silver-silver oxide core-shell (Ag@AgO) nanocomposite that immobilizes a binding agent capable of specifically binding to the analyte form a binding agent-analyte complex and wherein the binding of the analyte to the binding agent causes electron transfer resistance at the sensing electrode surface thereby providing a change in the electrochemical response at the sensing electrode surface proportional to the number of binding agent-analyte complexes,
• B) a detection solution comprising a soluble redox probe, and
• C) a test equipment which measures the electrochemical response at the sensing electrode surface,
• b) contacting the sensing electrode with the fluid under conditions that allow the formation of the binding agent-analyte complexes for a period of time sufficient to allow the formation of the binding agent-analyte complexes,
• c) washing the sensing electrode with a washing solution to remove unbound and non-specifically bound chemicals from the sensing electrode,
• d) introducing a detection solution over the sensing electrode,
• e) measuring the electrochemical response at the sensing electrode surface, and
• f) calculating the concentration of the analyte in the fluid based on the electrochemical response at the sensing electrode surface.

A method of detecting the presence of a cortisol-related chronic condition in a subject, the method comprising:

• a) providing an electrochemical biosensor comprising:
• A) a sensing electrode having attached to its surface a binding agent capable of specifically binding to cortisol to form a binding agent-cortisol complex and wherein the binding of cortisol to the binding agent causes electron transfer resistance at the sensing electrode surface thereby providing a change in the electrochemical response at the sensing electrode surface proportional to the number of binding agent-cortisol complexes, B) a detection solution comprising a soluble redox probe, and
• C) a test equipment which measures the electrochemical response at the sensing electrode surface,
• b) contacting the sensing electrode with the biofluid under conditions that allow the formation of the binding agent-cortisol complexes for a period of time sufficient to allow the formation of the binding agent-cortisol complexes,
• c) washing the sensing electrode with a washing solution to remove unbound and non-specifically bound chemicals from the sensing electrode,
• d) introducing a detection solution over the sensing electrode,
• e) measuring the electrochemical response at the sensing electrode surface,
• f) calculating the concentration of cortisol in the biofluid based on the electrochemical response at the sensing electrode surface, and
• g) identifying the subject as having a cortisol-related chronic condition;
• if the concentration of cortisol in the biofluid is higher or lower than the concentration of cortisol in the biofluid of a subject not having the cortisol-related chronic condition, wherein the biofluid of the subject and the subject not having the cortisol-related chronic condition are collected at similar times of day.