Methods for measuring an amount of an analyte in a complex sample

A method for detecting an amount of an analyte in a solution comprising:

• providing an assay chamber including an electrode positioned at the assay chamber and a capture molecule attached to the electrode via a linker;
• providing to the assay chamber a solution including an analyte, a binding partner of the analyte, at least one electrochemically active agent, and a detecting probe having a pH-dependent signaling tag attached thereto, the solution is in contact with the electrode and has a first pH value at which the pH-dependent signaling tag does not produce a signal and the analyte is not bound to the binding partner of the analyte;
• applying a first electrical signal to the electrode to change the pH of the solution near the surface of the electrode from the first pH value to a second pH value, while the pH of other areas of the solution remains at the first pH value;
• binding the analyte to the capture molecule and to the detecting probe at the first end of the assay chamber, wherein the pH-dependent signaling tag near the surface of the electrode produces a first signal at the second pH value and wherein the pH-dependent signaling tag in the other areas of the solution at the first pH value does not produce a signal;
• applying a second electrical signal to the electrode to change the pH of the solution near the surface of the electrode from the second pH value to a third pH value, while the pH of other areas of the solution remains at the first pH value;
• and
• measuring a second signal produced by the pH-dependent signaling tag at the third pH value near the surface of the electrode, wherein the pH-dependent signaling tag in the other areas of the solution at the first pH value does not produce a signal.

A method for detecting an amount of an analyte in a solution comprising:

• providing an assay chamber including an electrode positioned at the assay chamber and a capture molecule attached to the electrode via a linker;
• providing to the assay chamber a solution including an analyte, a binding partner of the analyte, at least one electrochemically active agent, and a detecting probe having a pH-dependent signaling tag attached thereto, the solution is in contact with the electrode and has a first pH value at which the pH-dependent signaling tag does not produce a signal and the analyte is bound to the binding partner of the analyte;
• applying a first electrical signal to the electrode to change the pH of the solution near the surface of the electrode from the first pH value to a second pH value, while the pH of other areas of the solution remains at the first pH value;
• dissociating the binding partner of the analyte from the analyte at the first end of the assay chamber at the second pH value;
• applying a second electrical signal to the electrode to change the pH of the solution near the surface of the electrode from the second pH value to the first pH value, while the pH of other areas of the solution are at the first pH value;
• binding the analyte to the capture molecule and to the detecting probe at the first end of the assay chamber;
• applying a third electrical signal to the electrode to change the pH of the solution near the surface of the electrode from the first pH value to the second pH value, while the pH of other areas of the solution remains at the first pH value;
• dissociating the binding partner of the analyte from the analyte at the first end of the assay chamber at the second pH value;
• applying a fourth electrical signal to the electrode to change the pH of the solution near the surface of the electrode from the second pH value to the first pH value, while the pH of other areas of the solution are at the first pH value;
• binding the analyte to the capture molecule and to the detecting probe at the first end of the assay chamber;
• applying a next electrical signal to the electrode to change the pH of the solution at the first end of the assay chamber from the second pH value to a third pH value, the pH-dependent signaling tag is in an on state at the third pH value;
• and
• measuring a signal produced by the pH-dependent signaling tag at the third pH value.