Depletion-limited analyte sensing with non-equilibrium binding probes

A method of determining an initial concentration of an analyte in a sample fluid, the method comprising:

• providing a plurality of irreversible probes, the plurality of irreversible probes each including at least one binding site configured to bind to an analyte;
• providing a sample fluid including the analyte;
• introducing the sample fluid to the plurality of irreversible probes to allow binding of the analyte to at least one of the plurality of irreversible probes at the binding site, wherein the analyte binding to the at least one of the plurality of irreversible probes produces a change in a signal having a signal strength;
• depleting the analyte from being unbound to at least one of the plurality of irreversible probes to the extent that the signal strength is unchanged for a period of time;
• and
• calculating the initial concentration of the analyte in the sample fluid based on the signal strength after depleting the analyte from being unbound to at least one of the plurality of irreversible probes to the extent that the signal strength is unchanged for the period of time.