Sensor constructs and detection methods

A method for determining the presence of an analyte in a sample, comprising the steps of:

• (a) providing a sensor construct comprising in a single molecule:
• (i) a molecular scaffold;
• (ii) a first label comprising an RET donor bound to the molecular scaffold;
• (iii) a second label comprising an RET acceptor bound to the molecular scaffold, wherein the RET acceptor is separated from the RET donor by a distance that allows a FRET interaction to occur between the RET donor and the RET acceptor;
• and
• (iv) a molecular recognition domain which specifically binds the analyte without producing a conformational change in the molecular scaffold, wherein the molecular recognition domain is bound to a component of the sensor construct selected from the group consisting of the molecular scaffold, the first label, and the second label;
• (b) contacting the sample with the sensor construct, wherein an altered FRET interaction between the RET donor and the RET acceptor occurs when the molecular recognition domain binds the analyte in the sample, thereby producing an optical signal directly in response to binding the analyte, wherein the first label, the second label, and the molecular recognition domain remain bound to the molecular scaffold as a single molecule when the molecular recognition domain binds the analyte;
• and
• (c) detecting the optical signal, thereby determining the presence of the analyte in the sample.