What are the challenges in the development and use of FRET-based biosensors?

One challenge of FRET-based biosensors is the low signal-to-noise ratio. This makes it hard to detect and measure subtle changes in FRET signals. Creating probes labeled with FRET that specifically target a certain substance is another challenge. It's vital for the probes to be highly selective to accurately detect and measure the target. Creating a FRET-based biosensor is also a costly and lengthy process, involving making, preparing and tagging the probes. Analyzing the vast datasets FRET biosensors produce needs special software and computing power, which is another limitation. These biosensors also typically aim to identify a single substance, but many experiments require detecting multiple substances simultaneously.

Environmental factors like pH, temperature, and salt levels can alter how well the probes bind and the FRET signal they produce. Also, other substances in the sample such as self-emitting molecules or chemicals that hinder fluorescence can alter the accuracy of the measurements. FRET-based biosensors are often used in mixtures like blood or tissue, which contain many different molecules that can alter the FRET signal.