QD-FRET-based biosensing of small molecule analytes using transcription factor-DNA binding

An alternative molecular recognition approach was developed for sensing small molecule analytes using the differential binding of an allosteric transcription factor (TF, specifically TetR) to its cognate DNA as the molecular recognition element coupled with fluorescent resonance energy transfer (FRET) to yield an internally calibrated optical signal transduction mechanism. Sensors were evaluated comprising Cy5-modified DNA (FRET acceptor) with either a tdTomato-TetR fusion protein (FP-TF) or quantum dot-TetR conjugate (QD-TF) as the FRET donor by measuring the ratio of acceptor and donor fluorescence intensities (F-A/F-D) with titrations of a derivative of the antibiotic tetracycline, anhydrous tetracycline (aTc). A proof-of-concept FRET-based biosensor was successfully demonstrated through the modulation of F-A/F-D signal intensities based on varying analyte concentrations. Sensor design parameters affecting overall signal-to-noise ratio and sensitivity of the sensors are also identified.