Fig. 2.
Development of smFRET based intramolecular SiMREPS approach for detection of miR-141. (A) Schematic of the optimal iSiMREPS sensor design for detecting miR-141 detection. (B) A representative single molecule kinetic trace and average dwell times of high- and low-FRET states for different sensor designs. Dwell time averages were calculated using an exponential decay fitting model. (C) A representative field of view, single-molecule kinetic trace, and Nb+d histogram with and without application of invaders and in presence of 0 and 10% formamide for detecting miR-141. (D, E) The average dwell times of high- and low-FRET states and the average number of candidate miR-141 bound molecules per FOV for different formamide v/v% conditions with a standard data acquisition of 10 s after applying optimized kinetic filtering parameters. (F) Standard curve for miR-141 showing an LOD of ~1.3 fM. Linear fits were constrained to a y-intercept of accepted counts at 0 fM and R2 = 0.9816. All data are presented as the mean ± s.d of n = ≥ 3 independent measurements. For each independent measurement, 10 FOVs were collected. Panels A, B, D and E were adapted with permission from reference 13.