Fig. 4.

The effects of formamide on iSiMREPS sensors for rapid detection of miRNA and ctDNA. (A) Representative single- molecule kinetic fingerprints and histograms of the number of candidate molecules per FOV showing a given number of binding and dissociation events (N_b+d) after applying thresholds for FRET intensity, S/N, and dwell times of bound and unbound states in presence of 5 pM miR-141, without (top) and with 10% (v/v) formamide (bottom). The Q₈C₆QS₁₈CS₃ sensor as depicted in Figure 2A was used for this study and pre-treated with a capture invader (5´TCCGCCATATAACACTGTCTG 3´) and query invader (5´GAGTGTCCCGCGGCCCAGGA 3´) to remove non-target-bound sensors from coverslip before imaging under an objective-TIRF microscope. (B) The average dwell times for miR-141 bound state (high-FRET $\left({\overline{\tau }}_{\text{on}}\right)$ and non-bound state (low-FRET) $\left({\overline{\tau }}_{\text{off}}\right)$ as a function of formamide (0–20%, v/v). (C) The number of candidate miR-141 bound molecules per FOV as a function of formamide after applying an optimized kinetic parameter (see SI, and Table S6). (D) Representative single- molecule kinetic fingerprints and N_b+d histograms per FOV in presence of 10 pM EGFRΔ_{exon_19} without (top) and with 10% formamide (bottom). Q₈C₆QS₁₈CS₁₉ sensor and invaders as depicted in Figure 3A were used for this study. (E) The ${\overline{\tau }}_{\text{on}}$ and ${\overline{\tau }}_{\text{off}}$ for EGFRΔ_{exon_19} as a function of formamide (0–20%, v/v). (F) The number of candidate EGFRΔ_{exon_19} bound molecules per FOV as a function of formamide after applying optimized kinetic parameters (see SI, and Table S7). All data are processed at a standard data acquisition of 10s. All data are presented as mean ± s.d., where n ⩾ 3 independent experiments.