fig3

Figure 3. Characterization and optimization of Cas12a-based assay. (A and B) Quantitative fluorescence intensity detected from cleaved FQ probes in various EV-to-LP ratios. The reaction volume was 50 µL; (C) Quantitative fluorescence intensity detected from EV-to-LP ratio of 5:1 after 30-min incubation at 25, 35, and 37 °C, respectively. The reaction volume was 50 µL; (D, G, J) LOD of L858R mutation in H1975 EVs suspended in PBS; (E, H, K) LOD of L858R mutation in H1975 EVs suspended in PBS followed by immunocapture onto surfaces; (F, I, L) LOD of L858R mutation in H1975 EVs suspended in human plasma followed by immunocapture onto surfaces; (M) L858R mutation detection in patient’s plasma EVs. Thirty patients with stage-IV NSCLC and ten healthy volunteers were enrolled. The red dot line indicates the FLt of mutation detection; (N) Detection of L858R by using RT-qPCR. DNA fragments were extracted from various amounts of H1975 EVs, followed by RT-qPCR detection; (O, P, Q, R) showed a zoomed-in view of the blanks for (A) and (B). The zoomed-in view of low FL for (D-F) was presented in (P-R). FQ: Fluorescence-quenching; EV: extracellular vesicle; LOD: limit of detection; NSCLC: non-small cell lung cancer; RT-qPCR: real-time quantitative PCR; FLt: fluorescence threshold; FL: fluorescence.