fig2

Figure 2. Characterization of EV-LP fusion and validation of ribonucleoprotein in mutation detection. (A-C) Electron microscope image of LP, H1975 EVs, and H441 EVs, respectively (scale bar: 100 nm); (D) Size distributions of LPs, EVs, and fused EV-LP; (E and F) Electron microscope image of EV-LP fusion; (G) Fluorescence signals of dyes-labeled LPs in the FRET assay; (H) Gel analysis of the FQ probes after cleavage (Lane 1-5: FQ probes were trans-cleaved by Cas12a in the presence of the L858R mutation; H1975 EV amount was 1 × 10⁵, 1 × 10⁶, 1 × 10⁷, 1 × 10⁸, and 1 × 10⁹, respectively. Lane 6: FQ probes were cleaved by Cas12a, which was used as a positive control. Lane 7: FQ probes were not cleaved by Cas12a in the absence of L858R mutation; H441 EV amount was 1 × 10¹⁰. Lane 8: FQ probes were not cleaved by Cas12a in the absence of EV DNA. Lane 9: FQ probes were not cleaved in the absence of Cas12a. Lane 10: FQ probes only; (I-K) Fluorescence co-localization analysis of fused EV-LP. (I) In the absence of the L858R mutation, only fluorescence signals emitted from PKH26 (red) were observed in H441 EV-LP (left; scale bar: 50 µm); (J) In the presence of the L858R mutation, dual fluorescence signals emitted from PKH26 and shredded FQ probes (green) were observed in H1975 EV-LP (right); (K) The insert shows a local zoom-in view. EV: Extracellular vesicle; FRET: förster resonant energy transfer; FQ: fluorescence-quenching; LP: liposomes.