Fig. 1: RT–RPA–CRISPR liposome design, characterization and functional evaluation. | Nature Nanotechnology

Fig. 1: RT–RPA–CRISPR liposome design, characterization and functional evaluation.

From: Liposome-mediated detection of SARS-CoV-2 RNA-positive extracellular vesicles in plasma

Fig. 1

a, Schematic of the proposed assay, indicating CD81-mediated capture of plasma EVs, their fusion with RT–RPA–CRISPR-loaded liposomes, RT–RPA-mediated target amplification, and signal generation by CRISPR-mediated cleavage of a quenched fluorescent probe in proportion to target amplicon concentration. Analysis sample types include cell culture media and plasma from non-human primate (NHP) COVID-19 disease models and patients with COVID-19. FAM, carboxyfluorescein. b, Schematic of the RT–RPA–CRISPR liposome synthesis workflow and reagents. DMPC, 1,2-dimyristoyl-sn-glycerol-3-phosphorylcholine. cf, Representative TEM images of liposomes at low (c) and high (d) magnification, and of liposome–EV fusion reactions (e,f). Two repeat experiments were performed. Scale bars, 500 nm (c), 100 nm (df). g,h, Schematic (g) and results (h) of an assay measuring the increase in FRET donor signal (588 nm) and decrease in FRET acceptor signal (665 nm) due to dye separation on labelled EVs (2 × 108) as a result of increased distance following membrane fusion after incubation with 1× (2 × 108) or 10× (2 × 109) molar ratios of unlabelled liposomes. The data represent the mean ± s.d. of three replicates. The schematics in a,b and g were created with BioRender.com.

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