Fig. 1: Schematic of the RCA–ExM method for in situ single-EV biomarker profiling.
From: Single extracellular vesicle imaging via rolling circle amplification–expansion microscopy
A Overview of the analytical workflow: 1) a diagnostic blood sample is collected and processed for analysis; 2) plasma EVs are captured on the assay chip, incubated with reagents to amplify specific EV biomarker signals in situ; then 3) EVs are released from the chip through Nb.BbvCI digestion, conjugated to hydrogel monomers, embedded in the resulting polymerized hydrogel matrix, and expanded by the osmotic swelling of this matrix. Single vesicles are then imaged to identify specific EV subpopulations, as determined by co-detection of distinct RCA and MB signal corresponding to specific biomarkers on individuals RCA-Lipo-EVs. In the signal amplification (step 1), captured EVs are labeled with protein-specific switch hairpin probes (SPs), which serve as templates for rolling circle amplification (RCA). These SP-labeled EVs are then fused with liposomes that encapsulate a miRNA-specific molecular beacon (MB) and a dual-specificity nuclease (DSN) (MB-DSN@Lipo) to generate Lipo-EV fusion vesicles that contain a signal-amplifying enzymatic circuit. Finally, RCA was initiated using the SPs on the Lipo-EV surface, producing RCA-Lipo-EVs. For the RCA/hydrogel-mediated EV expansion process (step 2), RCA products on target EVs interact with added hydrogel monomers, fixing them into the resulting hydrogel matrix, causing them to expand when this matrix is subjected to an osmotic swelling procedure, to permit single-EV resolution by fluorescence microscopy. B RCA-ExM single-EV biomarker profiles can be used to identify cancer patients and classify their immunotherapy response, while resolving multiple EV subpopulations with distinct EV biomarker profiles.
