Rapid and efficient fluorescent aptasensor for PD-L1 positive extracellular vesicles isolation and analysis: EV-ANCHOR

Programmed death ligand-1 (PD-L1) positive extracellular vesicles (EVs) subpopulation have recently emerged as potent candidates for clinical cancer diagnosis and immunotherapy monitoring. However, rapid and efficient isolation and analysis of plasma PD-L1 EVs is still challenging in various cancers. Herein, we developed an integrated isolation and analysis system for the PD-L1 EV subpopulation in plasma using a fluorescent aptasensor, EV-ANCHOR, based on aptamer functionalized metal-organic frameworks and cholesterol-trigger signal amplification. First, EV-ANCHOR quickly anchors PD-L1 positive EVs with a specific aptamer and separates them without using ultracentrifugation. Then, taking advantage of cholesterol anchoring EV lipid bilayers, a lager mount of trigger strand oligonucleotides anchor on the surface of EVs, triggering the single strand's nicking activity of restriction endonucleases (Nt.BstNBI), producing amplified fluorescence signals. By integrating efficient separation and sensitive fluorescence measurement, EV-ANCHOR detects PD-L1 EVs from 2.5 x 105 to 1.0 x 108 particles/mu L with the limit of detection (LOD) reaching 9.40 x 104 particles/mu L. In clinical cohorts of cancer, EV-ANCHOR exhibits excellent clinical diagnostic efficacy in distinguishing cancer patients from healthy donors by quantifying circulating PD-L1 positive EVs. In addition, it also be proved to offer a potential tool for cancer immunotherapy efficacy monitoring in the immune checkpoint inhibitor (ICI) therapy group. Overall, EVANCHOR brings a novel approach to isolate and detect PD-L1 EVs and expands the analysis strategy for EV subpopulations, offering information for cancer diagnosis and real-time immunotherapy evaluation for various cancers.