Ultrasensitive detection of tumor-derived small extracellular vesicles based on nonlinear hybridization chain reaction fluorescence signal amplification and immunomagnetic separation

Abstract

Small extracellular vesicles (sEVs) have attracted wide attention as a promising tumor biomarker. However, sensitive and selective detection of sEVs is challenging due to the low levels of sEVs in the early stage of cancers. Herein, a novel fluorescent sensor was developed for the detection of sEVs with high sensitivity and selectivity based on nonlinear hybridization chain reaction (nHCR) signal amplification and immunomagnetic separation. Firstly, sEVs were captured and enriched by CD63 antibody conjugated magnetic beads via antibody–antigen reactions. Then, cholesterol-modified DNA probes were anchored spontaneously on lipid membranes of sEVs through efficient hydrophobic interactions between the cholesterol moiety and the phospholipid bilayer of sEVs. The simultaneous recognition of the transmembrane protein and the phospholipid bilayer structure of the sEVs could effectively eliminate interferences from free proteins. The sticky ends of the cholesterol-modified DNA probes acted as the initiator to trigger nHCR to form a hyperbranched network of DNA structure that could recruit more fluorescent signal molecules for signal amplification. Under the optimal conditions, the nHCR-based strategy showed high sensitivity for the detection of sEVs with a limit of detection of 80 particles per μL. In addition, the as-constructed method was successfully applied for the analysis of clinical samples. It provides a sensitive and selective platform for the isolation and detection of sEVs in the early diagnosis of cancers.