Electrochemical detection of cancer cell-derived small extracellular vesicles based on three aptamer recognition and dual signal amplification

Small extracellular vesicles (sEVs) are nanoscale particles secreted by a variety of cells and are present in all body fluids. They play a crucial role in intercellular communication and are closely associated with diseases, such as cancer, making cancer cell-derived sEVs key biomarkers for liquid biopsies. However, accurately detecting sEVs is challenging due to their phenotypic diversity. In this study, we developed a bioelectrochemical platform for the highly selective detection of sEVs derived from CCRF-CEM tumor cells. The platform uses three specific proteins-CD63, PTK7, and PSMA-as inputs, with changes in the electrochemical signal of an electroactive label (ferrocene, Fc) as the output. To accomplish this, three hairpin DNA molecules were designed, each containing an aptamer sequence targeting one of these proteins. When binding occurs, these aptamer sequences induce conformational changes in the hairpin structure, causing the release of specific sequences. These released sequences trigger an endonuclease-assisted enzymatic shear cycle. The resulting sheared sequences activate a cyclic MNAzyme shearing process, leading to a decrease in the voltammetric response of Fc. Under optimal conditions, the bioelectrochemical assay showed a linear detection range of 1.0 x 102-1.0 x 107 particles/mu L, with a detection limit of 15 particles/mu L for sEVs derived from CCRF-CEM tumor cells. Notably, this assay only signals a response when all three proteins are present simultaneously, highlighting its significant potential for precise cancer detection in clinical diagnostics.