CRISPR-Based Homogeneous Electrochemical Strategy for Near-Zero Background Detection of Breast Cancer Extracellular Vesicles via Fluidity-Enhanced Magnetic Capture Nanoprobe

Precise identification and analysis of multiple protein biomarkers on the surface of breast cancer cell-derived extracellular vesicles (BC-EVs) are of great significance for noninvasive diagnosis of the breast cancer subtypes, but it remains a major challenge owing to their high heterogeneity and low abundance. Herein, we established a CRISPR-based homogeneous electrochemical strategy for near-zero background and ultrasensitive detection of BC-EVs. To realize the high-performance capture and isolation of BC-EVs, fluidity-enhanced magnetic nanoprobes were facilely prepared. After capturing BC-EVs, the AND logic gate-based catalytic hairpin assembly (CHA) and the trans-cleavage activity of CRISPR-Cas12a against the magnetic signal nanoprobes were triggered successively, generating a significant electrochemical signal. Notably, the as-developed metal-mediated magnetic signal nanoprobes could efficiently decrease the background signal by magnetic separation, endowing the method with a high signal-to-noise ratio. Consequently, by ingeniously integrating DNA logic gate-based CRISPR-CHA signal amplification with dual magnetic nanoprobes in a homogeneous electrochemical strategy, precise identification and ultrasensitive detection of BC-EVs was successfully achieved through simultaneous and specific recognition of dual protein markers on the BC-EVs surface. More importantly, this approach could effectively discriminate specific subgroups of BC-EVs in clinical serum samples, which may provide great opportunities for the accurate diagnosis and prognosis evaluation of breast cancer in a noninvasive manner.