CRISPR-Based Homogeneous Electrochemical Strategy for Near-Zero Background Detection of Breast Cancer Extracellular Vesicles via Fluidity-Enhanced Magnetic Capture Nanoprobe
Limin Yang, Jingang Zhang, Jing Zhang, Ting Hou, Qian Gao, Xiaojuan Liu, Feng Li
Abstract
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.