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Article Abstract
Protein biomarkers on breast cancer-derived small extracellular vesicles (BC-sEVs) hold great promise in liquid biopsy. However, it remains challenging due to their inherent heterogeneity and low abundance. Herein, we developed an AND logic gate-based DNA cascade signal amplification strategy, termed Alternating Primer Exchange Reaction-activated Cas12a (Alt-PER-Cas12a), for the ultrasensitive detection of BC-sEVs in clinic samples. This dual-protein recognition system employs EpCAM/MUC1-specific capture probes to release two DNA hairpins (Hep and Hmu) as AND gate inputs in Alt-PER. The corresponding Hep and Hmu hairpins can initiate the Alt-PER with a large amount of primers to generate long single-stranded DNA products with alternating repeat units. Each repeating unit serves as a CRISPR activator, inducing the trans-cleavage activity of Cas12a and enabling cascade signal amplification. The as-constructed strategy exhibits excellent sensitivity with LOD of 2.6 × 10 particles/mL. It has been successfully used to discriminate breast cancer patients from healthy donors (AUC = 0.992) in clinical validation, and shows great potential for liquid biopsy.
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| http://dx.doi.org/10.1016/j.talanta.2025.128411 | DOI Listing |
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