Aptamer-directed tyramide signal amplification for ultrasensitive detection of small extracellular vesicles for temporally heterogenous colorectal cancer

Abstract

Colorectal cancer (CRC) is the most common gastrointestinal neoplasm, with a significant global disease burden. Current diagnostic modalities for CRC largely involve invasive methods, limiting the early diagnosis of CRC. Tumor-derived small extracellular vesicles (sEVs) are extracellularly secreted nanoscale vesicles carrying various biomolecules with intercellular messages, which regulate tumor progression and metastasis. In this study, TEV6RB, a novel aptamer with a 37-nucleotide sequence specific to CRC sEVs, was developed via CRC Toggle sEVs Systematic Evolution of Ligands by EXponential enrichment (TEV-SELEX) using various Dukes' staging CRC cell-derived sEVs. TEV6RB, with a parallel G-quadruplex structure, exhibited high binding affinities for CRC sEVs with low nanomolar dissociation constants (K_d; 3.848, 5.904, and 5.234 nM for SW620, LS 174 T, and HT29 cells, respectively). By using TEV6RB aptamer and tyramide signal amplification, our aptamer-directed tyramide signal amplification system (APTSA) achieved ultrasensitive detection of CRC sEVs with limit-of-detection values of 3.6 × 10², 3.5 × 10³, and 8.4 × 10² particles/μL for SW620, LS 174 T, and HT29 cells, respectively. This system further validated its high specificity in distinguishing CRC sEVs from healthy and normal colon samples. Our system presents a potential liquid biopsy approach for sEVs-mediated diagnosis of CRC.