Rapid and ultrasensitive fluorescent screening of extracellular vesicles using dual-aptamer recognition-triggered RCA

Abstract

Extracellular vesicles (EVs), as a crucial marker of liquid biopsy, are important for early diagnosis and prognostic assessment of cancer. However, the cumbersome sample purification process and the false-positives results from interfering proteins present challenges to the rapid and precise identification of EVs. Herein, a simple, convenient, non-purified fluorescent strategy is proposed for detecting surface proteins on EVs based on a dual-binding mode coupled with rolling circle amplification (RCA). This strategy depends on the dual-recognition of double-positive surface proteins on EVs and their corresponding aptamers, which induces the formation of the ligation probe that subsequently initiates the RCA reaction, leading to amplified fluorescent signal output. Under the optimal reaction conditions, this method demonstrates high specificity and sensitivity, exhibiting a detection range of 1 × 10³ to 1 × 10¹⁰ particles per mL with improved detection limit as low as 7.64 × 10² particles per mL. On the basis of this dual-binding mode, our strategy enables the occurrence of false-positive results arising from interfering proteins in the serum to be avoided. Furthermore, this strategy requires only a simple centrifugation process and omits complex purification processes, providing the benefits of simplified operation, short analysis time, and efficient preservation of protein activity. Therefore, with the superiority of rapidness, accuracy, simplicity, sensitivity, and specificity, this strategy holds the potential to provide a valuable tool for EV identification, and the related early diagnosis of cancer.