The expression levels of small extracellular vesicles (sEVs) are closely associated with several significant biological processes, which can be used as a crucial biomarker for cancer diagnosis, such as colorectal cancer. More efforts are still necessary to amplify sEVs detection sensitivity, as their expression is minimal during the early stages of colorectal cancer. Through the integration of a catalytic assembly-triggered DNAzyme motor and gold nanoparticles (AuNP) aggregation, we have developed a triple signal amplified biosensor for the detection of sEVs. In this method, catalytic assembly triggered DNAzyme motor continuously cleaved on the hairpin probe which is fixed on the surface of AuNPs, leaving a single-strand sequence on the surface of AuNPs to induce the aggregation. This approach employs a triple signal amplification process to enhance the efficiency of the reaction and circumvent the issue of expensive and readily degradable proteases. Secondly, the signal output system is based on dynamic light scattering technology, which enables ultra-sensitive detection of sEVs with a detection limit of 3.08 particles/μL. The present strategy exhibits significant potential for the analysis of a variety of additional analytes in clinical research disciplines due to its appealing analytical capabilities.

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