Spherical DNA Nanomotors Enable Ultrasensitive Detection of Active Enzymes in Extracellular Vesicles for Cancer Diagnosis

Abstract Enzymes encapsulated in extracellular vesicles (EVs) hold promise as biomarkers for early cancer diagnosis. However, precise measurement of their catalytic activities within EVs remains a notable challenge. Here, we report an enzymatically triggered spherical DNA nanomotor (EDM) that enables one‐pot, cascaded, and highly sensitive analysis of the activity of EV‐associated or free apurinic/apyrimidinic endonuclease 1 (APE1, a key enzyme in base excision repair) across various biological samples. The EDM capitalizes on APE1‐triggered activation of DNAzyme (Dz) and its autonomous cleavage of substrates to achieve nonlinear signal amplification. Using EDM, we demonstrate a strong correlation between APE1 activity in EVs and that of their parental cancer cells. Additionally, EV APE1 mirrors the fluctuation of cellular APE1 activity in response to chemotherapy‐induced DNA damage. In a pilot clinical study (n=63), the EDM‐based assay reveals that more than 80 % of active APE1 in serum samples is EV‐encapsulated. Notably, EV APE1 can differentiate early prostate cancer (PCa) patients from healthy donors (HDs) with an overall accuracy of 92 %, outperforming free APE1 in sera. We anticipate that EDM will become a versatile tool for quantifying EV‐associated enzymes.