Aptamer-bivalent-cholesterol-mediated proximity entropy-driven exosomal protein reporter for tumor diagnosis.

Exosomal proteins from the parental cells are considered to be promising biomarker sets for precise tumor diagnostics and monitoring. However, the accurate quantitative analysis of low-abundance exosomal proteins remains challenging due to the heterogeneity of clinical samples. Here, we standardized the exosomal concentration with a fluorogenic membrane probe and developed an aptamer-bivalent-cholesterol-mediated Proximity Entropy-driven Exosomal Protein Reporter (PEEPR). The proposed PEEPR enables the in-situ analysis of multiple exosomal proteins by integrating bivalent cholesterol anchor (exosomal lipid bilayer) and aptamer (exosomal proteins) with a proximity entropy-driven circuit. Based on this strategy, we successfully achieved detection limits of 3.9 pg/mL exosomal GPC-3 and 3.4 pg/mL exosomal PD-L1. Notably, the standardization of exosome concentrations is designed to avoid errors due to biological heterogeneity. The results showed that evaluating the levels of exosomal GPC-3 and PD-L1 in clinical samples via this strategy could accurately differentiate healthy individuals, hepatitis B patients, and hepatocellular carcinoma patients. In summary, PEEPR is a promising clinical diagnostic strategy for the quantitative analysis of a variety of tumor-associated exosomal proteins for the precise diagnosis and personalized treatment monitoring of tumors.