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The proteins expressed on exosomes have emerged as promising liquid-biopsy biomarkers for cancer diagnosis. However, molecular profiling of exosomal proteins remains technically challenging. Herein, we report a nanozyme-assisted immunosorbent assay (NAISA) that enables sensitive and rapid multiplex profiling of exosomal proteins. This NAISA system is based on the installation of peroxidase-like nanozymes onto the phospholipid membranes of exosomes, thus avoiding the need for post-labelling detection antibodies. The exosomal proteins are determined by a sensitive nanozyme-catalyzed colorimetric assay less than 3 h, without the need for multi-step incubation and washing operations. Using NAISA to profile exosomal proteins from different cell lines and clinical samples, we reveal that tumor-associated exosomal proteins can serve as promising biomarkers for accurate cancer diagnosis in a cooperative detection pattern. Methods: Exosomes were engineered with DSPE-PEG-SH through hydrophobic interaction, and then were assembled with gold nanoparticles (2 nm) to produce Exo@Au nanozyme. The proteins on Exo@Au could be selectively captured by their specific antibodies seeded into a 96-well plate. The immobilized Exo@Au shows peroxidase-like activity to perform colorimetric assays by reaction with 3,3',5,5'-tetramethylbenzidine (TMB) and H 2 O 2 . The protein levels of exosomes were recorded on a microplate reader. Results: The NAISA platform is capable of profiling multiple exosomal proteins from both cancer cell lines and clinical samples. The expression levels of exosomal proteins, such as CD63, CEA, GPC-3, PD-L1 and HER2, were used to classify different cancer cell lines. Moreover, the protein profiles have been applied to differentiate healthy donors, hepatitis B patients, and hepatic cell carcinoma (HCC) patients with high accuracy. Conclusion: The NAISA nanozyme was allowed to rapidly profile multiple exosomal proteins and could have great promise for early HCC diagnosis and identification of other cancer types.

Nanozyme-assisted sensitive profiling of exosomal proteins for rapid cancer diagnosis