An orthogonal barcoding enabled smart nanodevice for highly efficient isolation and proteomic profiling of tumor-derived extracellular vesicles.

Tumor-derived extracellular vesicles (T-EVs) are small, membrane-bound particles secreted by cancer cells into the extracellular environment. These vesicles carry tumor-specific molecules, making them promising candidates as biomarkers for cancer diagnosis and monitoring. Among the various molecular components of T-EVs, such as nucleic acids and lipids, proteins stand out due to their unique characteristics and functional significance in cancer progression, diagnosis, and therapy. However, the heterogeneity of T-EVs poses a significant challenge to their effective utilization. Herein, we developed an orthogonal barcoding enabled smart nanodevice for the isolation of T-EVs and proteomic profiling. The T-EVs subpopulations were recognized from complex clinical samples, specifically through an orthogonal labeling barcode, which was created using two allosteric aptamers against the exosomal marker CD63 and the tumor marker EpCAM. Simultaneously, the labeled barcode on T-EVs initiated targeted binding with the DNA complementary tag modified mesoporous silica foam (MOSF-tag), achieving in situ exosomal protein extraction and digestion within the nanopores of the MOSF-tag. This integrated strategy not only streamlines the process by eliminating complex steps and minimizing sample loss but also significantly enhances protein identification efficiency. Compared to traditional methods for T-EVs isolation and protein digestion, the smart nanodevice has demonstrated a remarkable improvement in the detection of exosomal proteins and specific proteins from the cell culture medium. As a proof of concept, we applied this strategy to serum samples from prostate cancer (PCa) patients, confirming its efficacy. A total of 832 proteins were identified, with 211 showing differential expression between patients and healthy controls. Among these, 113 proteins were significantly upregulated in the PCa group. These uniquely expressed proteins are likely associated with PCa development, invasion, and metastasis, highlighting their potential as biomarkers for the early diagnosis and prognosis of PCa in the future. This innovative approach not only advances the field of T-EVs research but also opens new avenues for the discovery of clinically relevant biomarkers in cancer.