Sequential Labeling-Assisted Precise and Multitarget Analysis of Surface Proteins on Extracellular Vesicles

Analysis of multiple surface proteins on extracellular vesicles (EVs) can reveal biological characteristics and potential therapeutic targets of cancer, particularly in highly heterogeneous breast cancer. However, due to the limited surface area of EVs, spatial hindrance remains a challenge for multiprotein assessment. Here, we present a sequential labeling-assisted electrochemical method for the precise and multitarget analysis of surface proteins on EVs, using breast cancer-related epidermal growth factor receptor and programmed death ligand-1 as examples. This sequential labeling is achieved through the use of a pair of aptamer probes functionalized with electroactive nanoparticles and an oxidative cleavage process facilitated by the bleomycin-Fe²⁺ complex. The results demonstrate that sequential labeling efficiently avoids the adverse effects of spatial hindrance, enabling accurate analysis of target surface proteins on as low as 341 particles/mL of standard EVs derived from triple-negative breast cancer (TNBC) cells. Moreover, this sequential labeling-assisted method is successfully applied to clinical blood samples from healthy individuals and TNBC patients, highlighting its potential utility in early diagnosis and disease-course monitoring of breast cancer. Therefore, this work offers a feasible tool for the precise identification and analysis of multiple surface proteins on individual EVs, providing valuable information at the protein level for the accurate diagnosis and personalized treatment of breast cancer.