Tracing Tumor-Derived Extracellular Vesicle Matrix Metalloproteinase 14 Using Dual-Target Orthogonal Barcoding-Based Microscale Thermophoretic Assays

Longitudinal monitoring of tumor progression and metastasis provides significant benefits to improve the treatment outcome of breast cancer. Matrix metalloproteinase 14 (MMP14) on extracellular vesicles (EVs) is highly relevant to tumor invasiveness, yet the sensitivity and specificity of detection are hindered by tremendous amounts of normal EVs amidst complex blood samples. Here we developed an ultrafast one-step dual-target orthogonal barcoding-based microscale thermophoretic extracellular vesicle (DOT-EV) assay to evaluate MMP14 levels on tumor-derived EVs (T-EVs). Leveraging the orthogonal barcoding of two allosteric aptamer probes targeting MMP14 and EpCAM, our assay specifically recognizes signals from MMP14 of tumor EVs and enhances the accuracy of clinical diagnosis. The microscale thermophoresis induces convection flow and accelerates DNA assembly on EVs, enabling ultrafast one-step detection of T-EV MMP14 within just 20 s, consuming 10 μL plasma samples. The T-EV MMP14 profiling on metastatic mouse models and patient samples of breast cancer demonstrated that the DOT-EV assay can monitor different development stages of the tumor and predict metastatic potential. Notably, triple-negative breast cancer was successfully differentiated from other subtypes. The DOT-EV assay offers a pioneering tool for noninvasive and precise diagnosis, progression monitoring, and metastasis prediction of cancer, showing great promise in advancing the clinical implementation of precision medicine.