Simultaneous quantification of exosomal MMP14 expression and proteolysis activity on a spherical dual-probe-based fluorescent nanosensor

Among various exosomal proteins, matrix metalloproteinases (MMPs) are a family of membrane associated endopeptidases and have been considered as potential biomarkers in liquid biopsy owing to their multiple roles in pathological processes. However, the potential of MMP14 expression (MMP14-E) and MMP14 proteolytic activity (MMP14-A) in clinical diagnosis is still not clear due to the lack of sensitive and simultaneous detection techniques. Herein, we propose a fluorescent nanosensor for the simultaneous detection of MMP14-E and MMP14-A using a spherical aptamer/peptide dual-probe strategy. The aptamer and peptide probes were sequentially immobilized on Fe 3 O 4 magnetic nanoparticles coated with gold nanoparticles (m-AuNPs) using a disulfide linker. MMP14 can be specifically recognized by the aptamer, while the proteolytic-active MMP14 can cleave the peptide probe. While achieving simultaneous detection, the proposed sensor provides better analytical performances than traditional MMP14 sensors owing to the m-AuNP-based spherical dual-probe strategy. This sensor has been successfully applied for the detection of exosomal MMP14 from cell culture media and real serum samples. Levels of both MMP14-E and MMP14-A increase in serum from cancer patients, indicating their potential applications as biomarkers in liquid biopsy for disease diagnosis and real-time surveillance. A spherical dual-probe sensor was developed for the simultaneous and sensitive detection of exosomal MMP14 expression and proteolytic activity. It has been successfully used in human serum, showing its good applicability in cancer diagnosis.