FO-SPR biosensor calibrated with recombinant extracellular vesicles enables specific and sensitive detection directly in complex matrices.J Extracell Vesicles. 2021 02; 10(4):e12059.JE
Extracellular vesicles (EVs) have drawn huge attention for diagnosing myriad of diseases, including cancer. However, the EV detection and analyses procedures often lack much desired sample standardization. To address this, we used well-characterized recombinant EVs (rEVs) for the first time as a biological reference material in developing a fiber optic surface plasmon resonance (FO-SPR) bioassay. In this context, EV binding on the FO-SPR probes was achieved only with EV-specific antibodies (e.g. anti-CD9 and anti-CD63) but not with non-specific anti-IgG. To increase detection sensitivity, we tested six different combinations of EV-specific antibodies in a sandwich bioassay. Calibration curves were generated with two most effective combinations (anti-CD9/Banti-CD81 and anti-CD63/Banti-CD9), resulting in 103 and 104 times higher sensitivity than the EV concentration in human blood plasma from healthy or cancer patients, respectively. Additionally, by using anti-CD63/Banti-CD9, we detected rEVs spiked in cell culture medium and HEK293 endogenous EVs in the same matrix without any prior EV purification or enrichment. Lastly, we selectively captured breast cancer cell EVs spiked in blood plasma using anti-EpCAM antibody on the FO-SPR surface. The obtained results combined with FO-SPR real-time monitoring, fast response time and ease of operation, demonstrate its outstanding potential for EV quantification and analysis.