The Second Near-Infrared Window Quantum Dot-Based Fluorescence Anisotropy Probes for Separation-Free, Sensitive and Rapid Detection of Small Extracellular Vesicle PD-L1 in Plasma Samples

Studies revealed that the levels of small extracellular vesicle (sEV) programmed cell death-ligand 1 (PD-L1) may serve as a predictive biomarker for the clinical responses to immunotherapy. While, the currently available enzyme-linked immunosorbent assay (ELISA)-based quantitative analysis of sEV PD-L1 is tedious and time-consuming and have low sensitivity. Additionally, to avoid the interference signal from free forms of PD-L1, sEVs were isolated from plasma by ultracentrifugation with low recovery and rigorous steps. Since the mass and volume of PD-L1-positive sEVs were significantly larger than free forms of PD-L1, we developed a free-separation anisotropy probe (FSAP) for sensitive and rapid quantitative detection of sEV PD-L1 in human plasma samples. It could avoid the interference of free forms of PD-L1 and shorten the whole process to 30 min. The level of PD-L1-positive sEVs was quantified by FSAP with high sensitivity and low detection limit (375 sEVs/µL). We studied plasma samples from 15 oral squamous cell carcinoma patients (OSCCP) and 15 healthy donors (HD), we found that cancer patients have higher levels of sEVs than healthy donors. Most importantly, FSAP can detect amounts of PD-L1-positive sEVs in plasma samples that diluted more than 70 times and distinguish OSCC patients from healthy donors well.