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.

研究表明，小细胞外囊泡 (sEV) 程序性细胞死亡配体 1 (PD-L1) 的水平可作为免疫疗法临床反应的预测性生物标志物。然而，目前可用的基于酶联免疫吸附测定 (ELISA) 的 sEV PD-L1 定量分析繁琐且耗时且灵敏度低。此外，为了避免来自游离形式的 PD-L1 的干扰信号，通过低回收率和严格步骤的超速离心从血浆中分离出 sEV。由于 PD-L1 阳性 sEV 的质量和体积明显大于游离形式的 PD-L1，我们开发了一种自由分离各向异性探针 (FSAP)，用于灵敏、快速地定量检测人血浆样本中的 sEV PD-L1。避免了PD-L1游离形式的干扰，整个过程缩短至30分钟。PD-L1 阳性 sEV 的水平由具有高灵敏度和低检测限 (375 sEVs/µL) 的 FSAP 进行量化。我们研究了 15 名口腔鳞状细胞癌患者 (OSCCP) 和 15 名健康供体 (HD) 的血浆样本，我们发现癌症患者的 sEV 水平高于健康供体。最重要的是，FSAP 可以检测稀释超过 70 倍的血浆样本中 PD-L1 阳性 sEV 的数量，并能很好地区分 OSCC 患者和健康供体。我们发现癌症患者的 sEV 水平高于健康捐赠者。最重要的是，FSAP 可以检测稀释超过 70 倍的血浆样本中 PD-L1 阳性 sEV 的数量，并能很好地区分 OSCC 患者和健康供体。我们发现癌症患者的 sEV 水平高于健康捐赠者。最重要的是，FSAP 可以检测稀释超过 70 倍的血浆样本中 PD-L1 阳性 sEV 的数量，并能很好地区分 OSCC 患者和健康供体。