Advanced Photonics Center, Southeast University, Nanjing, 210096, Jiangsu, China.
Mikrochim Acta. 2022 Apr 8;189(5):182. doi: 10.1007/s00604-022-05278-6.
Programed cell death ligand 1 (PD-L1) is a protein biomarker overexpressed on exosomes derived from tumor cells. It plays an important role in tumor diagnosis, screening, evaluation of therapeutic efficacy, and prognosis. In this study, a facile method is presented to detect PD-L1-overexpressing cancer exosomes with high specificity and sensitivity. First, gold nanospheres (GNSs) were attached to the bottom of an eight-well chambered slide by electrostatic adsorption, forming the detection substrate. Then, Cy5-labeled CD63 aptamers (i.e., the capture probes) were modified on the GNSs by Au-S bond. After adding samples containing target exosomes which were stained by membrane dyes DiI in advance, FAM-labeled PD-L1 aptamers (i.e., the immunoprobes) were added to recognize PD-L1 on the target exosomes. By triple-color fluorescence co-localization (TFC) of the Cy5, DiI, and FAM channels, highly sensitive and reliable detection of the PD-L1-overexpressing exosomes was achieved in the concentration range 7.78 × 10 to 7.78 × 10 particles/mL with a detection limit down to 6 particles/mL. The advantages of the proposed detection method include the following; first, the detection substrate is easy to prepare and convenient to clean. Second, the TFC strategy can completely exclude nonspecific reaction sites and thus significantly improves the accuracy. Such a facile and reliable detection method holds a great potential in exosome-based cancer theranostics. In this paper, we proposed a triple-color fluorescence co-localization (TFC) strategy to significantly improve the reliability of exosome detection and the detection substrate is easy to prepare and convenient to clean. In addition, the LOD is down to 6 particles/mL, which is quite low compared with other detection methods.
程序性死亡配体 1（PD-L1）是一种在肿瘤细胞衍生的外泌体上过表达的蛋白质生物标志物。它在肿瘤诊断、筛查、疗效评估和预后中起着重要作用。在这项研究中，提出了一种简便的方法来检测过表达 PD-L1 的癌症外泌体，具有高特异性和灵敏度。首先，通过静电吸附将金纳米球（GNS）附着在八孔室载玻片的底部，形成检测基底。然后，通过 Au-S 键将 Cy5 标记的 CD63 适体（即捕获探针）修饰在 GNS 上。加入预先用膜染料 DiI 染色的含有靶向外泌体的样品后，加入 FAM 标记的 PD-L1 适体（即免疫探针）以识别靶向外泌体上的 PD-L1。通过 Cy5、DiI 和 FAM 通道的三重荧光共定位（TFC），在 7.78×10 至 7.78×10 个颗粒/mL 的浓度范围内实现了对过表达 PD-L1 的外泌体的高灵敏度和可靠检测，检测限低至 6 个颗粒/mL。所提出的检测方法的优点包括：首先，检测基底易于制备且便于清洁。其次，TFC 策略可以完全排除非特异性反应位点，从而显著提高准确性。这种简便可靠的检测方法在外泌体癌症治疗中有很大的应用潜力。在本文中，我们提出了一种三重荧光共定位（TFC）策略，可显著提高外泌体检测的可靠性，且检测基底易于制备且便于清洁。此外，LOD 低至 6 个颗粒/mL，与其他检测方法相比相当低。
