Triple-color fluorescence co-localization of PD-L1-overexpressing cancer exosomes

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 × 10¹ to 7.78 × 10⁴ 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.