Fluorescence Super-Resolution Imaging Chip for Gene Silencing Exosomes.

Tumor cell-derived extracellular vesicles and their cargo of bioactive substances have gradually been recognized as novel biomarkers for cancer diagnosis. Meanwhile, the PD-L1 (Programmed Death-Ligand 1) protein, as an immune checkpoint molecule, is highly expressed on certain tumor cells and holds significant potential in immune therapy. In comparison to PD-L1 monoclonal antibodies, the inhibitory effect of PD-L1 siRNA (small interfering RNA) is more advantageous. In this article, we introduced a microfluidic chip integrating cell cultivation and exosome detection modules, which were intended for the investigation of the gene silencing effect of PD-L1 siRNA. Basically, cells were first cultured with PD-L1 siRNA in the chip. Then, the secreted exosomes were detected via super-resolution imaging, to validate the inhibitory effect of siRNA on PD-L1 expression. To be specific, a "sandwich" immunological structure was employed to detect exosomes secreted from HeLa cells. Immunofluorescence staining and DNA-PAINT (DNA Point Accumulation for Imaging in Nanoscale Topography) techniques were utilized to quantitatively analyze the PD-L1 proteins on HeLa exosomes, which enabled precise structural and content analysis of the exosomes. Compared with other existing PD-L1 detection methods, the advantages of our work include, first, the integration of microfluidic chips greatly simplifying the cell culture, gene silencing, and PD-L1 detection procedures. Second, the utilization of DNA-PAINT can provide an ultra-high spatial resolution, which is beneficial for exosomes due to their small sizes. Third, qPAINT could allow quantitative detection of PD-L1 with better precision. Hence, the combination of the microfluidic chip with DNA-PAINT could provide a more powerful integrated platform for the study of PD-L1-related tumor immunotherapy.