Fluorescence Super-Resolution Imaging Chip for Gene Silencing Exosomes

微泡 基因沉默 赫拉 细胞生物学 外体 免疫荧光 小干扰RNA RNA干扰 细胞培养 转染 细胞 小RNA 分子生物学 生物 化学 基因 核糖核酸 抗体 免疫学 生物化学 遗传学
作者
Gaoqiang Yin,Tongsheng Qi,Jinxiu Wei,Tingyu Wang,Zhuyuan Wang,Yiping Cui,Shenfei Zong
出处
期刊:Sensors [MDPI AG]
卷期号:24 (1): 173-173 被引量:1
标识
DOI:10.3390/s24010173
摘要

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.
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