Institute of Geriatrics, Affiliated Nantong Hospital of Shanghai University, The Sixth People's Hospital of Nantong, School of Medicine, Shanghai University, Nantong 226011, P. R. China.
Center for Molecular Recognition and Biosensing, Shanghai Engineering Research Center of Organ Repair, School of Life Sciences, Shanghai University, Shanghai 200444, P. R. China.
Anal Chem. 2022 Jun 21;94(24):8748-8755. doi: 10.1021/acs.analchem.2c01318. Epub 2022 Jun 1.
Molecular diagnostics devoted to discover and monitor new biomarkers is gaining increasing attention in clinical diagnosis. In this work, a programmable DNA-fueled electrochemical analysis strategy is designed for the determination of an emerging biomarker in lung cancer, PD-L1-expressing exosomes. Specifically, PD-L1-expressing exosomes are first enriched onto magnetic beads functionalized with PD-L1 antibody and are able to interact with cholesterol-modified hairpin templates. Then, programmable DNA synthesis starts from the hairpin template-triggered primer exchange reaction and generates a large number of extension products to activate the trans-cleavage activity of CRISPR-Cas12a. After that, CRISPR-Cas12a-catalyzed random cleavage boosts the degradation of methylene blue-labeled signaling strands, so electro-active methylene blue molecules can be enriched onto a cucurbit[7]uril-modified electrode for quantitative determination. Our method demonstrates high sensitivity and specificity toward electrochemical analysis of PD-L1-expressing exosomes in the range from 10 to 10 particles mL with a low detection limit of 708 particles mL. When applied to clinical samples, our method reveals an elevated level of circulating PD-L1-expressing exosomes in lung cancer patients, especially for those at the advanced stages. Therefore, our method may provide new insight into liquid biopsy for better implementation of immunotherapy in lung cancer in the future.
致力于发现和监测新型生物标志物的分子诊断在临床诊断中受到越来越多的关注。在这项工作中，设计了一种可编程 DNA 驱动的电化学分析策略，用于测定肺癌中新兴的生物标志物——表达 PD-L1 的外泌体。具体来说，首先将表达 PD-L1 的外泌体富集到用 PD-L1 抗体功能化的磁性珠上，并且能够与胆固醇修饰的发夹模板相互作用。然后，可编程 DNA 合成从发夹模板触发的引物交换反应开始，并产生大量的延伸产物以激活 CRISPR-Cas12a 的转切割活性。之后，CRISPR-Cas12a 催化的随机切割增强了亚甲蓝标记的信号链的降解，因此电活性亚甲蓝分子可以被富集到修饰有葫芦[7]脲的电极上进行定量测定。我们的方法在 10 到 10 个粒子 mL 的范围内对表达 PD-L1 的外泌体的电化学分析表现出高灵敏度和特异性，检测限低至 708 个粒子 mL。当应用于临床样本时，我们的方法显示出肺癌患者循环表达 PD-L1 的外泌体水平升高，特别是在晚期患者中。因此，我们的方法可能为液体活检提供新的见解，以更好地实施肺癌的免疫治疗。
