Wang Liangliang, Wu Xiufeng, Chen Guanyu, Chen Yawen, Xu Lilan, Wang Jianmin, Chen Jinghua
Innovation Center for Cancer Research, Clinical Oncology School of Fujian Medical University, Fujian Cancer Hospital, Fuzhou, Fujian Province, 350014, China; Key Laboratory of Advanced Technology for Cancer Screening and Early Diagnosis, Fuzhou, Fujian Province, 350014, China.
Department of Breast Surgical Oncology, Clinical Oncology School of Fujian Medical University, Fujian Cancer Hospital, Fuzhou, Fujian Province, 350014, China.
Biosens Bioelectron. 2023 Oct 23;243:115783. doi: 10.1016/j.bios.2023.115783.
Exosomal microRNA (miRNA) are important biomarkers for liquid biopsy, and display clinical molecular signatures for cancer diagnosis. Although advanced detection methods have been established to detect exosomal miRNAs, they are faced with certain challenges. Therefore, we aimed to establish a dual amplification-based electrochemical method for detecting exosomal miRNA. This method combined a two-hairpins-based ternary hybridization structure (thTHS)-initiated single-stranded DNA (ssDNA) amplification reaction (ssDAR) and sodium perchlorate (NaClO)-assisted electrocatalytic cycle. Two DNA hairpins were designed to hybridize with target miRNA, forming thTHS. Next, ssDAR was triggered by thTHS to produce long ssDNA on magnetic beads. The long ssDNA, complementary to the signal probes, was subsequently released onto a methylene blue (MB)-labeled double-stranded DNA-modified electrode for strand displacement reaction. This led to a quantitative change in MB and a change in electrocatalytic reduction current from the electrocatalytic cycle of MB-ferricyanide. An amplified electrocatalytic reduction current was produced by adding NaClO to the electrocatalytic system, which substantially improved the signal response range and detection sensitivity. Ultimately, exosomal miRNA detection was achieved by recording changes in the electrocatalytic reduction current before and after miRNA addition. This electrochemical method exhibited a sensitive concentration response with a detection limit of 45 aM and selective miRNA recognition, and successfully used to detect exosomal miRNA derived from cells and serum. Additionally, this method exhibited better discrimination ability between patients with breast cancer (BC) and those people without BC (patients with benign breast disease and healthy people), providing a promising strategy for detecting and monitoring cancer biomarkers.
外泌体微小RNA（miRNA）是液体活检的重要生物标志物，并显示出用于癌症诊断的临床分子特征。尽管已经建立了先进的检测方法来检测外泌体miRNA，但它们仍面临一定挑战。因此，我们旨在建立一种基于双重扩增的电化学方法来检测外泌体miRNA。该方法结合了基于两个发夹的三元杂交结构（thTHS）引发的单链DNA（ssDNA）扩增反应（ssDAR）和高氯酸钠（NaClO）辅助的电催化循环。设计两个DNA发夹与靶miRNA杂交，形成thTHS。接下来，thTHS触发ssDAR在磁珠上产生长链ssDNA。与信号探针互补的长链ssDNA随后被释放到亚甲基蓝（MB）标记的双链DNA修饰电极上进行链置换反应。这导致MB的定量变化以及MB-铁氰化物电催化循环中电催化还原电流的变化。通过向电催化系统中添加NaClO产生放大的电催化还原电流，这大大提高了信号响应范围和检测灵敏度。最终，通过记录添加miRNA前后电催化还原电流的变化来实现外泌体miRNA的检测。这种电化学方法表现出灵敏的浓度响应，检测限为45 aM，并且能够选择性识别miRNA，并成功用于检测源自细胞和血清的外泌体miRNA。此外，该方法在乳腺癌（BC）患者与非BC患者（乳腺良性疾病患者和健康人）之间表现出更好的区分能力，为检测和监测癌症生物标志物提供了一种有前景的策略。
