Qi Yan, Yu Jiangtao, Lou Ming, Yu Yameng, Li Ruohan, Zhang Zhenhao, Dai Yuxuan, Lao Kejing, Cao Meng, Gou Xingchun
Institute of Basic and Translational Medicine & Shaanxi Key Laboratory of Brain Disorders, Xi'an Medical University, Xi'an, 710021, Shaanxi Province, PR China; Engineering Research Center of Brain Diseases Drug Development, Universities of Shaanxi Province, Xi'an Medical University, Xi'an, 710021, Shaanxi Province, PR China.
Stomatology College of Xi'an Medical University, Xi'an Medical University, Xi'an, 710021, Shaanxi Province, PR China.
Anal Chim Acta. 2025 Feb 22;1340:343659. doi: 10.1016/j.aca.2025.343659. Epub 2025 Jan 12.
Accurate quantification of microRNA (miRNA) is of great significance because it provides opportunities for the accurate early diagnosis of a series of human diseases including cancers. Currently, complicated nucleic acid amplification technologies are always required for the highly sensitive miRNA detection. The introduction of nucleic acid signal amplification coupled with various enzymes will inevitably lead to tedious work and increase the complexity of the analysis process. It is still urgently desired to develop enzyme-free yet sensitive assays that enable the sensitive analysis of miRNA in complicated biological samples.
A single microbead (MB)-based localized catalytic hairpin assembly (CHA) strategy is proposed for the sensitive analysis of microRNA (miRNA). This rationally designed CHA strategy allows target miRNA to walk only on a single MB which can create a micro-amplification zone, initiating a highly efficient localized CHA reaction, generating a large number of fluorescent DNA duplexes on the surface of single MB. The efficient localized CHA on single MB can not only greatly suppress the nonspecific reaction between two hairpin probes, thus decreasing the background signal, but also greatly enhance the brightness of MB owing to the highly-concentrated fluorescence enrichment on only one MB. Therefore, highly sensitive quantification of miRNA has been achieved by measuring the fluorescence signal on MB using a confocal fluorescence microscope. This new strategy exhibits a detection limit of 1.09 pM for let-7a detection, and enables high specificity of distinguishing homologous miRNA family members.
This is the first report by only using one single MB as a carrier to conduct localized CHA, rendering highly-concentrated fluorescence enrichment on only one MB and a dramatic increase in sensitivity. This single MB-based localized CHA strategy has been successfully applied to the accurate analysis of miRNA target in complex biological sample.
微小RNA（miRNA）的准确定量具有重要意义，因为它为包括癌症在内的一系列人类疾病的准确早期诊断提供了机会。目前，高灵敏度的miRNA检测总是需要复杂的核酸扩增技术。引入核酸信号放大并结合各种酶将不可避免地导致工作繁琐，并增加分析过程的复杂性。仍然迫切需要开发无酶但灵敏的检测方法，以便能够在复杂的生物样品中灵敏地分析miRNA。
提出了一种基于单个微珠（MB）的局部催化发夹组装（CHA）策略用于微小RNA（miRNA）的灵敏分析。这种合理设计的CHA策略允许目标miRNA仅在单个MB上移动，该MB可以创建一个微放大区，启动高效的局部CHA反应，在单个MB表面产生大量荧光DNA双链体。单个MB上的高效局部CHA不仅可以极大地抑制两个发夹探针之间的非特异性反应，从而降低背景信号，而且由于仅在一个MB上的高浓度荧光富集，还可以极大地增强MB的亮度。因此，通过使用共聚焦荧光显微镜测量MB上的荧光信号，实现了miRNA的高灵敏度定量。这种新策略对let-7a检测的检测限为1.09 pM，并能够高度特异性地区分同源miRNA家族成员。
这是首次仅使用单个MB作为载体进行局部CHA的报道，在单个MB上实现了高浓度荧光富集并显著提高了灵敏度。这种基于单个MB的局部CHA策略已成功应用于复杂生物样品中miRNA靶标的准确分析。
