Wang Weiya, Peng Yuan, Wu Jin, Zhang Man, Li Qiaofeng, Zhao Zunquan, Liu Mingzhu, Wang Jiu, Cao Gaofang, Bai Jialei, Gao Zhixian
Department of Public Health and Management, Binzhou Medical University, Yantai 264003, People's Republic of China.
Tianjin Key Laboratory of Risk Assessment and Control Technology for Environment and Food Safety, Tianjin Institute of Environmental and Operational Medicine, Tianjin 300050, People's Republic of China.
Anal Chem. 2021 Mar 16;93(10):4488-4496. doi: 10.1021/acs.analchem.0c04681. Epub 2021 Mar 2.
17β-Estradiol (E2) can cause an adverse effect on the human endocrine system even at the nanomolar level. Measurements of very low levels of E2 remain a critical challenge due to insufficient sensitivity. In this study, a multistep isothermal amplification fluorescence strategy was constructed, which could realize the exponential amplification of target E2. Specifically, strand displacement reaction (SDA), rolling circle amplification (RCA), and multiprimed rolling circle amplification (MRCA) were combined in a series to quantify trace complementary strand of E2 (cDNA). The E2 aptamer and cDNA were hybridized and modified on the magnetic beads. E2 could bind to its aptamer and cause the release of the cDNA. Then, cDNA would combine with the template DNA, initiating the SDA-RCA-MRCA. The molecular beacons, possessing low background signal, whose fluorescence was quenched in the state of chain folding, could be specifically recognized by the long single-stranded DNA (L-ssDNA) generated by the multistep isothermal amplification triggered by cDNA, and then the fluorescence of the molecular beacons could be restored. Therefore, the E2 could be quantitatively detected by the recovery fluorescence intensity. The fluorescence value showed a good linear relationship with the concentration of E2 in the range of 0.001836-183.6 nM, and the limit of detection (LOD) was as low as 63.09 fM. In addition, the recovery rates of this method spiked in milk and water were 80.8-107.0%, respectively. This method has the advantage of multistep isothermal amplification to obtain abundant fluorescence signals, which may provide a new possibility for highly sensitive detection of other small-molecule targets.
17β-雌二醇（E2）即使在纳摩尔水平也会对人体内分泌系统产生不良影响。由于灵敏度不足，对极低水平的E2进行测量仍然是一项严峻挑战。在本研究中，构建了一种多步等温扩增荧光策略，可实现目标E2的指数扩增。具体而言，链置换反应（SDA）、滚环扩增（RCA）和多引物滚环扩增（MRCA）串联组合，以定量E2的微量互补链（cDNA）。E2适配体与cDNA杂交并修饰在磁珠上。E2可与其适配体结合并导致cDNA释放。然后，cDNA会与模板DNA结合，启动SDA-RCA-MRCA。分子信标具有低背景信号，其荧光在链折叠状态下被淬灭，可被由cDNA触发的多步等温扩增产生的长单链DNA（L-ssDNA）特异性识别，然后分子信标的荧光得以恢复。因此，可通过恢复的荧光强度对E2进行定量检测。荧光值与0.001836 - 183.6 nM范围内的E2浓度呈现良好的线性关系，检测限（LOD）低至63.09 fM。此外，该方法在牛奶和水中加标的回收率分别为80.8 - 107.0%。该方法具有多步等温扩增以获得丰富荧光信号的优势，可能为其他小分子靶标的高灵敏度检测提供新的可能性。
