College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua 321004, China.
College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua 321004, China.
Spectrochim Acta A Mol Biomol Spectrosc. 2020 Jan 5;224:117415. doi: 10.1016/j.saa.2019.117415. Epub 2019 Jul 22.
A simple fluorescent strategy based on the formation of triple-helix molecular switch (THMS) between a signal transduction probe (STP) and an aptamer (Apt) was constructed for the determination of chloramphenicol (CAP). A weak fluorescence intensity was observed for STP solution due to the proximity of fluorophore and quencher through intramolecular DNA hybridization, causing the fluorescence quenching. The fluorescence intensity of the system was significantly enhanced after the addition of Apt. It was attributed to the formation of THMS between the Apt and STP through the Watson-Crick and Hoogsteen base pairing, resulting in the restoration of fluorescence because of the long distance between the fluorophore and quencher of STP. The fluorescence intensity of the system decreased due to the release of STP caused by the specific binding between Apt and CAP. The quantitative analysis of CAP could be achieved based on the decreased fluorescence intensity. The parameters affecting the performance of THMS including the Apt arm length, pH of buffer solution, Mg concentration and the formation time of THMS were investigated in detail. Under the optimal conditions (Apt arm length of 9 bases, pH of 6.5, 2.5 × 10 μmol L Mg, THMS formation time of 30 min), the decreased fluorescence intensity and the concentration of chloramphenicol were linear in the range of 5.0 × 10-2.0 × 10 μmol L with the correlation coefficient of 0.9963. The limit of detection was 1.2 nmol L. Subsequently, the developed method was applied to the analysis of chloramphenicol in honey sample, and the recovery was between 84.5% and 103.0% with relative standard deviation less than 4.6%.
基于信号转导探针（STP）与适体（Apt）之间形成三螺旋分子开关（THMS），构建了一种简单的荧光测定氯霉素（CAP）的策略。由于 STP 溶液中荧光团和猝灭剂通过分子内 DNA 杂交接近，导致荧光猝灭，因此观察到 STP 溶液的荧光强度较弱。加入 Apt 后，该体系的荧光强度显著增强。这归因于 Apt 与 STP 之间通过 Watson-Crick 和 Hoogsteen 碱基配对形成 THMS，由于 STP 的荧光团和猝灭剂之间的距离较远，从而恢复了荧光。由于 Apt 与 CAP 之间的特异性结合导致 STP 的释放，该体系的荧光强度降低。可以基于降低的荧光强度进行 CAP 的定量分析。详细研究了影响 THMS 性能的参数，包括 Apt 臂长、缓冲溶液的 pH 值、Mg 浓度和 THMS 的形成时间。在最佳条件下（Apt 臂长为 9 个碱基，pH 值为 6.5，2.5×10 μmol·L Mg，THMS 形成时间为 30 min），降低的荧光强度与氯霉素的浓度在 5.0×10-2.0×10 μmol·L 范围内呈线性关系，相关系数为 0.9963。检测限为 1.2 nmol·L。随后，将所开发的方法应用于蜂蜜样品中氯霉素的分析，回收率在 84.5%至 103.0%之间，相对标准偏差小于 4.6%。
