基于截短适配体的无标记结构切换荧光偏振检测氯霉素。
Label free structure-switching fluorescence polarization detection of chloramphenicol with truncated aptamer.
机构信息
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, China; School of Food Science and Technology, Jiangnan University, Wuxi, 214122, China; International Joint Laboratory on Food Safety, Jiangnan University, Wuxi, 214122, China.
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, China; School of Food Science and Technology, Jiangnan University, Wuxi, 214122, China; Three Squirrels Inc., Wuhu, 241000, China.
出版信息
Talanta. 2021 Aug 1;230:122349. doi: 10.1016/j.talanta.2021.122349. Epub 2021 Mar 23.
In this study, the original chloramphenicol aptamer containing 80 bases was truncated to 30 bases with high affinity by the SYBR Green I assay. It was found that the ionic strength and type affect the recognition of aptamers, especially magnesium ion played a vital role in the binding process. Furthermore, the binding performance of aptamer, including binding mode, key binding sites and conformational changes were further investigated by circular dichroism spectroscopy, UV-vis absorption spectrum and molecular docking. Based on these research data, we inferred that chloramphenicol bound to the minor groove region in the aptamer double helix. Finally, the optimized aptamer LLR10 was used to develop a novel label free fluorescence polarization assay to detect chloramphenicol within SYBR Green I as the source of fluorescence polarization signal. Under optimal conditions, the designed method showed a linear detection range of 0.1-10 nM with a detection limit of 0.06 nM. Additionally, the aptasensor exhibited a high accuracy to the detection of chloramphenicol in milk samples with a recovery rate from 93.7% to 98.4%. Therefore, the developed label free fluorescence polarization aptasensor provides a new idea for the rapid, reliable and sensitive detection of chloramphenicol, which can be applied to food safety control.
在这项研究中，通过 SYBR Green I 检测，将原始的含 80 个碱基的氯霉素适体截断为具有高亲和力的 30 个碱基。结果发现，离子强度和类型会影响适体的识别，尤其是镁离子在结合过程中起着至关重要的作用。此外，通过圆二色光谱、紫外-可见吸收光谱和分子对接进一步研究了适体的结合性能，包括结合模式、关键结合位点和构象变化。基于这些研究数据，我们推断氯霉素结合到适体双螺旋的小沟区域。最后，优化后的适体 LLR10 被用于开发一种新型的无标记荧光偏振测定法，以检测 SYBR Green I 中的氯霉素作为荧光偏振信号的来源。在最佳条件下，该设计方法的线性检测范围为 0.1-10 nM，检测限为 0.06 nM。此外，该适体传感器对牛奶样品中氯霉素的检测具有较高的准确性，回收率为 93.7%至 98.4%。因此，开发的无标记荧光偏振适体传感器为快速、可靠和灵敏检测氯霉素提供了新的思路，可应用于食品安全控制。
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