He Fan, Shen Yudong, Liu Juewen
College of Food Science, Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural University, Guangzhou 510642, Guangdong, P. R. China.
Department of Chemistry, Waterloo Institute for Nanotechnology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.
Analyst. 2021 Mar 7;146(5):1642-1649. doi: 10.1039/d1an00102g. Epub 2021 Feb 17.
Melamine binding to polythymine (poly-T) DNA has been widely used to develop biosensors for the detection of melamine. In this work, SYBR Green I (SGI) was used to stain the binding reaction, and using DNA melting experiments, it was confirmed that poly-T DNA formed intramolecular binding complexes to bind multiple melamine molecules. In addition, while this system was insensitive to ionic strength, longer DNA and lower pH favored the binding of melamine. A fluorescence resonance energy transfer (FRET) sensor was designed by labeling a TAMRA and a Cy5 fluorophore on the two ends of T DNA, respectively. In the presence of SGI, FRET-based ratiometric detection was achieved with an apparent K of 85 μM and a limit of detection of 10.7 μM melamine. Without SGI, the sensitivity of detection was decreased by 47-fold. Interference from Hg can be masked by adding EDTA. Detection of melamine in milk was achieved with recovery rates from 87.0 to 101.7%. This study has provided both basic biochemical insights and a ratiometric sensor for highly sensitive and robust detection of melamine.
三聚氰胺与聚胸腺嘧啶（poly-T）DNA的结合已被广泛用于开发检测三聚氰胺的生物传感器。在这项工作中，使用SYBR Green I（SGI）对结合反应进行染色，并通过DNA解链实验证实，poly-T DNA形成分子内结合复合物以结合多个三聚氰胺分子。此外，虽然该系统对离子强度不敏感，但较长的DNA和较低的pH值有利于三聚氰胺的结合。通过分别在T DNA的两端标记TAMRA和Cy5荧光团，设计了一种荧光共振能量转移（FRET）传感器。在SGI存在的情况下，实现了基于FRET的比率检测，三聚氰胺的表观K为85 μM，检测限为10.7 μM。没有SGI时，检测灵敏度降低了47倍。添加EDTA可以掩盖汞的干扰。牛奶中三聚氰胺的回收率为87.0%至101.7%。这项研究既提供了基本的生化见解，又提供了一种用于高灵敏度和稳健检测三聚氰胺的比率传感器。
