SYBR Green I promotes melamine binding to poly-thymine DNA and FRET-based ratiometric sensing,Analyst

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SYBR Green I promotes melamine binding to poly-thymine DNA and FRET-based ratiometric sensing
Analyst ( IF 3.6 ) Pub Date : 2021-2-9 , DOI: 10.1039/d1an00102g
Fan He _{1,

2,

3,

4,

5} , Yudong Shen _{1,

2,

3,

4,

5} , Juewen Liu _{6,

7,

8,

9,

10}

Affiliation

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 T₃₀ DNA, respectively. In the presence of SGI, FRET-based ratiometric detection was achieved with an apparent K_d 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 Hg²⁺ 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.

中文翻译：

SYBR Green I促进三聚氰胺与聚胸腺嘧啶DNA的结合和基于FRET的比率传感

三聚氰胺与聚胸腺嘧啶（poly-T）DNA的结合已被广泛用于开发用于检测三聚氰胺的生物传感器。在这项工作中，SYBR Green I（SGI）用于染色结合反应，并使用DNA熔解实验，证实了poly-T DNA形成了分子内结合复合物以结合多个三聚氰胺分子。另外，尽管该系统对离子强度不敏感，但是更长的DNA和更低的pH有利于三聚氰胺的结合。通过分别在T ₃₀ DNA的两端标记TAMRA和Cy5荧光团来设计荧光共振能量转移（FRET）传感器。在存在SGI的情况下，基于表观K _d的基于FRET的比率检测得以实现85μM的检测限和10.7μM三聚氰胺的检测限。没有SGI，检测灵敏度降低了47倍。可以通过添加EDTA掩盖来自Hg ^2+的干扰。牛奶中三聚氰胺的检出率为87.0至101.7％。这项研究提供了基本的生化见解和用于高度敏感和鲁棒性检测三聚氰胺的比例传感器。

更新日期：2021-02-17

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