Key Laboratory of Chem-Biosensing, Anhui Province, PR China; Key Laboratory of Functional Molecular Solids, Anhui Province, PR China; College of Chemistry and Materials Science, Center for Nano Science and Technology, Anhui Normal University, Wuhu 241000, PR China.
Key Laboratory of Chem-Biosensing, Anhui Province, PR China; Key Laboratory of Functional Molecular Solids, Anhui Province, PR China; College of Chemistry and Materials Science, Center for Nano Science and Technology, Anhui Normal University, Wuhu 241000, PR China; State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha 410082, PR China.
Talanta. 2018 Aug 1;185:106-112. doi: 10.1016/j.talanta.2018.02.102. Epub 2018 Feb 27.
This paper reported a novel colorimetric assay strategy for avidin and biotin interactions based on terminal protection of the biotinylated single-stranded DNA and the surface plasmon resonance adsorption of gold nanoparticles (AuNPs). In this assay, it was firstly found that biotin-ssDNA specifically bound to the target protein avidin with strong affinity could be protected from hydrolysis by exonuclease I (Exo I). Furthermore, a colorimetric strategy was designed for the detection of avidin and biotin interactions. In the process, in the presence of avidin, the interaction of avidin and biotin protected the digestion of Exo I towards the biotin-ssDNA. The biotin-ssDNA with negatively charged would attach to the surface of AuNPs with positively charge in high salt solution through electrostatic interactions, which prevented AuNPs to aggregate. With the increased addition of avidin, the absorbance of AuNPs in 520 nm increased gradually and the color showed gradually wine red. By taking advantage of terminal protection, the developed strategy could offer high sensitivity for detecting small molecule-protein interactions. The results revealed that the developed strategy was highly sensitive for detecting avidin in the concentration ranging from 0.01 to 0.2 μg/mL with the detection limit of 4 × 10 μg/mL.The developed assay also showed highly specific, cost-efficient and convenient. Moreover, this strategy only required labeling the small molecule on a single-stranded DNA, circumventing protein modifications that might be harmful for activity. In view of these advantages, this new colorimetric method could have potential to become a universal, sensitive, and selective platform for detection of small molecule-protein interactions.
本文报道了一种基于末端保护生物素化单链 DNA 和金纳米粒子（AuNPs）表面等离子体共振吸附的新型生物素与亲和素相互作用的比色分析策略。在该测定法中，首次发现生物素-ssDNA 特异性结合具有强亲和力的靶蛋白亲和素有能够被核酸外切酶 I（Exo I）水解保护。此外，设计了一种用于检测亲和素和生物素相互作用的比色策略。在此过程中，在存在亲和素的情况下，亲和素与生物素的相互作用保护 Exo I 对生物素-ssDNA 的消化。带负电荷的生物素-ssDNA 通过静电相互作用在高盐溶液中与带正电荷的 AuNPs 结合，从而阻止 AuNPs 聚集。随着亲和素的增加，AuNPs 在 520nm 处的吸光度逐渐增加，颜色逐渐变为酒红色。通过末端保护，所开发的策略可以提供用于检测小分子-蛋白相互作用的高灵敏度。结果表明，该方法对浓度范围为 0.01 至 0.2μg/mL 的亲和素有很高的灵敏度，检测限为 4×10μg/mL。所开发的测定法还具有高度特异性、成本效益高和方便等特点。此外，该策略仅需要在单链 DNA 上标记小分子，避免了可能对活性有害的蛋白质修饰。鉴于这些优点，这种新的比色方法有可能成为一种通用的、敏感的、选择性的小分子-蛋白相互作用检测平台。
