Key Laboratory of Applied Surface and Colloid Chemistry of Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an, China.
Anal Chem. 2012 Mar 20;84(6):2830-6. doi: 10.1021/ac300436m. Epub 2012 Feb 29.
The magnitude of fluorescence enhancement was found to depend strongly on the distance between fluorophores and metal nanostructures in metal-enhanced fluorescence (MEF). However, the precise placement of the particle in front of the molecule with nanometer accuracy and distance control is a great challenge. We describe a method using acetylcholinesterase (AChE) to modulate the distance between a gold nanoparticle (AuNP) and the fluorophore 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) (DDAO). We found that DDAO is a reversible mixed type-I AChE inhibitor. DDAO binds to the peripheral anionic site and penetrates into the active gorge site of AChE via inhibition kinetics test and molecular docking study. The affinity ligand DDAO bound to AChE which was immobilized onto AuNPs, and its fluorescence was sharply enhanced due to MEF. The fluorescence was reduced by distance variations between the AuNP and DDAO, which resulted from other inhibitors competitively binding with AChE and partly or completely displacing DDAO. Experimental results show that changes in fluorescence intensity are related to the concentration of inhibitors present in the solution. In addition, the nanobiosensor has high sensitivity, with detection limits as low as 0.4 μM for paraoxon and 10 nM for tacrine, and also exhibits different reduction efficiencies for the two types of inhibitor. Thus, instead of an inhibition test, a new type of affinity binding-guided fluorescent nanobiosensor was fabricated to detect AChE inhibitors, determine AChE inhibitor binding mode, and screen more potent AChE inhibitors. The proposed strategy may be applied to other proteins or protein domains via changes in the affinity ligand.
荧光增强的幅度被发现强烈依赖于荧光团和金属纳米结构之间的距离，这在金属增强荧光（MEF）中是如此。然而，以纳米精度和距离控制精确地将粒子放置在分子前面是一个巨大的挑战。我们描述了一种使用乙酰胆碱酯酶（AChE）来调节金纳米粒子（AuNP）和荧光团 7-羟基-9H-（1,3-二氯-9,9-二甲基吖啶-2-酮）（DDAO）之间距离的方法。我们发现 DDAO 是一种可逆的混合型 I AChE 抑制剂。通过抑制动力学测试和分子对接研究，DDAO 通过结合到外周阴离子位点并穿透 AChE 的活性峡谷位点与 AChE 结合。亲和配体 DDAO 与固定在 AuNPs 上的 AChE 结合，由于 MEF，其荧光被急剧增强。由于其他抑制剂与 AChE 竞争性结合并部分或完全取代 DDAO，因此 AuNP 和 DDAO 之间的距离变化导致荧光减少。实验结果表明，荧光强度的变化与溶液中存在的抑制剂浓度有关。此外，该纳米生物传感器具有高灵敏度，对马拉硫磷的检测限低至 0.4 μM，对他克林的检测限低至 10 nM，并且对两种类型的抑制剂表现出不同的还原效率。因此，我们制造了一种新型的基于亲和结合的荧光纳米生物传感器来检测 AChE 抑制剂，确定 AChE 抑制剂的结合模式，并筛选更有效的 AChE 抑制剂，而不是进行抑制测试。通过改变亲和配体，该策略可能适用于其他蛋白质或蛋白质结构域。
