Chowdhury Mustafa H, Lakowicz Joseph R, Ray Krishanu
Center for Fluorescence Spectroscopy, Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 725 West Lombard Street, Baltimore, MD, 21201, USA.
J Phys Chem C Nanomater Interfaces. 2011 Apr 21;115(15):7298-7308. doi: 10.1021/jp112255j.
We present a strategy for enhancing the intrinsic emission of the enzyme cofactors flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) and nicotinamide adenine dinucleotide (NADH). Ensemble studies show that silver island films (SIFs) are the optimal metal enhanced fluorescence (MEF) substrates for flavins and gave emission enhancements of over 10-fold for both FAD and FMN. A reduction in the lifetime of FAD and FMN on SIFs was also observed. Thermally evaporated aluminum films on quartz slides were found to be the optimal MEF substrate for NADH and gave a 5-fold increase in the emission intensity of NADH. We present finite-difference time-domain (FDTD) calculations that compute the enhancement in the radiated power emitting from an excited state dipole emitting in the wavelength range of NADH in close proximity to an aluminum nanoparticle, and a dipole emitting in the emission wavelength of flavins next to a silver nanoparticle. These calculations confirm that aluminum serves as the optimal MEF substrate for NADH and silver was the optimal MEF substrate for flavins. This is because the plasmon resonance properties of aluminum lie in the UV-blue regime and that of silver lie in the visible region. We also present the results of single molecule studies on FMN which show SIFs can both significantly enhance the intrinsic emission from single FMN molecules, significantly reduce their lifetimes and also significantly reduce FMN blinking. This is the first report of the observation of MEF from cofactors both at the ensemble and single molecule level. We hope this study will serve as a platform to encourage the future use of metallic nanostructures to study cofactors using their intrinsic fluorescence to directly monitor enzyme binding reactions without the need of extrinsic labeling of the molecules.
我们提出了一种增强酶辅因子黄素腺嘌呤二核苷酸（FAD）、黄素单核苷酸（FMN）和烟酰胺腺嘌呤二核苷酸（NADH）固有发射的策略。整体研究表明，银岛膜（SIFs）是用于黄素的最佳金属增强荧光（MEF）底物，FAD和FMN的发射增强均超过10倍。还观察到FAD和FMN在SIFs上的寿命缩短。发现石英载玻片上热蒸发的铝膜是用于NADH的最佳MEF底物，使NADH的发射强度增加了5倍。我们给出了有限时域差分（FDTD）计算结果，该计算计算了在靠近铝纳米颗粒处发射波长在NADH范围内的激发态偶极子辐射功率的增强，以及在银纳米颗粒旁边发射波长在黄素发射波长处的偶极子辐射功率的增强。这些计算证实铝是用于NADH的最佳MEF底物，银是用于黄素的最佳MEF底物。这是因为铝的等离子体共振特性位于紫外-蓝光区域，而银的等离子体共振特性位于可见光区域。我们还给出了对FMN的单分子研究结果，结果表明SIFs既能显著增强单个FMN分子的固有发射，又能显著缩短其寿命，还能显著减少FMN的闪烁。这是关于在整体和单分子水平上观察到辅因子的MEF的首次报告。我们希望这项研究将成为一个平台，鼓励未来使用金属纳米结构来研究辅因子，利用它们的固有荧光直接监测酶结合反应，而无需对分子进行外部标记。
