Fu Yi, Zhang Jian, Lakowicz Joseph R
Center for Fluorescence Spectroscopy, Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 725 West Lombard Street, Baltimore, Maryland 21201, United States.
J Phys Chem C Nanomater Interfaces. 2011 Mar 24;115(15):7202-7208. doi: 10.1021/jp109617h.
The enzyme cofactors are intrinsically fluorescent and participate directly in the single molecule enzymology studies. Due to photobleaching, one cannot follow kinetics continuously by cofactor fluorescence for more than several minutes typically. Modification of spectral properties of fluorophores, such as the amplification of emission intensity, can be achieved through coupling with surface plasmons in close proximity to metallic nanostructures. This process, referred to as metal-enhanced fluorescence, offers promise for a range of applications, including bioassays, sensor technology, microarrays, and single-molecule studies. Here, we demonstrated up to a 100-fold increase in the emission of the single cofactors and flavoenzymes near silver nanostructures. Amplified fluorescence of different types of flavins and flavoenzymes has been interpreted by using time-resolved single molecule fluorescence data. The results show considerable promise for the studies of enzyme kinetics using the intrinsic fluorescence from the cofactors.
酶辅因子具有内在荧光，并直接参与单分子酶学研究。由于光漂白，通常情况下，通过辅因子荧光无法连续跟踪动力学超过几分钟。通过与靠近金属纳米结构的表面等离子体耦合，可以实现荧光团光谱特性的改变，如发射强度的放大。这个过程被称为金属增强荧光，在一系列应用中具有前景，包括生物测定、传感器技术、微阵列和单分子研究。在这里，我们展示了在银纳米结构附近单辅因子和黄素酶的发射增强高达100倍。通过使用时间分辨单分子荧光数据解释了不同类型黄素和黄素酶的放大荧光。结果表明，利用辅因子的内在荧光研究酶动力学具有很大的前景。
