Nienhaus G Ulrich, Maffre Pauline, Nienhaus Karin
Institute of Applied Physics and Center for Functional Nanostructures (CFN), Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany.
Methods Enzymol. 2013;519:115-37. doi: 10.1016/B978-0-12-405539-1.00004-X.
Engineered nanoparticles (NPs) have found widespread application in technology and medicine. Whenever they come in contact with a living organism, interactions take place between the surfaces of the NPs and biomatter, in particular proteins, which are currently not well understood. We have introduced fluorescence correlation spectroscopy (FCS) and dual-focus FCS (2fFCS) to measure protein adsorption onto small NPs (~10-30 nm diameter). FCS allows us to measure, with subnanometer precision and as a function of protein concentration, the increase in hydrodynamic radius of the NPs due to protein adsorption. Investigations of the adsorption of a number of important serum proteins onto negatively charged, carboxyl-functionalized NPs revealed a stepwise increase of the NP size due to protein binding, clearly indicating that a protein monolayer enshrouds the NP. Structure-based calculations of the protein surface potentials reveal positively charged patches through which the proteins interact electrostatically with the negatively charged NP surfaces; the observed protein layer thickness is correlated with the molecular dimensions of the proteins binding in suitable orientations.
工程纳米颗粒（NPs）已在技术和医学领域得到广泛应用。每当它们与生物体接触时，纳米颗粒表面与生物物质（特别是蛋白质）之间就会发生相互作用，而目前人们对这种相互作用还了解甚少。我们引入了荧光相关光谱（FCS）和双焦点FCS（2fFCS）来测量蛋白质在小纳米颗粒（直径约10 - 30纳米）上的吸附。FCS使我们能够以亚纳米精度并作为蛋白质浓度的函数，测量由于蛋白质吸附导致的纳米颗粒流体动力学半径的增加。对多种重要血清蛋白在带负电荷的羧基功能化纳米颗粒上的吸附研究表明，由于蛋白质结合，纳米颗粒尺寸呈逐步增加，这清楚地表明蛋白质单层包裹了纳米颗粒。基于结构的蛋白质表面电位计算揭示了带正电的斑块，蛋白质通过这些斑块与带负电的纳米颗粒表面发生静电相互作用；观察到的蛋白质层厚度与以合适方向结合的蛋白质的分子尺寸相关。
