National Institute of Standards and Technology, Material Measurement Laboratory, Gaithersburg, MD 20899-8520, USA.
Nanoscale. 2012 May 21;4(10):3208-17. doi: 10.1039/c2nr30415e. Epub 2012 Apr 5.
We report on a systematic investigation of molecular conjugation of tumor necrosis factor-α (TNF) protein onto gold nanoparticles (AuNPs) and the subsequent binding behavior to its antibody (anti-TNF). We employ a combination of physical and spectroscopic characterization methods, including electrospray-differential mobility analysis, dynamic light scattering, polyacrylamide gel electrophoresis, attenuated total reflectance-Fourier transform infrared spectroscopy, fluorescence assay, and enzyme-linked immunosorbent assay. The native TNF used in this study exists in the active homotrimer configuration prior to conjugation. After binding to AuNPs, the maximum surface density of TNF is (0.09 ± 0.02) nm(-2) with a binding constant of 3 × 10(6) (mol L(-1))(-1). Dodecyl sulfate ions induce desorption of monomeric TNF from the AuNP surface, indicating a relatively weak intermolecular binding within the AuNP-bound TNF trimers. Anti-TNF binds to both TNF-conjugated and citrate-stabilized AuNPs, showing that non-specific binding is significant. Based on the number of anti-TNF molecules adsorbed, a substantially higher binding affinity was observed for the TNF-conjugated surface. The inclusion of thiolated polyethylene glycol (SH-PEG) on the AuNPs inhibits the binding of anti-TNF, and the amount of inhibition is related to the number ratio of surface bound SH-PEG to TNF and the way in which the ligands are introduced. This study highlights the challenges in quantitatively characterizing complex hybrid nanoscale conjugates, and provides insight on TNF-AuNP formation and activity.
我们报告了一种系统的研究方法，即将肿瘤坏死因子-α（TNF）蛋白分子缀合到金纳米粒子（AuNPs）上，然后研究其与抗体（抗-TNF）的结合行为。我们采用了一系列物理和光谱学表征方法，包括电喷雾-差分迁移分析、动态光散射、聚丙烯酰胺凝胶电泳、衰减全反射-傅里叶变换红外光谱、荧光测定和酶联免疫吸附测定。本研究中使用的天然 TNF 在缀合之前以活性三聚体构型存在。与 AuNPs 结合后，TNF 的最大表面密度为（0.09 ± 0.02）nm(-2)，结合常数为 3 × 10(6)（mol L(-1))(-1)。十二烷基硫酸钠离子诱导 TNF 从 AuNP 表面解吸，表明 AuNP 结合的 TNF 三聚体之间存在相对较弱的分子间结合。抗-TNF 与 TNF 缀合和柠檬酸稳定的 AuNPs 均结合，表明非特异性结合非常显著。基于吸附的抗-TNF 分子数量，观察到 TNF 缀合表面具有更高的结合亲和力。在 AuNPs 上加入巯基化聚乙二醇（SH-PEG）会抑制抗-TNF 的结合，抑制程度与结合到表面的 SH-PEG 与 TNF 的数量比以及配体的引入方式有关。本研究强调了定量表征复杂混合纳米级缀合物所面临的挑战，并提供了关于 TNF-AuNP 形成和活性的见解。
