Material Measurement Laboratory, National Institute of Standards and Technology , Gaithersburg, Maryland 20899-8520, United States.
Langmuir. 2011 Mar 15;27(6):2464-77. doi: 10.1021/la104124d. Epub 2011 Feb 22.
The adsorption and conformation of bovine serum albumin (BSA) on gold nanoparticles (AuNPs) were interrogated both qualitatively and quantitatively via complementary physicochemical characterization methods. Dynamic light scattering (DLS), asymmetric-flow field flow fractionation (AFFF), fluorescence spectrometry, and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy were combined to characterize BSA-AuNP conjugates under fluid conditions, while conjugates in the aerosol state were characterized by electrospray-differential mobility analysis (ES-DMA). The presence of unbound BSA molecules interferes with DLS analysis of the conjugates, particularly as the AuNP size decreases (i.e., below 30 nm in diameter). Under conditions where the γ value is high, where γ is defined as the ratio of scattering intensity by AuNPs to the scattering intensity by unbound BSA, DLS size results are consistent with results obtained after fractionation by AFFF. Additionally, the AuNP hydrodynamic size exhibits a greater proportional increase due to BSA conjugation at pH values below 2.5 compared with less acidic pH values (3.4-7.3), corresponding with the reversibly denatured (E or F form) conformation of BSA below pH 2.5. Over the pH range from 3.4 to 7.3, the hydrodynamic size of the conjugate is nearly constant, suggesting conformational stability over this range. Because of the difference in the measurement environment, a larger increase of AuNP size is observed following BSA conjugation when measured in the wet state (i.e., by DLS and AFFF) compared to the dry state (by ES-DMA). Molecular surface density for BSA is estimated based on ES-DMA and fluorescence measurements. Results from the two techniques are consistent and similar, but slightly higher for ES-DMA, with an average adsorbate density of 0.015 nm(-2). Moreover, from the change of particle size, we determine the extent of adsorption for BSA on AuNPs using DLS and ES-DMA at 21 °C, which show that increasing the concentration of BSA increases the measured change in AuNP size. Using ES-DMA, we observe that the BSA surface density reaches 90% of saturation at a solution phase concentration between 10 and 30 μmol/L, which is roughly consistent with fluorescence and ATR-FTIR results. The equilibrium binding constant for BSA on AuNPs is calculated by applying the Langmuir equation, with resulting values ranging from 0.51 × 10(6) to 1.65 × 10(6) L/mol, suggesting a strong affinity due to bonding between the single free exterior thiol on N-form BSA (associated with a cysteine residue) and the AuNP surface. Moreover, the adsorption interaction induces a conformational change in BSA secondary structure, resulting in less α-helix content and more open structures (β-sheet, random, or expanded).
通过互补的物理化学特性分析方法，从定性和定量两个方面研究了牛血清白蛋白（BSA）在金纳米粒子（AuNPs）上的吸附和构象。动态光散射（DLS）、不对称流场流分离（AFFF）、荧光光谱和衰减全反射-傅里叶变换红外光谱（ATR-FTIR）相结合，对流体条件下的 BSA-AuNP 缀合物进行了表征，而气溶胶状态下的缀合物则通过电喷雾-差分迁移率分析（ES-DMA）进行了表征。未结合的 BSA 分子的存在会干扰缀合物的 DLS 分析，特别是当 AuNP 尺寸减小（即直径小于 30nm 时）时。在γ值较高的情况下，其中γ定义为 AuNPs 的散射强度与未结合的 BSA 的散射强度之比，DLS 尺寸结果与通过 AFFF 分离后获得的结果一致。此外，AuNP 水动力尺寸在 pH 值低于 2.5 时比在较低酸性 pH 值（3.4-7.3）时由于 BSA 缀合而表现出更大的比例增加，这与 pH 值低于 2.5 时 BSA 的可逆变性（E 或 F 构象）构象相对应。在 pH 范围从 3.4 到 7.3 时，缀合物的水动力尺寸几乎保持不变，表明在此范围内构象稳定。由于测量环境的差异，与在干燥状态（即通过 ES-DMA 和荧光测量）相比，在湿状态（即通过 DLS 和 AFFF）下测量时，BSA 缀合后观察到 AuNP 尺寸的更大增加。基于 ES-DMA 和荧光测量，估计了 BSA 的分子表面密度。两种技术的结果一致且相似，但 ES-DMA 略高，平均吸附物密度为 0.015nm(-2)。此外，根据粒径的变化，我们使用 DLS 和 ES-DMA 在 21°C 下确定 BSA 在 AuNPs 上的吸附程度，结果表明 BSA 浓度的增加会导致 AuNP 尺寸的测量变化。通过 ES-DMA，我们观察到 BSA 表面密度在 10 到 30μmol/L 的溶液相浓度之间达到 90%的饱和，这与荧光和 ATR-FTIR 结果大致一致。通过应用 Langmuir 方程计算 BSA 在 AuNPs 上的平衡结合常数，得到的值范围为 0.51×10(6)到 1.65×10(6)L/mol，表明由于 N 型 BSA（与半胱氨酸残基相关的一个胱氨酸残基）的单个游离外巯基与 AuNP 表面之间的键合，存在很强的亲和力。此外，吸附相互作用会诱导 BSA 二级结构发生构象变化，导致α-螺旋含量减少，开放结构（β-片层、无规或扩展）增多。
