pH Impacts the Orientation of Antibody Adsorbed onto Gold Nanoparticles
pH Impacts the Orientation of Antibody Adsorbed onto Gold Nanoparticles
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DOI：
10.1021/acs.bioconjchem.9b00123
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发表时间：
2019-04-01
影响因子：
4.7
通讯作者：
Driskell, Jeremy D.
中科院分区：
文献类型：
--
作者：
Ruiz, Guadalupe;Tripathi, Kiran;Driskell, Jeremy D.
利用金纳米颗粒（AuNPs）独特性质的新型检测策略为诊断测试的进步带来了巨大的希望。许多这些纳米粒子使能技术的基础是将抗体固定到AuNP表面上，以提供与靶物质的选择性结合。固定化抗体的方向和上样密度决定了F-ab的可及性，对分析性能至关重要。在这里，我们使用pH来系统地控制抗体上的表面电荷分布，并研究蛋白质电荷对金纳米颗粒吸附的影响。纳米粒子跟踪分析（NTA）被用来测量在不同的pH值的抗辣根过氧化物酶抗体（抗HRP）到金纳米粒子的吸附动力学。NTA能够通过测量作为抗体浓度的函数的AuNP的流体动力学直径（D-H）的增加来原位测量抗体在AuNP上的吸附。吸附亲和力，蛋白质层厚度，并在每个pH值的结合协同性提取的吸附等温线的最佳拟合的希尔修改的朗缪尔方程。我们的数据显示在pH 7.5、8.0和8.5下在饱和时形成单层抗体，并且在该pH范围内没有观察到抗HRP-AuNP结合常数的差异（K-d类似于11 nM）。然而，在单层覆盖下具有吸附蛋白质的AuNP的D-H的增加是pH依赖性的，对于pH 7.5、8.0和8.5，分别测量为13.2 +/- 1.1 nm、9.8 +/- 1.0 nm和7.4 +/- 0.6 nm。此外，酶介导的测定结果显示，当在pH 7.5、8.0和8.5下吸附时，固定化抗HRP抗体的抗原结合能力分别为33 +/-2%、23 +/-7%和18 +/- 2%。我们的数据证实，抗体电荷可以随pH改变，以调节和优化抗体在AuNP上的取向。这些研究描述了我们为建立设计标准所做的持续努力，以制备具有最大抗原结合活性的缀合物，从而增强生物功能纳米材料的性能。
Novel detection strategies that exploit the unique properties of gold nanoparticles (AuNPs) hold great promise for the advancement of diagnostic testing. Fundamental to many of these nanoparticle-enabled techniques is the immobilization of antibodies onto the AuNP surface to afford selective binding to target species. Orientation and loading density of the immobilized antibodies govern F-ab accessibility and are critical to the analytical performance. Here, we use pH to systematically control the surface charge distribution on an antibody and investigate the impact of protein charge on adsorption to AuNPs. Nanoparticle tracking analysis (NTA) is used to measure the adsorption dynamics of anti-horseradish peroxidase antibody (anti-HRP) onto AuNPs at different pHs. NTA enables in situ measurement of antibody adsorption on AuNP by measuring the increase in hydrodynamic diameter (D-H) of the AuNPs as a function of antibody concentration. The adsorption affinity, protein layer thickness, and binding cooperativity at each pH are extracted from the best fit of the adsorption isotherms to the Hill-modified Langmuir equation. Our data show a monolayer of antibody is formed at saturation at pHs 7.5, 8.0, and 8.5, and no difference in anti-HRP-AuNP binding constants is observed in this pH range (K-d similar to 11 nM). However, the increase in D-H of the AuNPs with adsorbed protein at monolayer coverage is pH-dependent, measuring 13.2 +/- 1.1 nm, 9.8 +/- 1.0 nm, and 7.4 +/- 0.6 nm for pHs 7.5, 8.0, and 8.5, respectively. Moreover, results of an enzyme-mediated assay reveal the antigen-binding capacity of the immobilized anti-HRP antibody is 33 +/- 2%, 23 +/- 7%, and 18 +/- 2% when adsorbed at pHs 7.5, 8.0, and 8.5, respectively. Our data confirm that antibody charge can be altered with pH to modulate and optimize antibody orientation on AuNP. These studies describe our continued efforts to establish design criteria to prepare conjugates with maximum antigen-binding activity that will enhance the performance of biofunctional nanomaterials.