Characterizing the Surface Coverage of Protein-Gold Nanoparticle Bioconjugates.
Characterizing the Surface Coverage of Protein-Gold Nanoparticle Bioconjugates.
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DOI：
10.1021/acs.bioconjchem.8b00366
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发表时间：
2018-08-15
影响因子：
4.7
通讯作者：
Dyer RB
中科院分区：
文献类型：
--
作者：
Kozlowski R;Ragupathi A;Dyer RB
功能性酶-纳米颗粒生物缀合物在生物医学和生物技术应用中越来越重要，例如药物递送和生物传感。这种生物缀合物的功能的优化需要对其结构和活性进行仔细的控制和表征，但目前的方法不足以实现这一目的。现有方法的一个关键缺点是缺乏一种精确的方法来定量低（单层）表面覆盖的生物缀合物的蛋白质含量。在这项研究中，开发了一种集成的表征方法，用于蛋白质-金纳米颗粒（AuNP）生物缀合物，重点是蛋白质在AuNP上的位点特异性附着和表面覆盖。二氢叶酸还原酶的含单半胱氨酸的突变体共价连接到直径为5、15和30 nm的金纳米颗粒上，提供了一系列的表面曲率。研究了与蛋白质表面不同区域的位点特异性连接，包括与柔性环和刚性α螺旋的连接。表征方法包括SDS-PAGE、紫外-可见分光光度法、动态光散射和一种新的基于荧光的方法，用于准确测定AuNP上的低蛋白浓度。准确测定缀合物样品中的蛋白质和AuNP浓度允许计算表面覆盖度。我们发现，表面覆盖率与表面曲率的金纳米粒子，与较高的表面覆盖率观察到较高的表面曲率。这些表征方法的组合对于理解蛋白质-AuNP生物缀合物的功能，特别是酶活性是重要的。
Functional enzyme—nanoparticle bioconjugates are increasingly important in biomedical and biotechnology applications such as drug delivery and biosensing. Optimization of the function of such bioconjugates requires careful control and characterization of their structures and activity, but current methods are inadequate for this purpose. A key shortcoming of existing approaches is the lack of an accurate method for quantitating protein content of bioconjugates for low (monolayer) surface coverages. In this study, an integrated characterization methodology for protein—gold nanoparticle (AuNP) bioconjugates is developed, with a focus on site-specific attachment and surface coverage of protein on AuNPs. Single-cysteine-containing mutants of dihydrofolate reductase are covalently attached to AuNPs with diameters of 5, 15, and 30 nm, providing a range of surface curvature. Site-specific attachment to different regions of the protein surface is investigated, including attachment to a flexible loop versus a rigid α helix. Characterization methods include SDS-PAGE, UV—vis spectrophotometry, dynamic light scattering, and a novel fluorescence-based method for accurate determination of low protein concentration on AuNPs. An accurate determination of both protein and AuNP concentration in conjugate samples allows for the calculation of the surface coverage. We find that surface coverage is related to the surface curvature of the AuNP, with a higher surface coverage observed for higher surface curvature. The combination of these characterization methods is important for understanding the functionality of protein—AuNP bioconjugates, particularly enzyme activity.
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影响因子：
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通讯作者：
Brust M