Optimization and Structural Stability of Gold Nanoparticle-Antibody Bioconjugates
Optimization and Structural Stability of Gold Nanoparticle-Antibody Bioconjugates
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DOI：
10.1021/acsomega.9b02276
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发表时间：
2019-09-17
期刊：
影响因子：
4.1
通讯作者：
Vasquez, Erick S.
中科院分区：
文献类型：
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作者：
Busch, Robert T.;Karim, Farzia;Vasquez, Erick S.
与生物分子结合的金纳米颗粒（AuNPs）已经成为利用独特的表面化学和光学性质的合适的生物传感器。许多努力集中在抗体与AuNP的生物缀合上，从而产生检测特定类型细菌的敏感生物缀合物。不幸的是，细菌在各种恶劣的环境下茁壮成长，需要了解生物缀合物的稳定性。在这里，我们展示了一种用于优化单核细胞增生李斯特菌多克隆抗体与金纳米颗粒的生物缀合机制的方法，该方法通过在不同pH值（从2至11）和2-（N-吗啉代）乙磺酸（MES）、3-（N-吗啉代）丙磺酸、NaOH、HCl条件下共价结合。通过将洛伦兹曲线拟合到酰胺I和II区域，我们分析了抗体二级结构的稳定性。这表明在生物缀合期间，随着pH从7.9降低至2，抗体二级结构的表观分解增加。我们发现可变的吸附效率，测量为抗体吸附到金纳米粒子表面的百分比，从17%到27%，作为pH值从2增加到6，然后在pH值7.9和11分别降低到8%和13%。透射电子显微镜（TEM）分析揭示了由于从抗体结合到单个纳米颗粒的冠层组装与聚集或簇自组装成大聚集体的冠层组装而导致的尺寸和形态变化之间的差异。从pH 2到6，电晕层形成尺寸从3.9增加到5.1 nm，在pH 7.9时，存在不完全的电晕形成，而在pH 11时，存在6.4 nm的电晕层形成。这些结果表明共价结合过程在较低pH值下更有效;然而，观察到抗体的聚集和失活。我们证明，最佳的生物缀合条件是在pH 6和MES缓冲液类型的共价键合和抗体二级结构的稳定性，使用傅立叶变换红外，形态特征和冠层形成，使用TEM，和低波长的紫外线-可见光的生物缀合后的指标确定。
Gold nanoparticles (AuNPs) bound with biomolecules have emerged as suitable biosensors exploiting unique surface chemistries and optical properties. Many efforts have focused on antibody bioconjugation to AuNPs resulting in a sensitive bioconjugate to detect specific types of bacteria. Unfortunately, bacteria thrive under various harsh environments, and an understanding of bioconjugate stability is needed. Here, we show a method for optimizing Listeria monocytogenes polyclonal antibodies bioconjugation mechanisms to AuNPs via covalent binding at different pH values, from 2 to 11, and 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid, NaOH, HCl conditions. By fitting Lorentz curves to the amide I and II regions, we analyze the stability of the antibody secondary structure. This shows an increase in the apparent breakdown of the antibody secondary structure during bioconjugation as pH decreases from 7.9 to 2. We find variable adsorption efficiency, measured as the percentage of antibody adsorbed to the AuNP surface, from 17 to 27% as pH increases from 2 to 6 before decreasing to 8 and 13% at pH 7.9 and 11, respectively. Transmission electron microscopy (TEM) analysis reveals discrepancies between size and morphological changes due to the corona layer assembly from antibody binding to single nanoparticles versus aggregation or cluster self-assembly into large aggregates. The corona layer formation size increases from 3.9 to 5.1 nm from pH 2 to 6, at pH 7.9, there is incomplete corona formation, whereas at pH 11, there is a corona layer formed of 6.4 nm. These results indicate that the covalent binding process was more efficient at lower pH values; however, aggregation and deactivation of the antibodies were observed. We demonstrate that optimum bioconjugation condition was determined at pH 6 and MES buffer-type by indicators of covalent bonding and stability of the antibody secondary structure using Fourier transform-infrared, the morphological characteristics and corona layer formation using TEM, and low wavelength shifts of ultraviolet-visible after bioconjugation.