Busch Robert T, Karim Farzia, Weis John, Sun Yvonne, Zhao Chenglong, Vasquez Erick S
Department of Chemical and Materials Engineering, Department of Electro-Optics and Photonics, Department of Biology, Integrative Science and Engineering Center, and Department of Physics, University of Dayton, 300 College Park, Dayton, Ohio 45469, United States.
ACS Omega. 2019 Sep 9;4(12):15269-15279. doi: 10.1021/acsomega.9b02276. eCollection 2019 Sep 17.
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 polyclonal antibodies bioconjugation mechanisms to AuNPs via covalent binding at different pH values, from 2 to 11, and 2-(-morpholino)ethanesulfonic acid (MES), 3-(-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.
与生物分子结合的金纳米颗粒（AuNP）已成为利用独特表面化学性质和光学特性的合适生物传感器。许多努力都集中在抗体与AuNP的生物共轭上，从而产生一种灵敏的生物共轭物来检测特定类型的细菌。不幸的是，细菌在各种恶劣环境中都能生存，因此需要了解生物共轭物的稳定性。在此，我们展示了一种通过在2至11的不同pH值以及2-（-吗啉代）乙磺酸（MES）、3-（-吗啉代）丙烷磺酸、NaOH、HCl条件下通过共价结合来优化多克隆抗体与AuNP生物共轭机制的方法。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。结果表明，随着pH从7.9降至2，生物共轭过程中抗体二级结构的表观破坏增加。我们发现，随着pH从2增加到6，吸附效率（以吸附到AuNP表面的抗体百分比衡量）从17%增加到27%，然后在pH 7.9和11时分别降至8%和13%。透射电子显微镜（TEM）分析揭示了由于抗体与单个纳米颗粒结合形成的冠层组装与聚集或簇自组装成大聚集体导致的尺寸和形态变化之间的差异。冠层形成尺寸从pH 2时的3.9 nm增加到pH 6时的5.1 nm，在pH 7.9时，冠层形成不完全，而在pH 11时，形成了6.4 nm的冠层。这些结果表明，共价结合过程在较低pH值下更有效；然而，观察到抗体发生聚集和失活。我们证明，通过使用傅里叶变换红外光谱法的共价键合和抗体二级结构稳定性指标、使用TEM的形态特征和冠层形成以及生物共轭后紫外可见光谱的低波长位移，确定了pH 6和MES缓冲液类型为最佳生物共轭条件。
