Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801, USA.
Langmuir. 2010 Oct 5;26(19):15430-5. doi: 10.1021/la1021824.
Aniline-catalyzed hydrazone ligation between surface-immobilized hydrazines and aldehyde-modified antibodies is shown to be an efficient method for attaching protein capture agents to model oxide-coated biosensor substrates. Silicon photonic microring resonators are used to directly evaluate the efficiency of this surface bioconjugate reaction at various pHs and in the presence or absence of aniline as a nucleophilic catalyst. It is found that aniline significantly increases the net antibody loading for surfaces functionalized over a pH range from 4.5 to 7.4, allowing derivatization of substrates with reduced incubation time and sample consumption. This increase in antibody loading directly results in more sensitive antigen detection when functionalized microrings are employed in a label-free immunoassay. Furthermore, these experiments also reveal an interesting pH-dependent noncovalent binding trend that plays an important role in dictating the amount of antibody attached onto the substrate, highlighting the competing contributions of the bioconjugate reaction rate and the dynamic interactions that control opportunities for a solution-phase biomolecule to react with a substrate-bound reagent.
固载在表面的腙与醛修饰的抗体之间的苯胺催化腙连接反应，被证明是一种将蛋白质捕获剂连接到模型氧化物涂层生物传感器基底上的有效方法。硅光子微环谐振器被用于直接评估在不同 pH 值、有无苯胺作为亲核催化剂的情况下，这种表面生物缀合反应的效率。研究发现，在 pH 值为 4.5 到 7.4 的范围内，苯胺显著增加了表面的净抗体负载量，从而可以减少孵育时间和样品消耗来对基底进行衍生化。当功能化的微环用于无标记免疫分析时，这种抗体负载的增加直接导致了更灵敏的抗原检测。此外，这些实验还揭示了一种有趣的 pH 依赖性非共价结合趋势，这种趋势在决定附着在基底上的抗体数量方面起着重要作用，突出了生物缀合反应速率和控制溶液相生物分子与基底结合试剂反应机会的动态相互作用的竞争贡献。
