Efficient Bioconjugation of Protein Capture Agents to Biosensor Surfaces Using Aniline-Catalyzed Hydrazone Ligation
Efficient Bioconjugation of Protein Capture Agents to Biosensor Surfaces Using Aniline-Catalyzed Hydrazone Ligation
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DOI：
10.1021/la1021824
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发表时间：
2010-10-05
期刊：
影响因子：
3.9
通讯作者：
Bailey, Ryan C.
中科院分区：
文献类型：
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作者：
Byeon, Ji-Yeon;Limpoco, F. T.;Bailey, Ryan C.
苯胺催化的氢援助在表面毫米弹性化的氢氮和醛改性抗体之间结扎是将蛋白质捕获剂连接到氧化物涂层生物传感器底物上的有效方法。硅光子微孔谐振器用于直接评估该表面生物偶联反应在各种pHS上的效率，并且在存在或不存在苯胺作为亲核催化剂的情况下。发现苯胺显着增加了在4.5到7.4范围内功能化的表面的净抗体加载，从而允许衍生底物的孵育时间和样品消耗减少。当在无标记的免疫测定中采用功能化的微孔时，抗体加载的增加直接导致更灵敏的抗原检测。此外，这些实验还揭示了一个有趣的pH依赖性非共价结合趋势，在决定附着在底物上的抗体量中起着重要作用相生物分子与底物结合的试剂反应。
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 arc 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.