Covalent attachment and non-specific binding of reactive probe molecules onto surfaces.
Covalent attachment and non-specific binding of reactive probe molecules onto surfaces.
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反应性探针分子在表面上的共价附着和非特异性结合。
DOI：
10.1163/156856295x00517
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Griesser,HJ
中科院分区：
文献类型：
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作者：
Chatelier,RC;Gengenbach,TR;Vasic,ZR;Griesser,HJ
蛋白质共价附着到聚合物表面的策略的优化需要开发能够区分共价附着的蛋白质和非共价结合（物理吸附）的蛋白质的分析方法。我们使用溶液相衍生反应来模拟和探索蛋白质固定化反应策略，探究活性胺、羰基和酰肼基团的表面密度。研究的标记化合物是荧光素衍生物（通过吸附光谱法定量）和氟化苯基化合物（通过 XPS 定量）。对照实验包括使用没有反应基团的表面进行相同的标记反应，或者通过溶液中的共价反应封闭标记化合物的反应基团后将聚合物表面浸入标记溶液中。我们总是发现衍生化标签的物理吸附产生不可忽视的贡献。研究了多个控制表面和一种新颖的“交叉衍生化-XPS”方法，目的是改善物理吸附的补偿。我们的记录表明，即使对于小分子标记，物理吸附成分也惊人地大，再加上已知的蛋白质吸附到聚合物上的倾向，表明在通过衍生化对表面基团进行定量分析以及解释聚合物表面上的共价蛋白质固定化时要谨慎。
Optimization of strategies for the covalent attachment of proteins onto polymer surfaces requires the development of analytical methods which can differentiate between proteins that are covalently attached and those that are non-covalently bound (physisorbed). We probed for the surface density of reactive amine, carbonyl, and hydrazide groups using solution phase derivatization reactions to mimic and explore protein immobilization reaction strategies. Labeling compounds investigated were fluorescein derivatives, which were quantified by adsorption spectroscopy, and fluorinated phenyl compounds which were quantified by XPS. Control experiments consisted of performing the same labeling reactions using surfaces without reactive groups, or immersing the polymer surface into the labeling solution after blocking the reactive group of the labeling compound by a covalent reaction in solution. We always found non-negligible contributions arising from physisorption of the derivatization labels. Multiple control surfaces and a novel 'crossover derivatization-XPS' method were studied with the aim of improving compensation for physisorption. Our documentation of surprisingly large physisorption components even for small molecule labels, together with the known propensity of proteins to adsorb onto polymers, suggests caution in quantitative analysis of surface groups by derivatization, and in interpreting covalent protein immobilizations onto polymeric surfaces.