Succinimidyl ester surface chemistry: implications of the competition between aminolysis and hydrolysis on covalent protein immobilization.
Succinimidyl ester surface chemistry: implications of the competition between aminolysis and hydrolysis on covalent protein immobilization.
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DOI：
10.1021/la503439g
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发表时间：
2014-11-04
期刊：
影响因子：
--
通讯作者：
Shimazu K
中科院分区：
文献类型：
--
作者：
Lim CY;Owens NA;Wampler RD;Ying Y;Granger JH;Porter MD;Takahashi M;Shimazu K
n -羟基琥珀酰亚胺（NHS）酯端基团通常用于通过酰胺键将含胺的生物分子（如蛋白质和肽）共价偶联到表面。这种一步氨解通常在接近生理pH值（pH 6至pH 9）的缓冲水溶液中进行。在这些条件下，酯基的水解与酰胺化过程竞争，可能降低偶联化学的效率。本文研究了在硼酸盐缓冲液（50 mM, pH 8.50）中，利用硫代酸酯（琥珀酰丙酸酯）（DSP）在金膜上的化学吸附形成的硫代酸单层固定共价蛋白的效率。通过红外光谱（IR）、x射线光电子能谱（XPS）、电化学还原脱附和接触角测量来评估这些涂层的结构和反应性。与整个反应的简单的伪一级反应速率定律相反，基于dsp的单分子膜的水解遵循具有初始成核步骤的反应机理，表明界面反应对层中填充和缺陷的存在有很强的依赖性。这一解释用于随后的IR-ERS动力学图分析，该图给出了非均相氨水解速率常数ka，比非均相水解速率常数kh低3个数量级以上。更重要的是，这些异质动力学速率对蛋白质固定化的预测表明，在使用低蛋白质浓度和接近生理pH值的缓冲液的耦合条件下，蛋白质更有可能被物理吸附而不是共价连接。由于非共价连接蛋白可能产生的影响，这一结果对于使用NHS化学进行蛋白质固定的生物传感器至关重要。
N-Hydroxysuccinimide (NHS) ester terminal groups are commonly used to covalently couple amine-containing biomolecules (e.g., proteins and peptides) to surfaces via amide linkages. This one-step aminolysis is often performed in buffered aqueous solutions near physiological pH (pH 6 to pH 9). Under these conditions, the hydrolysis of the ester group competes with the amidization process, potentially degrading the efficiency of the coupling chemistry. The work herein examines the efficiency of covalent protein immobilization in borate buffer (50 mM, pH 8.50) using the thiolate monolayer formed by the chemisorption of dithiobis (succinimidyl propionate) (DSP) on gold films. The structure and reactivity of these adlayers are assessed via infrared spectroscopy (IR), X-ray photoelectron spectroscopy (XPS), electrochemical reductive desorption, and contact angle measurements. The hydrolysis of the DSP-based monolayer is proposed to follow a reaction mechanism with an initial nucleation step, in contrast to a simple pseudo first-order reaction rate law for the entire reaction, indicating a strong dependence of the interfacial reaction on the packing and presence of defects in the adlayer. This interpretation is used in the subsequent analysis of IR-ERS kinetic plots which give a heterogeneous aminolysis rate constant, ka, that is over 3 orders of magnitude lower than that of the heterogeneous hydrolysis rate constant, kh. More importantly, a projection of these heterogeneous kinetic rates to protein immobilization suggests that under coupling conditions in which low protein concentrations and buffers of near physiological pH are used, proteins are more likely physically adsorbed rather than covalently linked. This result is paramount for biosensors that use NHS chemistry for protein immobilization due to effects that may arise from noncovalently linked proteins.
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