IMMOBILIZATION OF PROTEINS TO A CARBOXYMETHYLDEXTRAN-MODIFIED GOLD SURFACE FOR BIOSPECIFIC INTERACTION ANALYSIS IN SURFACE-PLASMON RESONANCE SENSORS
IMMOBILIZATION OF PROTEINS TO A CARBOXYMETHYLDEXTRAN-MODIFIED GOLD SURFACE FOR BIOSPECIFIC INTERACTION ANALYSIS IN SURFACE-PLASMON RESONANCE SENSORS
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DOI：
10.1016/0003-2697(91)90424-r
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发表时间：
1991-11-01
影响因子：
2.9
通讯作者：
LINDQUIST, G
中科院分区：
文献类型：
--
作者：
JOHNSSON, B;LOFAS, S;LINDQUIST, G
描述了一种用于将蛋白质快速且简单地共价固定到用于表面等离子体共振传感器的羧甲基葡聚糖修饰的金表面的方法。该方法利用羧甲基葡聚糖基质的一部分羧基通过与N-羟基琥珀酰亚胺和N-乙基-N′-（二甲基氨基丙基）碳二亚胺盐酸盐在水中反应形成N-羟基琥珀酰亚胺酯。在第二步中，蛋白质在pH值低于蛋白质等电点的低离子强度溶液中通过表面。蛋白质因此通过静电吸引力浓缩在基质中，同时与活性酯发生反应。在最后一步中，剩余的活性酯通过与乙醇胺反应转化为酰胺。该序列在包括集成的微流体盒和自动进样器的系统中自动执行。在70 fmol mm-2范围内的表面浓度下，固定蛋白质所需的典型反应时间小于30 min。可以改变参数如蛋白质浓度、蛋白质溶液离子强度、pH、反应时间和试剂浓度以控制配体的固定量。固定化配体与其生物对应物的生物特异性相互作用通过对于不同量的蛋白A对免疫球蛋白与固定化金黄色葡萄球菌蛋白A的相互作用的影响来说明。
A method for fast and simple covalent immobilization of proteins to a carboxymethyldextran-modified gold surface intended for surface plasmon resonance sensors is described. The method utilizes the formation of N-hydroxysuccinimide esters from a fraction of the carboxyl groups of the carboxymethyldextran matrix via reaction with N-hydroxysuccinimide and N-ethyl-N′-(dimethylaminopropyl) carbodiimide hydrochloride in water. In a second step the protein is passed over the surface in a solution of low ionic strength with a pH value below the isoelectric point of the protein. The protein is thereby concentrated in the matrix by electrostatic attraction forces and a simultaneous reaction with the active esters takes place. In a final step, the remaining active esters are transformed into amides via reaction with ethanolamine. This sequence is performed automatically in a system comprising an integrated microfluidic cartridge and an autosampler. Typical reaction times of less than 30 min are required for the immobilization of proteins at surface concentrations in the region of 70 fmol mm−2. Parameters such as protein concentration, protein solution ionic strength, pH, reaction times, and reagent concentration can be varied in order to control the immobilized amount of ligand. The biospecific interaction of the immobilized ligand with its biological counterpart is illustrated by the effects on the interaction of immunoglobulins with immobilized Staphylococcus aureus protein A for various amounts of protein A.