基于拟糖蛋白的电化学阻抗生物传感器用于糖与蛋白质相互作用的研究

摘要

以D-甘露糖和伴刀豆球蛋白(ConA)的相互作用为研究对象,利用美拉德反应将D-甘露糖共价结合到负载蛋白牛血清蛋白(BSA)的表面形成拟糖蛋白,然后将拟糖蛋白固定到玻碳电极表面,以拟糖蛋白表面的D-甘露糖为分子识别物质,构建了检测伴刀豆球蛋白(ConA)的电化学阻抗传感器.拟糖蛋白制备过程简单,D-甘露糖负载量大,在空间中提供多个结合位点,因此能与ConA形成多价复合物,提高了传感器的灵敏度.该传感器的响应值与ConA浓度的对数在5.0×10-11～5.0×10-9 mol/L之间呈良好的线性关系,检出限为1.7×10-11 mol/L,D-甘露糖和ConA之间的结合常数为2.6×106 L/mol.该方法简单,可适用于不同糖和蛋白质相互作用的研究,为构建高灵敏度的电化学阻抗传感器提供了新思路.%The interaction between D-mannose and concanavalin (ConA) was always used as a model target for protein-carbohydrate interactions.A simple,sensitive electrochemical impedance spectroscopy (EIS) biosensor was herein reported for the detection of ConA based on neoglycoproteins.D-mannose was covalently attached to a carrier protein,bovine serum albumin (BSA) to generate multivalent neoglycoproteins by the Maillard reaction.The biosensor was designed by immobilizing the neoglycoproteins on the surface of glassy carbon electrode,whose structures were recognized by concanavalinA (ConA).Since the neoglycoconjugates were easily prepared,a large number of D-mannoses could format multivalent complexs with ConA.The biosensor was highly sensitive.The increase of the electron transfer resistance of the biosensor was in logarithmically direct proportion to the concentration of ConA in the range ofS.0 × 10-11-5.0 × 10-9 mol/L.The detection limit for ConA was 1.7 × 10-11 mol/L,with an affinity constant Ka of 2.6 × 106 L/mol.The method developed in this study may be a promising approach,and could be extended to the design of an EIS biosensor for highly sensitive and rapid detection of other desired carbohydrate-protein interactions.