蛋白A与联硫基二(琥珀酰亚胺丙酸盐)修饰电化学免疫传感器测定雌二醇

摘要

将抗体结合蛋白A和交联剂联硫基二(琥珀酰亚胺丙酸盐)( DTSP)组成的骨架修饰到电极表面,再将单克隆雌二醇抗体与骨架中的蛋白A相结合,制备出电化学免疫传感器.利用样品雌二醇与辣根过氧化酶所标记的雌二醇同传感器表面抗体的竞争性结合,将该传感器用于测定溶液中样品雌二醇的浓度；利用方波伏安法监测电化学还原辣根过氧化酶催化产生的苯醌分子的电流,以还原电流为纵坐标,以雌二醇浓度为横坐标绘制标准曲线.结果表明,雌二醇浓度在50～1 500ng·L-1范围内与方波伏安还原电流呈良好的线性关系,灵敏度为0.51 μA ·ng 1 ·L,检测限为50 ng·L-1.所制备的免疫传感器良好的分析性能得益于蛋白A和DTSP所组成的骨架,该骨架能够增加免疫分子组分在电极表面的修饰量,并能够控制抗体在电极上的结合方位,使其抗原结合位点朝向电极外端,减少结合空间阻碍.%Hormone estradiol was detected at a dithiobis(succinimidyl propionate)/protein A-scaffold (DTSP│protein A-scaffold) modified electrochemical immunosensor.A monoclonal anti-estradiol capture antibody was captured by the scaffold to conduct a competitive immunoassay between sample estradiol and a horseradish peroxidase-labelled estradiol conjugate.Square wave voltammetry was used to monitor the reduction current of benzoquinone produced from the catalytic reaction of horseradish peroxidase.It has been found that the calibration plot of the reduction currents versus estradiol concentration shows a linear response within a concentration range of 50-1 500 ng ·L 1,and a sensitivity of 0.51 μA ·ng-1 ·L and a detection limit of 50 ng·L-1 are obtained.The desired characteristics of the immunosensor are attributed to the DTSP│ protein A-scaffold which is able to increase the dosage of immunization molecules on the surface of the electrode and control the binding sites of antibodies on the electrode,resulting in reduced steric hindrance while the binding sites of the antibodies are oriented opposite to the immunosensor surface.