The Study Of A Novel Renewable Surface Electrochemical Immunosensors For Estradiol

In this work, β-estradiol was chosen as the target, and a renewable surface electrochemical immunosensors was constructed by hydrophobic treating the antibody, implemented the rapid refreshment of sensitive membrane on the electrochemical immunosensors’ working electrode. Some disadvantages as the elaboration of antibody immobilization, bad reproducibility of surface membrane and difficulty in refreshment were overcome. A new method was provided for the construction of electrochemical sensors sensitive membrane and on-line detection. The study contents were listed as below:Estradiol-6-CMO was covalently attached through its carboxylic acid to the lysine groups of OVA by the mixed anhydride method, and identified by BCA protein assay kit, UV spectrometry and SDS-PAGE. The results demonstrated that molar extinction coefficient of hapten to carrier proteins was approx.93238, and exceeded hapten and carrier protein obviously. The coating antigen was synthesized successfully. Based on monoclonal antibody and coating antigen, ELISA standard curve for β-estradiol was drawn. The limit of detection (LOD) was approx.0.52ng/mL, and the CR of the other two structure analogues were both much lower than0.01%. Thus we believed that the specificity of this antibody worked well. Recovery of β-estradiol(500,50,5and0.5ng/mL) from tap water averaged between78%～134%.Antibody was covalently attached through its carboxylic acid to the amino-group of nonylamine by the active ester method, several factors were studied on the experimental result such as reaction time, reaction medium, and amount of PC. The effect and activity of hydrophobic antibody were assessed by SDS-PAGE and electrochemical method. The results indicated that hydrophobic antibody and antigen responsed well when the reaction time is3h at room temperature.Three electrode detection system was used in the sensors, Ag/AgCl as reference electrode, Pt as counter electrode, glass supported lipid membrane modified with antibody as working electrode and KCl as electrolyte. A renewable surface electrochemical immunosensors for detecting estradiol was established initially by using sequential injection system to implement the refreshment of liquid sensitive membrane. Based on it, the electrochemical characteristics of the modified electrode at different stages of modification were studied by linear sweep voltammetry, and optimized the antibody immobilization condition. The result indicated that the method have good response when p-estradiol was added, and detection range is0.3～4.2μg/mL. This method is suitable for detecting estradiol in water in the further study with good quality of simple operation, short reaction time and remarkable selectivity.