溶胶-凝胶-HBsAb膜免疫传感器的研制与应用

Preparation and Application of Sol-gel-HBsAb Immunosensor

采用溶胶-凝胶(Sol-gel)技术,成功地将乙肝表面抗体(HBsAb)包埋于Sol-gel中,再滴涂于铂盘电极表面,制成溶胶-凝胶-HBsAb膜非标记免疫传感器.根据抗原与抗体特异性结合形成的免疫复合物使敏感膜有效扩散截面积减小的特性,提出了利用铁氰化钾作为氧化还原探针间接检测乙肝表面抗原(HBsAg)的新方法.用循环伏安法(CV)对电极逐层修饰过程进行了表征,并探讨了对HBsAg定量检测的可行性及其响应机理.采用差示脉冲伏安法(DPV)检测人体血清中的 HBsAg.线性范围5～320μg/L,线性相关系数r=0.997.该传感器响应迅速,灵敏度高,稳定性好.于4℃干态保存14d,其响应信号基本不变.将其用于108例临床血清检验,与酶联免疫吸附法(ELISA)的符合率为87.5％.

A new type of nonlabbled immunosensor for the determination of hepatitis B surface antigen (HBsAg) was made by utilizing Sol-gel techniques to encapsulate hepatitis B surface antibody (HBsAb) on tetraethylorthosilicate(TEOS) derived Sol-gel films and then to coat the Sol-gel-HBsAb layer onto the surface of platinum disk electrode. The effective diffusion section area of K3Fe(CN)6 attained to the platinum electrode has been decreased along with the increase of the singularity reaction immunocompound of HBsAb and HBsAg, the respond current of K3Fe(CN)6 on the surface of electrode was directly proportional to the effective diffusion section area of K3Fe(CN)6 attained to the platinum electrode and inversely proportional to the concentration of HBsAg, so HBsAg could be determined indirectly by utilizing K3Fe(CN)6 as the oxidation-reduction probe. Cyclic voltammetry (CV) was employed to investigate the electrochemical character of the immunosensor in 5 mmol/L K3Fe(CN)6-0. 1 mol/L KNO3-PBS system. The linearity of HBsAg in the range of 5-320 μg/L with the correlation coefficient of 0. 997 was obtained from differential pulse voltammetry (DPV) by using the three systems. The dependences of the current response on incubation time, sensitivity and reproducibility were studied, and the stability of the sensor was also evaluated. The immunosensor was free from 30 μg/L CEA and 100 μg/L AFP. More than 87. 5% of the results of the human serum samples obtained by this methode were in agreement with those obtained by enzyme-linked assay (ELISA). The immunosensor was found to be stable for about 2 weeks when storing in a dry state at 4 ℃.