SpvC特异性电化学型双层壳聚糖纳米金免疫传感器的研究

Based on the amplification system of hydrogen peroxide biosensor based on horseradish peroxidase/GNPs-thionine/chitosan, the electrochemical immunosensor for anti-SpvC (phosphothreonine lyase) monoclonal antibody will be fabricated using double-layer gold nanoparticles and chitosan. The sensing element of the immunosensor was comprised of gold nanoparticles fixed with the SpvC monoclonal antibodies. To examine the quality of the synthesized gold nanoparticles, the size, shape and dispersion of the gold nanoparticles will be characterized by transmission electron microscopy (TEM), atomic force microscope (AFM) and UV-visible spectral scanning. The interactional affinities between the monoclonal antibodies and SpvC will be determined and optimized by BioLayer Interferometry (ForteBio), To assess the quality of the different steps in immunosensor fabrication, each procedure in its assembly was monitored by cyclic voltammetric and alternating current impedance. Chronoamperometry was used to measure the concentration of SpvC. In order to evaluate the immunosensor, it will be used to detect SpvC quantificationally for its specificity, cross reaction and application prospect for the selection of Treatment medicine and research for the pathogenic mechanism of Shigella and Salmonella. To valuate the nanoimmunosensor for food safety control, Chronoamperometry will also be used to measure the concentration of Shigella and Salmonella for their SpvC products. Various key parameters will be optimized for the platform of fast, specific, high sensitivity dectection to target antigens or haptens, and bacterial contamination and foodborne pathogenic microorganisms. Moreover, these may be the key step to set up a series of immunosensor instruments for food quality and safety control.

本项目拟在已经建立的纳米金-硫堇-壳聚糖吸附辣根过氧化物酶电化学生物传感放大系统的基础上，通过纳米金溶胶吸附抗苏氨酸磷酸裂合酶Balb/c小鼠单克隆抗体，利用光谱扫描、原子力显微镜、生物薄膜干涉、循环伏安及交流阻抗法表征其吸附和组装的各阶段，研制出电化学型纳米金双膜免疫传感电极。与此同时，将该电极用于SpvC的定量测定，建立各种参数，为进一步运用该传感器筛选治疗由志贺氏、沙门氏菌等OspF家族三型分泌系统效应蛋白所引起的疾病的药物和致病机理提供特异性、高敏感、定量检测方法；在此基础上建立分别和同时测定分泌SpvC的志贺氏和沙门氏菌的各项参数，从而研制出检测这两种主要食源性致病菌的高灵敏、特异性纳米金双膜电化学型免疫传感器；进一步优化其参数和工艺，提高测定灵敏度、稳定性、重复性、半衰期和再生性能，为相关仪器设备、电极的研制和商业化奠定基础。

本研究首先分离并纯化了沙门氏菌苏氨酸磷酸裂合酶（phosphothreonine lyase, SpvC），并以此为抗原制备单克隆抗体，获得了5个单克隆抗体细胞株。借助于生物分子相互作用仪（Octet® biolayer interferometry）从这5株抗SpvC单克隆抗体中筛选出与SpvC亲和力最强的1个单克隆抗体细胞株¬¬。采用以纳米金-硫堇-壳聚糖吸附辣根过氧化物酶为生物传感的信号放大系统，通过纳米金溶胶吸附抗SpvC Balb/c小鼠单克隆抗体，利用紫外光谱扫描、透射电镜表征纳米金，原子力显微镜、循环伏安及交流阻抗法表征其吸附和组装的各阶段，成功研制出电化学型纳米金双层膜免疫传感电极。利用电流-时间曲线法分别测定PBS稀释的沙门氏菌和志贺氏菌悬液，结果表明响应电流与两种菌在10~1.0×104 cfu/mL范围内线性相关，最低检测限为5cfu/mL，线性方程分别为：△I=0.2103C+0.0256 (R2=0.9943)，△I=0.2044C+0.0263 (R2=0.9904)，由方程斜率表明：抗体与沙门氏菌有更好地亲和力，这可能起因于SpvC来源于沙门氏菌。再利用时间电流曲线法测定PBS稀释的两种菌混合液，线性方程为：△I=0.2078C+0.026 (R2=0.9913)，表明该传感器可以检测这两种混合菌液中菌的总浓度。对其寿命、稳定性、重现性及选择性进行了研究，结果证实：该传感器具有一定的使用价值，为进一步用于食品安全检测奠定了基础。在此基础上，本项目进一步探讨了将该信号放大系统用于受体胞外结构域和配体识别后所导致的胞内信号放大和传导作用，取得了突破进展，在辣味（辣椒素、姜辣素）、甜味（阿斯巴甜、索马甜）、鲜味（味精、鸟苷酸）和苦味（蔗糖八乙酸酯、苯甲地钠胺、斛皮素）受体传感与动力学测定方面取得一批较有水平的研究成果。该项目为我们进一步进行受体传感器研究与应用奠定了坚实基础。