基于磁性纳米粒子的农药残留生物条形码免疫分析方法研究

Immunoassay was one of the most important monitoring methods for the detection of pollutant residue. Because of the existence of sample matrix interference, the limit of quantitation of immunoassay could not meet the requirement of monitoring of pesticide residue very well. To solve the bottleneck, the bio-bar code immunoassay with high sensitivity was prepared to investigated in the field of pesticide residue detection. The colloidal gold nanoparticle probe was developed by the combination of bio-bar code with the function of signal amplification and the colloidal gold nanoparticle coupling with monoclonal antibody against pesticide. Magnetic nanoparticle probe was developed by the combination of magnetic nanoparticle and ovalbumin coupled with pesticide hapten. Two DNA fragments which could be hybridized with bio-bar code were designed and synthesized, based on the synthesization, DNA chip probe and detection probe were developed. On the basis of development of colloidal gold nanoparticle probe and magnetic nanoparticle probe, pesticide competitive immunoassay system was generated. Subsequently, the released bio-barcode was combined with DNA chip probe and detection probe, with the method of gold-label silver-staining detection, the concentration of bio-bar code was detected, and the concentration of pesticide was tested indirectly. As the principle introduced above, the bio-bar code immunoassay based on the magnetic nanoparticle for the detection of pesticide residue was developed. The work will enrich the basic theory of bio-bar code immunoassay, broaden the application scope, greatly improve the detection sensitivity of immunoassay for pesticide residue, and is positive to improve the level of China's food safety.

免疫分析方法是污染物残留检测的重要技术手段，但由于基质效应的存在使得现有免疫分析方法的定量限仍不能很好满足农药残留监管要求。为解决这一现实瓶颈，本项目拟开展高灵敏度的生物条形码免疫分析方法研究。设计并合成具信号放大作用的生物条形码，与偶联有农药抗体的胶体金纳米颗粒结合，制备得胶体金纳米探针；筛选并活化磁性纳米粒子，与农药半抗原及OVA偶联物结合，制备得磁性纳米探针；设计并合成能与同一生物条形码杂交结合的2段DNA片段，制备得DNA芯片探针和检测探针；在制备胶体金纳米探针和磁性纳米探针基础上，建立农药竞争型免疫分析体系；利用DNA芯片探针和检测探针，结合解离的生物条形码，并使用金标银染法测定生物条形码含量，从而间接测得农药含量，建立农药生物条形码免疫分析方法。上述研究将丰富生物条形码免疫分析方法的基础理论，拓宽其应用范围，大大提高农药残留免疫分析方法的检测灵敏度，有利于保障我国食品安全。

建立了一种基于生物条形码探针和金纳米颗粒对农药残留检测的新方法。设计并合成具信号放大作用的生物条形码，与偶联有农药（三唑磷和克百威）抗体的胶体金纳米颗粒结合，制备得胶体金纳米探针；筛选并活化磁性纳米粒子，与农药（三唑磷和克百威）半抗原及OVA偶联物结合，制备得磁性纳米探针；在制备的胶体金纳米探针和磁性纳米探针基础上，建立农药竞争型免疫分析体系；利用DNA芯片探针和检测探针，结合解离的生物条形码，并使用金标银染法测定生物条形码含量，从而间接测得农药含量，建立农药生物条形码免疫分析方法。该方法三唑磷在水样品中最低检测限为0.55 ng/mL，克百威的检测限是0.50ng/mL。.上述研究将丰富生物条形码免疫分析方法的基础理论，拓宽其应用范围，大大提高农药残留免疫分析方法的检测灵敏度，有利于保障我国食品安全。