农药多残留上转发光纳米晶技术免疫层析快速检测

Lateral-flow immunochromatography (LFIC) is one of the idea techniques for the rapid and on-site detection of small contaminants such as pesticides. The present colloidal gold-labeled immunostrip assays, however, have some drawbacks, e.g., the low sensitivity, non-precise determination and so on. For purpose of solving the above bottleneck problems, in this research proposal, we attempt to utilize up-concerting nanophosphors (UCNPs) as novel labels and reporters for highly sensitive measurement of pesticide residues in complex agri-food smaples. Because of UCNPs’ unique optical ability to up-convert low-energy infrared radiation to high-energy visible light, the background fluorescence from biological samples would be effectively circumvented. In the present study, various kinds of UCNPs with different sizes and colors will be synthesized by diverse rare earth components, several types of nano-probes will be prepared, and different formats of competitive LFIC methods will be compared. The technical platform of UPT-LFIC for pesticides with diverse characteristics will be established, and hence the common theoretical rules will be concluded. Furthermore, the impact of different vegetable and fruit matrixes on the assay sensitivity of the competitive UPT-LFIC will be evaluated, as well as the method tolerance to matrix interference. The research findings from this project can provide scientific and technical foundation for the new rapid tests of trace contaminants in the fields of food safety control and environmental monitoring.

侧流免疫层析技术是实现农药等小分子污染物快速现场检测的理想手段之一。但常见的胶体金标记免疫试纸法存在灵敏度欠高、检测结果不够精确等缺点。为解决上述瓶颈问题，本项目拟利用稀土上转发光纳米晶“低能红外光激发、高能可见光发射”的独特光学性质，有效规避生物样本产生的荧光背景干扰，以实现复杂农产品基质中农药残留高灵敏度快速检测。通过合成不同颗粒尺寸、稀土组分的多种、多色上转发光纳米晶，制备多种类型的纳米免疫探针，比较不同竞争型免疫层析反应模式，构建适用于不同特性农药单残留和多残留检测的上转发光免疫层析技术平台并阐明共性规律，评价该方法对蔬果等农产品基质干扰的耐受能力。预期研究成果能为环境和食品中痕量化学污染物的快速检测提供科学依据和技术支撑。

基于抗原-抗体反应的免疫化学分析技术是实现农产品中农药残留快速筛查的有效手段之一。近年来，采用各种新型纳米颗粒作为标记材料建立高效、高灵敏的侧流免疫层析技术研究日趋增多。其中，上转换纳米粒子（upconversion nanoparticles，UCNPs）因其“长波长激发，短波长发射”的独特发光性质，能有效规避生物样本自身的背景荧光干扰，已在医学检验、生物诊断等方面得到了广泛应用。. 本项目以常用的新烟碱类、有机磷类和氰基菊酯类农药为主要研究对象，将合成的核壳结构UCNPs进行功能化修饰，制备了各类农药的上转发光免疫识别探针，比较了小分子竞争型侧流免疫层析反应的多种模式；成功研制了适用于分别检测吡虫啉等4种新烟碱类农药和对硫磷等3种有机磷类农药的上转换免疫试纸条，其测定时间为30-40 min, 方法灵敏度IC50值达到3-30 ng/mL，能满足我国食品中这些农药最大残留限量的检测要求，定量判断结果与仪器检测结果一致性达到90％以上，可应用于环境和农产品等复杂基质样品中农药多残留快速筛查；明确了构建出农药等小分子竞争型上转免疫层析技术的关键是采用高亲和力、解离速度慢的农药抗体与亲水性UCNPs形成高活性的免疫探针，并需要选择合适的人工抗原参与竞争反应，以及根据不同农药极性特征来优化反应体系参数。此外，以竞争抗原标记的胶体金和吡虫啉抗体偶联的UCNPs作为分子识别探针，建立了均相一步法上转换荧光共振能量转移免疫分析方法，该方法具有操作简便、无需洗涤、试剂用量少等优点。. 本项目拓展了UCNPs新型标记材料的应用范围，丰富了农药免疫分析技术体系，为研究其他农药等小分子竞争型上转换免疫检测方法提供了参考依据，并为探索其他模式的上转换荧光免疫分析技术奠定了研究基础。