贵金属荧光纳米簇生物识别探针在高通量免疫分析中的应用基础研究

A large number of veterinary drugs are used widely in the animal husbandry industry, resulting in a variety of veterinary drug residues in animal derived food, which have brought great challenges to the monitoring work. The development of high sensitive and high throughput immunoassays based on noble metal fluorescent nanoclusters is an effective approach to above problems, due to the characteristics of sensitization, broad spectrum, stable, and nontoxic of the nanomaterials, which can detect multiclass veterinary drugs residues in the same time. In this project, the preparation, purification and characterization of noble metal (Au or Ag) fluorescent nanoclusters are the primary task. Extraction of heavy chain and light chain amino acid sequence of the monoclonal antibody against tetracyclines is performed to determine the target gene, following cloning and expression. Then, under the optimized reaction system and condition, the noble metal fluorescent nanoclusters will be conjugated with the biological recognition materials such as monoclonal antibodies, single chain antibodies. Biological recognition probes with different excitation wavelengths and emission wavelengths can be prepared. Besides, the recognition mechanisms between veterinary drugs and single chain antibodies or relative probes also is explored via establishing the three-dimensional quantitative structure-activity relationship and molecular docking, and is used to find the key binding sites. Ultimately, the high-throughput chemiluminescent and fluorescent polarization immunoassays with high sensitivity will be successfully developed for multiresidue determination, which can provide the theoretical and technical basis for the monitoring of veterinary drug residues in animal-derived food.

动物养殖业用药广、种类多造成了动物性食品中兽药残留的多样复杂，给监控工作带来了极大挑战。贵金属荧光纳米材料具有增敏、宽光谱、稳定、无毒等特性，研究建立基于贵金属荧光纳米材料的高灵敏高通量免疫分析技术是解决这一问题的有效途径，该技术在残留限量标准下可实现同时检测不同类数十种兽药残留。本项目以BSA、DNA等为模板分子制备金、银等贵金属荧光纳米簇，纯化和表征；提取四环素类等单克隆抗体重链和轻链氨基酸序列，确定目的基因，克隆和表达；将贵金属荧光纳米簇与兽药小分子单链抗体等生物识别材料偶联，优化反应体系和反应条件，制备具有不同激发波长和发射波长的生物识别探针；建立三维定量构效关系和分子对接互作模型探索兽药小分子和单链抗体、生物识别探针之间的识别作用机制，确定关键结合位点；建立高灵敏高广谱的化学发光免疫分析、荧光偏振免疫分析等高通量快速检测技术，为我国动物性食品中兽药残留检测提供理论依据和技术保障。

免疫分析检测技术是动物源性食品中兽药残留高通量检测的一个有效途径，它具有方便快速、灵敏便捷、特异准确等优点。标记材料和生物识别原件是免疫分析检测技术的两个关键点。因此，在本项目中，我们合成了新型纳米材料，制备了单链抗体，探索了抗原和抗体间的分子识别相互作用机制，并在此基础上建立了基于新型纳米材料的兽药残留等快速检测方法及高通量筛查平台。主要研究内容包括：（1）新型纳米材料及生物识别探针的合成及表征：以蛋白质、氨基酸等配体分别合成了红色和绿色荧光金纳米簇，采用一步法合成有机/无机杂化荧光纳米花和二氧化硅二氧化锰复合纳米材料，对其形貌、粒径、元素组成、价态等性质进行表征评价，同时优化纳米材料与抗体的偶联条件，成功制备了特异性强的生物识别探针。（2）单链抗体制备：提取已有交链孢酚等抗体可变区、重链和轻链的氨基酸序列，筛选和确定目的基因，加入连接臂基因定向结构改造，筛选出高亲和性单链抗体。（3）分子识别相互作用：研究了单链抗体与抗原分子识别机制，建立了抗原-抗体结合的定量构效关系模型和互作虚拟模型，根据获得的作用力类型和关键氨基酸结合位点，指导了金刚烷胺和交链孢酚单链抗体的定点突变，获得的突变体相比亲本亲和力均提高了4倍。（4）方法建立：建立基于红色金纳米簇和芬顿反应介导的荧光免疫分析方法，用于快速灵敏检测饲料样品中的喹乙醇残留；利用绿色超强发光金纳米簇作为标记载体，结合链霉亲和素生物素放大系统，建立了两种典型瘦肉精残留（莱克多巴胺、克伦特罗）的快速检测的高灵敏荧光免疫层析方法；利用二氧化硅二氧化锰纳米复合材料标记广谱性单克隆抗体，构建了同时检测三类（磺胺类、喹诺酮类和酰胺醇类）兽药残留的高通量快速筛查平台，可同时筛查35种兽药，初步实现了对兽药残留的高灵敏、高通量、特异的定量分析，为兽药残留高通量筛查提供了新方法和新思路，具有重要的理论意义和广阔的应用前景。