多组分兽药残留的多重标记荧光偏振免疫分析研究

In recent years, the residue of veterinary drugs have been considered as an issue of worldwide concern due to its potential threat to human health. With the increasing demand for rapid screening programs, multi-analyte screening is required. However, multi-analyte detection is difficult to realize by general immunoassays. And the current trend is to develop immunoassays capable of measuring multiple targets in a single test. Fluorescence polarization immunoassay (FPIA) is a promising and reliable homogeneous immunoassay in solution phase which can reach equilibrium in minutes or even seconds, and no separation or washing steps are required, and advances in fluorescence-based detection techniques allow researchers to perform multiplexed analysis using multiple labeled assays. The present study is going to develop the multi-analyte determination of veterinary drug residue based on multi-labelled FPIA (Multi-FPIA). First, fluorescent probes will be synthetized by linking drug haptens with two different class of fluorescent materials, one is small molecules, the other is nano quantum dots. The best probes combination is depended on their fluorescence spectras easily distinguishable from each other. The optimal combination of probe/antibody is finally selected by FPIA based on the highest sensitivity and lowest non-specific binding, then the Multi-FPIA is developed by combination of different fluorescent probes, with corresponding specific antibodies for simultaneous analysis of multiple analytes in veterinary medicine. Animal-origin food is a very complex matrix consisting of different components (fats, proteins, various sugars, etc.), which can strongly interfere with the analytical determination. These analytes should be simultaneously extracted from the samples such as milk, honey and chicken muscle by a suitable pre-treatment, and the recovery studies should be investigaed. The present work will provide a fast, high-throughout screening method for multi-analyte determination of veterinary drug residues in food sample.

动物性食品中多种兽药残留并存的情况相当普遍，现已成为兽药残留监控工作的一大挑战。但传统免疫分析方法通常仅能对单一组分进行检测，研究建立基于多标记技术的荧光免疫分析方法是解决此问题的有效途径之一。然而，由于同种荧光染料间的干扰，现有报道仅能实现双标记的定量检测。前期研究发现，选择异种荧光材料很可能有助于减少干扰因素的累积。本项目拟将多种小分子荧光染料和量子点分别标记兽药分子合成荧光探针，以研究这两类探针在多标记定量检测中的兼容性；再通过正交结合试验筛选最佳的探针-抗体组合，将快速的荧光偏振免疫分析方法与多标记技术相整合来建立多标记荧光偏振免疫分析方法(Multi-FPIA)；最后评价不同前处理方法对牛奶、蜂蜜和鸡肉等样品的提取净化效果，以满足对Multi-FPIA检测的要求。本研究预期可实现同时检测动物性食品中四种兽药的残留，为多组分兽药残留检测的发展提供新的思路和技术储备。

动物性食品中的兽药残留严重危害人体健康，已成为全世界共同关注的公共卫生问题之一。目前，多种兽药残留并存的情况已经相当普遍，但由于抗体识别能力和检测模式的限制，传统免疫分析方法通常仅能对单一组分进行检测。为了实现对多组分兽药的同时检测，迫切需要新思路和新技术的引入。以荧光偏振免疫分析方法为代表的均相免疫分析方法是目前兽药残留免疫检测领域的研究热点。荧光技术易于实现多标记，本项目通过比较不同荧光材料间光谱的差异，选择常用的小分子有机荧光染料和量子点分别与多种兽用抗菌药物进行共价偶联，合成了氨基糖苷类，磺胺类，喹诺酮类，β-内酰胺类、氯霉素类等的荧光标记物，并筛选出与抗体配对最佳的荧光标记探针分别为：STR-FITC用于检测链霉素（Streptomycin, STR）；SD-TRITC用于检测磺胺噻唑（Sulfathiazole, ST）；SAR-TRX用于检测恩诺沙星（Enrofloxacin, ENR）；CEP-CF633用于检测头孢氨苄（Cephalexin, CEP）。本研究用上述四种荧光标记物及其特异性抗体建立了基于多重标记技术的四色标记荧光偏振免疫分析方法（Quad-fluorophore FPIA），可用于同时检测牛奶中链霉素、磺胺噻唑、恩诺沙星和头孢氨苄四种不同类抗菌药物的残留。本方法可在同一个检测孔中实现对四种抗菌药物的同时检测，对STR、ST、ENR和CEP的检测限分别为7.3、2.7、2.5和3.8 ng mL-1。空白牛奶中同时添加四种抗菌药物共同提取后，检测的平均回收率为59.8 ~ 88.1％，变异系数低于25.5％。因此，本方法可基本满足牛奶中这四种抗菌药物快速筛选检测的要求，大大提高检测的效率，并具有将其推广到牛奶中其他小分子污染物检测的应用前景。