量子点荧光猝灭免疫亲和凝胶检测柱检测番茄酱中罗丹明B的研究

Quantum Dots-based Fluorescence Quenching Immunoaffinity Test Column for the Quick Detection of Rhodamine B in Tomato Sauce

目的:本研究根据抗原-抗体特异性识别反应和荧光共振能量转移(fluorescence resonance energy transfer,FRET)的原理,构建一种基于量子点(Quantum dots,QDs)的新型荧光猝灭免疫亲和凝胶检测柱检测番茄酱中罗丹明B(Rhodamine B,RB)残留。方法:通过罗丹明B的单克隆抗体与溴化氢活化琼脂糖凝胶-4B偶联制备分析物抗体胶,设计单检测层的凝胶检测柱。基于抗原抗体的特异性结合反应,并利用罗丹明B和量子点之间的静电引力作用,构建一种新型的荧光猝灭免疫亲和凝胶检测柱。通过优化抗体添加量、量子点稀释倍数、孵育时间以及上样量等参数,最终实现番茄酱样品中罗丹明B的快速灵敏检测。结果:当1.0 g溴化氢活化的琼脂糖凝胶添加2.0 mg罗丹明B单克隆抗体,量子点溶液稀释3000倍,孵育时间控制在50 s,罗丹明B上样量为3.0 mL,该荧光猝灭免疫亲和凝胶检测柱针对罗丹明B的方法检测限(limit of detection,LOD)能够达到最小值1.0μg/L。样品分析结果表明,该方法针对番茄酱样品中罗丹明B的检测限为5.0μg/kg,并且与HPLC方法测定的结果表现出很好的一致性。结论:该方法操作简便,灵敏度高,检测时间短,结果易于判断,可以满足番茄酱中罗丹明B的现场快速检测的要求。

Objective:A novel fluorescence quenching immunoaffinity test column(FQ-ITC)assay,based on the principles of antigen-antibody specific reactions and fluorescence resonance energy transfer(FRET),was developed to determine rhodamine B(RB)residues in tomato sauce.Methods:The FQ-ITC assay was composed of one detection layer made of RB-antibody gel,which was prepared by the conjugation of RB-specific antibody with CNBr activated sepharose-4B.Based on the specific reaction of antigen-antibody and the electrostatic attraction between RB and quantum dots,a novel fluorescence quenching of immune affinity gel column was established,with optimizing the addition amount of antibody,the dilution ratio of quantum dots,incubation time and the addition amount of RB.And then the column was applied in the detection of rhodamine B in tomato paste sample.Results:When 1.0 g hydrobromide activated sepharose 4B was reacted with 2.0 mg monoclonal antibody,the quantum dots was diluted 3000-fold,the incubation time was 50 s,and the addition amount of RB was 3.0 mL,the limit of detection(LOD)of this assay in assay buffer reached the minimum 1.0μg/L,and the limit of detection of assay for RB in tomato sauce samples was 5.0μg/L.Besides,the results of the established method were in good agreement with HPLC.Conclusion:The FQ-ITC using QDs allows for simple,sensitive,time-saving,rapid detection of RB in tomato sauce samples on-site.