基于磁分离-上转换荧光标记的副溶血性弧菌免疫检测新方法研究

Novel Immunodetection Method for Vibrio parahaemolyticus Based on Magnetic Separation and Upconversion Nanoparticles Labeling

将上转换荧光标记与磁分离富集技术相结合,通过免疫识别成功构建了一种检测副溶血性弧菌（Vibrio parahaemolyticus）的新方法.首先通过水/溶剂热法合成NaY0.78F4：Yb0.20,Er0.02上转换荧光纳米颗粒,并对其表面进行氨基功能化修饰,同时一步法合成了氨基化Fe304磁性纳米颗粒.分别将副溶血性弧菌全菌抗体与磁性纳米颗粒连接作为捕获探针,将副溶血性弧菌的鞭毛蛋白A抗体与上转换荧光纳米颗粒连接作为信号探针.通过免疫识别形成捕获探针-目标菌-显示探针的“三明治”结构复合物,利用980 nm激光诱导荧光进行检测.在实验优化条件下,上转换荧光强度与副溶血性弧菌浓度在5×103～5×105 cfu/mL范围内呈良好线性关系,检测限为1×103 cfu/mL.用鲫鱼进行加标回收实验,结果表明,本方法准确性良好.

Based on upeonversion fluorescence labeling,magnetic separation and immunological recognition,a novel detection method of Vibrio parahaemolyticus was constructed.Primarily,the NaY0.78F4 ∶ Yb0.20,Er0.02 upconversion nanoparticles （UCNPs） were synthesized through hydrothermal method,and then amino group was modified on the surface of UCNPs.Meanwhile,amino-modified MNPs were synthesized by one-step process.Secondly,the antibodies of Vibrio parahaemolyticus binding with MNPs and antibodies of flagellin binding with UCNPs were used as capture probes and signal probes,respectively.The composite of MNPs-target bacteria-UCNPs was obtain,and detected by 980 nm laser.Under the optimum conditions,the upconversion fluorescence was a good linear in the range of 5×103～5×105 cfu/mL of Vibrio p arahaemolyticus,and the detection of limit was 1 × 103 cfu/mL.This method was accurate in the recovery text in the crucian.