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纳米金信号放大石英晶体微天平快速检测己烯雌酚
作者:刘小燕 彭媛 白家磊 郄志伟 宁保安 高志贤

摘要:

目的  构建具有高灵敏度和高选择性的石英晶体微天平(QCM)免疫传感器,实现对己烯雌酚(DES)的快速检测。方法  利用葡聚糖还原法制备粒径为40 nm的修饰有氨基的纳米金颗粒,将其与DES抗体偶联,制备纳米金-抗体偶联物。将晶片浸泡于5 mmol/L的巯基十一烷酸(MUA)溶液中16 h,用1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDC·HCl)和N-羟基琥珀酰亚胺(NHS)活化后,取20 μl 2.2 mg/ml的DES完全抗原(DES-HS-BSA),滴加于晶片表面,实现DES-HS-BSA在晶片表面的固定。另取50 μl 1 mol/L的乙醇胺溶液(pH 8.5)进行封闭,制备能够特异性识别DES的敏感膜。将QCM免疫传感器作为检测平台对反应条件进行优化,在优化条件的基础上,将10 μl 28 μg/ml的纳米金-抗体偶联物和10 μl 0.032~2.5 μg/ml的DES溶液混合均匀后滴加于制备有敏感膜的晶片表面,用QCM免疫传感器进行检测并绘制标准曲线。根据标准曲线方程计算最低检出限。对相同浓度的DES及其结构功能类似物进行检测,评估QCM免疫传感器的特异性。结果  DES-HS-BSA的最佳固定浓度为2.2 mg/ml;7 μg/ml的DES抗体和15 nmol/L的纳米金颗粒为两者的最佳偶联浓度;纳米金-抗体偶联物的最佳浓度为14 μg/ml。在0.16~500 ng/ml范围内,DES浓度的对数值与频移呈线性关系,标准曲线方程为Δf=-24.17 lgCDES+69.72,R2=0.998。该方法的检出限为0.13 ng/ml。DES结构功能类似物不会对DES的检测造成明显的干扰,该传感器具有很好的特异性。结论  纳米金信号放大QCM免疫传感器能够快速灵敏的对DES进行检测。

关键词:金属纳米粒子;生物传感技术;己烯雌酚;石英晶体微天平;自组装

Abstract:

Objective  To develop a quartz crystal microbalance (QCM) immunosensor with high sensitivity and selectivity for the rapid detection of diethylstilbestrol.Methods  Dextran was used as reducing agent for preparing gold nanoparticles (AuNPs) with the size of 40 nm. The AuNPs were coupled with anti-DES antibody after amination. A monolayer was generated after immersing the quartz crystal into the solution of 5 mmol/L 11-mercaptoundecanoic acid(MUA) for 16 hours. After the monolayer was activated by 1-ethyl-3-(3-dimethylaminopropry) carbodiimide hydrochloride (EDC·HCl) and N-hydrosuccinimide (NHS), 20 μl of 2.2 mg/ml DES-HS-BSA was dropped onto the surface of crystal to prepare a sensitive membrane which can recognize DES specifically. Then, 50 μl of 1 mol/L ethanolamine (pH 8.5) was used to seal the carboxylic groups to make the sensitive membrane which could identify DES specifically. QCM immunosensor was used as detection platform to optimize the reaction conditions. Under the optimized conditions, 10 μl of 28 μg/ml AuNPs-antibody was mixed with 10 μl of 0.03-2.5 μg/ml DES, and the mixture was added on the sensitive membrane. QCM immunosensor was used to detect the signals and the standard curve was obtained at the same time. The detection limit was calculated based on the standard curve. The specificity was evaluated by testing DES and its analogues with the same concentration.Results  The optimized concentration for the immobilization of DES-HS-BSA on the surface of QCM was 2.2 mg/ml. The optimized concentration for coupling anti-DES antibody with AuNPs was 7 μg/ml and 15 nmol/L, respectively. The optimized concentration of AuNPs-antibody was 14 μg/ml. The logarithm of DES concentration was proportional to the frequency shift in the range of 0.16-500 ng/ml, Δf=-24.170 lgCDES+69.71, R2=0.998. The detection limit of this method was 0.13 ng/ml. DES analogues could not influence the detection of DES obviously, so the sensor had good specificity.Conclusion  The quartz crystal microbalance immunosensor with gold nanoparticals amplification could detect DES sensitively and rapidly.

Key words: Metal nanoparticles;Biosensing techniques;Dierhylstilbestrol;Quartz crystal microbalance;Self-assembled monolayer

发表日期:2016/3

引用本文:

图/表:

  • 10.3760/cma.j.issn.0253-9624.2016.03.016.F001:图1 己烯雌酚与其结构功能类似物的信号响应;

    10.3760/cma.j.issn.0253-9624.2016.03.016.F001:图1 己烯雌酚与其结构功能类似物的信号响应;

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