头孢氨苄免疫层析表面增强拉曼光谱检测方法研究

doi:10.16431/j.cnki.1671-7236.2022.04.040

摘要/Abstract

摘要： 【目的】建立一种简便、快速、灵敏度高、特异性强的头孢氨苄免疫层析表面增强拉曼光谱(SERS)检测新方法。【方法】制备金银复合核壳结构的纳米贵金属,用来标记头孢氨苄抗体和拉曼信号分子5,5’-二硫代双2-硝基苯甲酸 (DTNB),合成拉曼免疫探针,用透射电子显微镜和双光束紫外分光光度计对纳米金和修饰拉曼信号分子的金银复合核壳结构进行表征。将合成的拉曼免疫探针固定在微孔板上,待测样品中的头孢氨苄与包被在硝酸纤维素膜上的头孢氨苄抗原竞争结合拉曼免疫探针,通过免疫层析试纸条结合共聚焦拉曼光谱仪读取DTNB的信号值,建立头孢氨苄定量检测技术,并应用于实际牛奶样品的检测。【结果】透射电子显微镜和双光束紫外分光光度计表征结果显示,纳米银成功包裹在金颗粒表面。用共聚焦拉曼光谱仪检测免疫层析试纸条上拉曼信号分子的特征峰,拉曼信号分子特征峰为1 062、1 154、1 334和1 558 cm^-1,其中1 334 cm^-1处特征峰信号最强,选取此处峰高定量测定头孢氨苄的含量。头孢氨苄免疫层析表面增强拉曼光谱检测的线性范围为0~1 ng/mL,检测限为0.001 ng/mL,头孢氨苄的半数抑制质量浓度为0.05 ng/mL。与头孢克洛交叉反应率为111.1%,与头孢曲松钠、头孢夫辛酯、头孢噻吩钠交叉反应率均低于0.1%。实际样品加标浓度为1、4和8 ng /mL,回收率分别为94.4%、103.3%和97.5%。【结论】本研究建立的头孢氨苄免疫层析表面增强拉曼光谱检测方法操作简便、快速、灵敏,耗材成本低廉,可满足奶牛养殖农场和乳品加工企业进行大批量样品初筛检测需求。

关键词: 头孢氨苄; 金银复合核壳; 表面增强拉曼光谱(SERS); 免疫层析

Abstract: 【Objective】 This study was aimed to establish a rapid,sensitive,specific and easy-to-use surface-enhanced Raman spectroscopy (SERS) combined with immunochromatographic assay for detecting cephalexin.【Method】 In this study,Raman molecule 5,5’-dithiobis-2-nitrobenzoic acid (DTNB) and cephalexin antibody were conjugated with Au (core)@Ag (shell) nanoparticle to serve as SERS nanoprobe.The gold nanoparticles and Au (core)@Ag (shell) nanoparticles were identified by transmission electron microscopy(TEM) and ultraviolet spectrophotometer.Using the combination of SERS and immunochromatography,the quantitative detection technology of cephalexin was established,and applied to the detection of milk samples.【Result】 The results of TEM and ultraviolet spectrophotometer showed that silver nanoparticles were successfully coated on the surface of gold particles.Raman spectrometer was used to detect the characteristic peaks of Raman signal molecules on the immunochromatographic strip,and the peak height was selected to determine the content of cephalexin.The molecular characteristic peaks of Raman signal were 1 062,1 154,1 334 and 1 558 cm^-1,of which the characteristic peak signal at 1 334 cm^-1 was the strongest.The peak height was selected to determine the content of cephalexin.The detection linear range was 0 to 1 ng/mL,the detection limit was 0.001 ng/mL,and the half inhibitory concentration was 0.05 ng/mL.The cross-reactivity with cefaclor was 111.1%,with ceftriaxone sodium,cefuroxime axetil and cephalotin sodium were all less than 0.1%,respectively.Recoveries of cephalexin from spiked milk at levels of 1,4 and 8 ng/mL were 94.4%,103.3% and 97.5%,respectively.【Conclusion】 The method of surface-enhanced Raman spectroscopy combined with immunochromatography for detecting cephalexin developed in this study was simple,rapid,sensitive and low-cost,which could be used for on-site mass sample screening and testing.

Key words: cephalexin; Au (core)@Ag (shell); surface-enhanced Raman spectroscopy (SERS); immunochromatographic assay

中图分类号:

S859.84

陈艳, 薛强, 金涌, 邹明强, 齐小花, 李博逸, 殷宏. 头孢氨苄免疫层析表面增强拉曼光谱检测方法研究[J]. 中国畜牧兽医, 2022, 49(4): 1577-1584.

CHEN Yan, XUE Qiang, JIN Yong, ZOU Mingqiang, QI Xiaohua, LI Boyi, YIN Hong. Devolopment of Surface-enhanced Raman Spectroscopy Combined with Immunochromatography for Detecting Cephalexin[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(4): 1577-1584.

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