基于巯基-烯烃点击化学快速可视化检测尿中黏糠酸

Rapid visual detection of trans,trans-muconic acid in urine based on mercapto-olefin click chemistry

目的建立尿中黏糠酸(t,t-MA)的巯基-烯烃点击化学反应的光度法。方法衍生化试剂5,5.-二硫硝基苯甲酸(DTNB)在中性或碱性pH条件下可生成呈现黄色的显色物质2-硝基-5-硫代苯甲酸(TNBA)。TNBA中的巯基通过与t,t-MA中的双烯结构发生巯基-烯烃点击化学反应可导致体系黄色消失,在2-硝基-5-硫代苯甲酸(TNBA)黄色体系中加入不同浓度的t,t-MA,体系黄色呈现不同程度的褪色,颜色变化可由肉眼直接分辨,通过吸光度值的变化,实现对尿中t,t-MA的定量检测。结果本法中t,t-MA在1.2μg/ml~14.4μg/ml浓度内线性良好,相关系数为0.9996,方法检出限为0.28μg/ml,加标回收率为88.2%~94.3%,批内精密度和批间精密度分别为7.13%~9.52%(n=6)与3.50%~4.62%(n=6)。结论本方法简便快捷,成本低廉,可用于职业接触苯人群尿中t,t-MA的快速可视化检测。

Objective To establish a spectrophotometric method for the determination of trans,trans-muconic acid(t,t-MA)in urine based on mercapto-olefin click chemistry.Methods The derivative reagent 5,5′-dithionitrobenzoic acid(DTNB)can produce the yellow color material 2-nitro-5-thionitrobenzoic acid(TNBA)at neutral or alkaline pH.The thiol group in TNBA and diene structure in t,t-MA can lead to the disappearance of the yellow color of the system due to the mercaptoolefins click chemical reaction.When different concentrations of t,t-MA were added to the yellow system of 2-nitro-5-thiobenzoic acid(TNBA),the yellow color of the system showed different degrees of fading.The color change can be visualized with the naked eye and the change of absorbance can be measured by the UV spectrophotometer to achieve the quantitative detection of t,t-MA in urine.Results The method showed good linearity in the range of 1.2μg/ml-14.4μg/ml(r=0.9996).The detect limit of the method was 0.28μg/ml.The average recovery ranged from 88.2%-94.3%and the intra-day and inter-day precisions(RSDs)were 3.50%-4.62%(n=6)and 7.13%-9.52%(n=6),respectively.Conclusion This method is simple,rapid and low-cost.It can be used for rapid and visual detection of trans,trans-muconic acid in the urine of the benzene occupationally exposed people.