一种高灵敏及高选择性荧光“关-开”分析测定谷胱甘肽的方法研究

A Highly Sensitive and Selective Fluorescence“off-on”Method for Glutathione Detection

本文基于无荧光的N-丁基-4-硝基-萘酰亚胺(BNNA)能够选择性地与谷胱甘肽(GSH)发生硝基-巯基亲核取代反应,并生成强荧光的芳香硫醚产物,而十六烷基三甲基溴化铵(CTAB)胶束对该反应具有明显的催化及荧光增敏作用的原理,建立了一种以BNNA为荧光探针,选择性荧光"关-开"分析测定GSH的新方法。在优化的实验条件下,BNNA-GSH-CTAB体系在470 nm波长处的荧光强度(激发波长390 nm)与GSH浓度在0.1~10.0μmol/L范围内呈线性关系,检测限为1.35×10^-9 mol/L,而且其他氨基酸,包括半胱氨酸(Cys)、高半胱氨酸(HCy)等对GSH的测定没有明显的影响。该方法具有荧光探针结构简单易得,Stockes位移大(80 nm),灵敏度高以及选择性强等特点,用于实际酵母样品中GSH的测定,加标回收率为94.4%~101.2%,相对标准偏差≤4.20%。

In this work,we reported a new fluorescence"off-on"method for the detection of glutathione(GSH)by using N-butyl-4-nitro-1,8-naphthalimide(BNNA)as fluorescence probe.This method was based on the thiol-NO2 nucleophilic substitution between non-fluorescent BNNA and GSH generating a strongly fluorescent thioether,and the presence of cetyltrimethylammonium bromide(CTAB)could not only catalyze the reaction but also improve the analysis sensitivity.Under optimal detection conditions,the fluorescence change of BNNA at 470 nm(excited at 390 nm)was linear over GSH concentrations ranging from 0.1μmol/L to 10.0μmol/L,and the limit of detection was estimated as 1.35×10^-9 mol/L.There were no obvious inferences from other common amino acids including cysteine and homocysteine.The proposed method had appealing properties of a simple and readily available fluorescence probe,highly selective and sensitive detection of GSH with large Stocks shift(80 nm).It has been successfully utilized to quantify GSH in actual yeast samples with recoveries of 94.4%-101.2%(RSD≤4.20%).