基于铜纳米簇模拟酶活性建立比色法测定鱼肉中的次黄嘌呤

Colorimetric Determination of Hypoxanthine in Fish Based on the Enzyme Mimetic Activity of Copper Nanoclusters

基于铜纳米簇(copper nanoclusters,CuNCs)的过氧化氢(H2O2)模拟酶活性,建立鱼肉中次黄嘌呤(hypoxanthine,Hx)含量快速检测的比色分析方法。通过室温搅拌法得到半胱氨酸(cysteine,Cys)-CuNCs,利用透射电镜、光电子能谱、傅里叶变换红外光谱和荧光光谱,分别表征Cys-CuNCs的形貌、元素组成、表面官能团和发光特征。紫外-可见光谱实验表明,Cys-CuNCs能灵敏催化H2O2氧化3,3’,5,5’-四甲基联苯胺显色,实现对H2O2含量的比色测定。在此基础上,利用Hx在黄嘌呤氧化酶的作用下生成H2O2,建立了一种检测Hx的比色分析方法。当孵育温度30℃、孵育时间1 h时,Hx浓度在4~100μmol/L范围内与吸光度呈良好的线性关系,检出限为1.2μmol/L。将该方法应用于鱼肉中Hx含量的测定,并通过与总挥发性盐基氮建立相关性,实现对鱼肉新鲜度的判断。

A colorimetric method for the rapid detection of hypoxanthine(Hx) content in fish was established using copper nanoclusters(CuNCs) with high intrinsic peroxidase-like activity. Cysteine-stabilized CuNCs were prepared by a simple stirring method at room temperature. The morphology, elemental composition, surface functional groups and luminescence property of Cys-CuNCs were characterized by transmission electron microscopy(TEM), photoelectron spectroscopy,Fourier transform infrared spectrometer(FTIR) spectroscopy and fluorescence spectroscopy, respectively. Ultraviolet-visible spectroscopic experiments showed that Cys-CuNCs sensitively catalyzed the oxidation of 3,3’,5,5’-tetramethylbenzidine by H2O2, allowing for the colorimetric sensing of H2O2content. On this basis, a colorimetric assay for Hx determination was established by generating H2O2 under the catalysis of xanthine oxidase. Under the optimal conditions as follows: incubation temperature 30 ℃ and incubation time 1 h, good linearity between Hx concentration in the range of 4–100 μmol/L and absorbance was observed and the detection of limit was 1.2 μmol/L. Finally, the proposed method was applied to the determination of Hx content in fish, and the freshness of fish was judged by establishing its correlation with total volatile basic nitrogen(TVB-N) content.