H2O2氧化-荧光分析法测定乌骨鸡黑色素细胞中黑色素含量  

A Fluorescence Detection Method for Melanin Content in Black-Bone Silky Fowl Melanocyte Based on H2O2 Oxidation

作　　者：陈露露 田颖刚[1,2] CHEN Lu-lu;TIAN Ying-gang(State Key Laboratory of Food Science and Technology,Nanchang University,Nanchang 330047,China;Engineering Research Center of Biomass Conversion,Ministry of Eduction,Nanchang University,Nanchang 330047,China)

机构地区：[1]南昌大学食品科学与技术国家重点实验室,江西南昌330047 [2]南昌大学生物质转化教育部工程研究中心,江西南昌330047

出　　处：《光谱学与光谱分析》2020年第5期1595-1600,共6页Spectroscopy and Spectral Analysis

基　　金：国家自然科学基金地区科学基金项目(21162018);食品科学与技术国家重点实验室自由探索课题(SKLF-ZZB-201520)资助。

摘　　要：建立一种简单、准确、灵敏度高的测定乌骨鸡黑色素细胞中黑色素含量的荧光分析法。黑色素不发荧光,一旦在强烈的氧化条件下(在碱性过氧化氢中加热)被氧化后就会发出强烈的荧光,通过测量其相对荧光强度来测定细胞中黑色素的含量。利用荧光分光光度计在带宽为5 nm,扫描速度为2400 nm·min^-1,扫描间距为1 nm的条件下确定乌骨鸡黑色素的激发波长和发射波长。通过单因素实验筛选浓度范围在10~100μg·mL^-1的乌骨鸡黑色素的最佳氧化条件,对该方法的线性范围、检测限、仪器精密度、重复性等结果进行分析评价。向A375细胞(无黑色素的人的黑色素瘤细胞)样品中分别加入25,40和80μg·mL^-1黑色素标准品,在最佳氧化条件下进行反应后测定细胞样品中黑色素含量。计算测定值与理论值的相对误差值,意在探究细胞中杂蛋白和脂质对黑色素定量结果的影响;向乌骨鸡黑色素细胞样品中分别加入5.00,7.50和10.00μg的黑色素标准品,按照最佳氧化条件测定其细胞样品中总黑色素含量,并计算其加标回收率。结果表明:乌骨鸡黑色素的激发波长为354 nm,发射波长为453 nm;乌骨鸡黑色素最佳氧化条件为:pH为8,氧化温度为55℃,过氧化氢浓度范围为24%~26%,氧化时间为2 h;黑色素浓度在10~100μg·mL^-1范围内与相对荧光强度呈良好线性关系,相关系数r=0.9974,线性回归方程为b=0.0147a+0.3138,荧光分析法的方法检出限为0.30μg·mL^-1,紫外分光光度法的方法检出限为3.68μg·mL^-1;精密度实验的RSD值为1.87%,重复性实验的RSD值为4.59%;在A375细胞中加入不同质量的黑色素标准品后,测量值与理论值的相对误差分别为2.78%,3.53%和0.25%;在乌骨鸡黑色素细胞中加入不同黑色素标准品后,测得的加标回收率分别为95.94%,92.14%和99.83%,RSD值为4.00%;综上所述,该方法简单、准确、稳定、检测限低,且测定结果不受细胞内杂�We established a simple,accurate and highly sensitive fluorescence analysis method for detecting the melanin content of Black-bone Silky Fowl(BSF)melanocytes for the first time.Melanins did not fluoresce but,once they were subjected to oxidative conditions(heating in alkaline hydrogen peroxide solution),they acquired strong fluorescence.Melanin content of melanocytes was determined by measuring its relative fluorescence intensity.In this paper,the excitation wavelength and emission wavelength of BSF melanin were determined by fluorescence spectrophotometer with a bandwidth of 5 nm,scanning speed of 2400 nm·min^-1 and scanning pitch of 1 nm.The optimal oxidation conditions of BSF melanin in the range of 10~100μg·mL^-1 were screened by a single factor experiment.The linear range,detection limit,instrument precision and repeatability of the method were analyzed and evaluated.25,40 and 80μg·mL^-1 melanin standards were added to samples of A375 cells(melanoma cells without melanin),and the melanin content in the cell samples was determined after that the reaction was carried out under optimal oxidation conditions.Calculate the relative error value of the measured value and the theoretical value for exploring the effect of heterologous proteins and lipids in cells on the quantitative results of melanin;After that 5.00,7.50 and 10.00μg melanin standards were added to BSF melanocyte samples,the total melanin content in the cell samples was determined under optimal oxidation conditions and then the spiked recovery was calculated.The results showed that the BSF melanin had an excitation wavelength of 354 nm and an emission wavelength of 453 nm;The optimal oxidation conditions for BSF melanin were as follows:the PH value was 8,oxidation temperature was 55℃,hydrogen peroxide concentration ranged from 24%to 26%and oxidation time was 2 h;The concentration of melanin in the range of 10~100μg·mL^-1 showed a good linear relationship with the relative fluorescence intensity.The correlation coefficient was 0.9974 and the

关 键 词：乌骨鸡 黑色素细胞 黑色素含量 荧光分析法 

分 类 号：O657.39[理学—分析化学]