基于碳点内滤效应快速检测鲜牛奶中碱性磷酸酶活性的荧光分析方法的构建  

Development of a Fluorescence Assay for Rapid Detection of Alkaline Phosphatase Activity in Fresh Milk Based on Inner Filter Effect of Carbon Dots

作　　者：贾宝珠 何镇熹 黄心洳 邱芷靖 郭琳苑 袁钰佩 王碧蔓 罗林[2] JIA Baozhu;HE Zhenxi;HUANG Xinru;QIU Zhijing;GUO Linyuan;YUAN Yupei;WANG Biman;LUO Lin(College of Biological and Food Engineering,Guangdong University of Education,Guangzhou 510303,China;Guangdong Provincial Key Laboratory of Food Quality and Safety,College of Food,South China Agricultural University,Guangzhou 510642,China)

机构地区：[1]广东第二师范学院生物与食品工程学院,广东广州510303 [2]广东省食品质量安全重点实验室,华南农业大学食品学院,广东广州510642

出　　处：《食品工业科技》2023年第17期334-341,共8页Science and Technology of Food Industry

基　　金：广州市科技计划(202102021002);广东省教育厅普通高校青年创新人才项目(2022KQNCX055);广东大学生科技创新培育专项资金“攀登计划”项目(pdjh2021b0368,pdjh2022b0382,pdjh2023b0386);华南农业大学大学生创新创业训练计划项目(202110564080,202110564077);广东第二师范学院大学生创新创业训练计划项目(202314278105)。

摘　　要：研究建立一种以氮掺杂碳点(Nitrogen-carbon dots,N-CDs)为荧光探针,可快速、准确、灵敏检测鲜牛奶中碱性磷酸酶(Alkaline phosphatase,ALP)的荧光分析方法。采用水热法,用乙二胺为氮源和邻苯二酚为碳源制备一种绿色荧光N-CDs,并采用透射电子显微镜(TEM)、紫外可见吸收光谱(UV-vis)、X射线电子能谱(XPS)和傅里叶红外光谱(FT-IR),对所合成的材料表面形貌、表面基团及光吸收特性进行表征,并通过对照试验对利用N-CDs构建检测ALP荧光分析方法的可行性进行验证。以检测体系中工作缓冲液的p H、底物硝基苯磷酸二钠(Disodium p-nitrophenyl phosphate,PNPP)的浓度、酶促反应时间为单因素,优化检测ALP的最佳条件,并建立相应的标准曲线。结果表明,材料表征结果也与以往报道的相符,N-CDs合成成功。最佳工作缓冲液为Tris-HCl(20 mmol/L,p H10),最佳PNPP浓度为1 mmol/L,最佳酶促反应时间为50 min。在最优条件下,ALP的活性浓度与N-CDs的荧光强度变化建立良好的线性关系,线性回归方程为:Y=15397.05X+70344.46(R~2=0.995),线性范围在0.01~25 U/L,检测限(LOD)为0.001 U/L(S/N=3)。在两种鲜牛奶中添加回收率在100.1%~107.2%之间,变异系数小于14.3%,表明该方法具有较好的准确性和可靠性。本研究为鲜牛奶中ALP的检测提供了一种准确高效的方法。This work aimed at developing a fluorescence assay for rapid and sensitive determination of alkaline phosphatase(ALP)activity in fresh milk using nitrogen doped carbon dots(N-CDs)as fluorescence probe.Green-emissive N-CDs were synthesized by using ethylenediamine as the nitrogen source and catechol as the carbon source through a hydrothermal method.The obtained N-CDs were characterized by transmission electron microscopy(TEM),UV-vis spectroscopy,X-ray photoelectron spectroscopy(XPS),and Fourier transform infrared(FT-IR)spectra.Control experiments were used to verify the feasibility for constructing the fluorescent assay using the N-CDs.Single-factor experiments were conducted to optimize the pH of working buffer,the concentration of disodium p-nitrophenyl phosphate(PNPP)and enzymatic reaction time for ALP detection.The results indicated that the as-synthesized N-CDs were successfully prepared with all the characterization results being consistent with those in previous works.The optimal working conditions for this assay were as follows:Tris-HCl(20 mmol/L,pH10)as working buffer,1 mmol/L of PNPP as enzymatic substrate,incubation for 50 min.Under the optimal conditions,it was found that fluorescence intensity of N-CDs linearly corelated with ALP concentration from 0.01 to 25 U/L with correlation coefficients of 0.995.The linear regression equation was Y=15397.05X+70344.46 with a limit of detection(LOD)of 0.001 U/L(S/N=3).Recoveries for two kinds of fresh milk were in the range from 100.1%to 107.2%,and the coefficients of variations were less than 14.3%,which indicated that the proposed method had good accuracy and reliability.This study provides an accurate and efficient method for the detection of ALP in fresh milk.

关 键 词：氮掺杂碳点 荧光探针 荧光内滤效应 碱性磷酸酶 鲜牛奶 

分 类 号：TS207.3[轻工技术与工程—食品科学]