牛乳酪蛋白及其水解物的纳米金荧光增强检测方法

Nano Gold Fluorescence Enhancement Detection Method for Milk Casein and Its Hydrolyzate

基于抗原抗体特异性反应和纳米金荧光增强效应构建了牛乳酪蛋白及其水解物的新型检测方法。实验以纳米金为载体,首先将抗体以非共价键结合于纳米金表面,根据抗原抗体特异性反应与异硫氰酸荧光素(FITC)标记蛋白结合,基于纳米金荧光增强效应使标记体系荧光达到最大,待测蛋白和FITC标记蛋白竞争性与金标抗体结合,通过检测荧光强度,从而间接测定蛋白质含量。通过一系列优化实验建立检测体系,结果为:酪蛋白荧光标记率为1.35,为充分标记;检测体系中抗原的最适加入量为30μg/mL。方法检测线性范围为0.2~1μg/mL,最低检测限为0.2μg/mL,可重复试验变异系数均小于6%。研究结果表明所建立的纳米金荧光增强检测方法具有简单快速、操作简单、重复性好,可用于牛乳酪蛋白及其水解物的定量检测。

A novel detection method for bovine milk protein and its hydrolysate was constructed based on antigen-antibody specific reaction and nanogold fluorescence enhancement effect.The experiment uses nano-gold as the carrier,firstly binds the antibody to the surface of the nano-gold by non-covalent bond,and binds to fluorescein isothiocyanate(FITC)labeled protein according to the specific reaction of the antigen-antibody.The fluorescence of the labeling system is maximized based on the enhancement effect of the nano-gold fluorescence,and the protein to be tested and The FITC-tagged protein competes with the gold-labeled antibody and indirectly measures the protein content by detecting the fluorescence intensity.The detection system was established through a series of optimization experiments.The results showed that the fluorescent labeling rate of casein was 1.35,which was sufficient for labeling;the optimum amount of antigen in the detection system was 30μg/mL.The linear range of the method was 0.2~1μg/mL,the minimum detection limit was 0.2μg/mL,and the coefficient of variation of the repeatable test was less than 6%.The results show that the established nano-gold fluorescence enhancement detection method is simple,rapid,easy to operate and reproducible,and can be used for quantitative detection of milk casein and its hydrolysate.