Department of Analytical Chemistry, University of Cordoba, Campus of Rabanales, Annex to Marie Curie Building, 14071 Cordoba, Spain.
J Agric Food Chem. 2011 Mar 23;59(6):2235-40. doi: 10.1021/jf104538a. Epub 2011 Mar 2.
A method for the determination of the antioxidant capacity using long-wavelength fluorescence measurements is described for the first time. This method is a modification of the conventional oxygen radical absorbance capacity (ORAC) method that uses fluorescein or phycoerythrin and the generator of peroxyl radicals, 2,2'-azo-bis-(2-methylpropionamidine) dihydrochloride (AAPH). The long-wavelength fluorophor nile blue is proposed as an analytical reagent alternative to these conventional fluorophores. Kinetic curves have been obtained by monitoring the fluorescence variation (λex, 620; λem, 680 nm) with time, using the 96-well microplate format. The vitamin E analogue 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) has been chosen as the model analyte, and the normalized area under the decay curve has been used as the analytical parameter. The dynamic range of the calibration curve is 0.8-8.0 μM, and the detection limit is 0.45 μM. The precision of the method, expressed as relative standard deviation and assayed using 1 and 5 μM Trolox concentrations, was 5.6 and 2.9%, respectively. The method has been applied to the analysis of fruit juices and wines, obtaining results that did not differ significantly from those provided using the ORAC method with fluorescein as reagent.
首次描述了一种使用长波长荧光测量来测定抗氧化能力的方法。该方法是对传统的使用荧光素或藻红蛋白和过氧自由基生成剂 2,2'-偶氮双（2-甲基丙脒）二盐酸盐（AAPH）的氧自由基吸收能力（ORAC）方法的改进。长波长荧光染料尼罗蓝被提议作为这些传统荧光染料的替代分析试剂。通过使用 96 孔微孔板格式随时间监测荧光变化（λex，620nm；λem，680nm），获得了动力学曲线。维生素 E 类似物 6-羟基-2,5,7,8-四甲基色满-2-羧酸（Trolox）被选为模型分析物，并且衰减曲线下的归一化面积被用作分析参数。校准曲线的动态范围为 0.8-8.0 μM，检测限为 0.45 μM。该方法的精密度表示为相对标准偏差，使用 1 和 5 μM Trolox 浓度进行测定，分别为 5.6%和 2.9%。该方法已应用于果汁和葡萄酒的分析，得到的结果与使用荧光素作为试剂的 ORAC 方法提供的结果没有显着差异。
