Ortega E, de Marcos S, Sanz-Vicente I, Ubide C, Ostra M, Vidal M, Galbán J
Analytical Biosensors Groups, Analytical Chemistry Department, Faculty of Sciences, Aragon Institute of Nanoscience, University of Zaragoza, 50009 Zaragoza, Spain.
Departamento de Química Aplicada, Facultad de Química, Universidad del País Vasco, Manuel de Lardizábal 3, 20018 San Sebastián, Spain.
Talanta. 2016 Jan 15;147:253-60. doi: 10.1016/j.talanta.2015.09.060. Epub 2015 Sep 30.
Choline oxidase (ChOx) is a flavoenzyme catalysing the oxidation of choline (Ch) to betaine aldehyde (BA) and glycine betaine (GB). In this paper a fundamental study of the intrinsic fluorescence properties of ChOx due to Flavin Adenine Dinucleotide (FAD) is presented and some analytical applications are studied in detail. Firstly, an unusual alteration in the excitation spectra, in comparison with the absorption spectra, has been observed as a function of the pH. This is ascribed to a change of polarity in the excited state. Secondly, the evolution of the fluorescence spectra during the reaction seems to indicate that the reaction takes place in two consecutive, but partially overlapped, steps and each of them follows a different mechanism. Thirdly, the chemical system can be used to determine the Ch concentration in the range from 5×10(-6)M to 5×10(-5)M (univariate and multivariate calibration) in the presence of BA as interference, and the joint Ch+BA concentration in the range 5×10(-6)-5×10(-4)M (multivariate calibration) with mean errors under 10%; a semiquantitative determination of the BA concentration can be deduced by difference. Finally, Ch has been successfully determined in an infant milk sample.
胆碱氧化酶（ChOx）是一种黄素酶，可催化胆碱（Ch）氧化为甜菜碱醛（BA）和甘氨酸甜菜碱（GB）。本文对黄素腺嘌呤二核苷酸（FAD）引起的ChOx的固有荧光特性进行了基础研究，并详细研究了一些分析应用。首先，与吸收光谱相比，观察到激发光谱随pH值的变化出现异常改变。这归因于激发态极性的变化。其次，反应过程中荧光光谱的演变似乎表明该反应分两个连续但部分重叠的步骤进行，且每个步骤遵循不同的机制。第三，该化学体系可用于在存在BA干扰的情况下，测定5×10⁻⁶M至5×10⁻⁵M范围内的Ch浓度（单变量和多变量校准），以及5×10⁻⁶至5×10⁻⁴M范围内的Ch + BA联合浓度（多变量校准），平均误差在10%以下；通过差值可推导出BA浓度的半定量测定结果。最后，已成功测定了婴儿奶粉样品中的Ch含量。
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