Study of a fluorometric-enzymatic method for bilirubin based on chemically modified bilirubin-oxidase and multivariate calibration
Study of a fluorometric-enzymatic method for bilirubin based on chemically modified bilirubin-oxidase and multivariate calibration
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DOI：
10.1016/s0039-9140(02)00023-1
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发表时间：
2002-05-16
期刊：
影响因子：
6.1
通讯作者：
Castillo, JR
中科院分区：
文献类型：
--
作者：
Andreu, Y;Ostra, M;Castillo, JR
胆红素氧化酶（Box）与荧光素衍生物（FS）的化学衍生化产生化学修饰的酶（BOx-FS），其激发和发射最大值分别在487和520 nm处。在胆红素的氧氧化反应期间，在修饰酶的存在下，修饰酶的荧光的变化取决于胆红素的浓度和类型。这种效应可用于分析目的。首先，对分析系统进行了理论-实验研究。阐明了荧光变化的机理，建立了数学模型，优化了影响荧光变化的变量。建立了三种胆红素的浓度范围（最高可达12 mg胆红素1（-1））和测量精度（约4%）。应用多元校正方法对整个动力学曲线进行校正，研究了该方法在直接和总胆红素同时测定中的应用。一个减少校准矩阵（来自一个53基地矩阵）提出了校准和不同的数值方法进行了测试：主成分回归（PCR），偏最小二乘回归（PLS）和人工神经网络（ANN）。直接胆红素和总胆红素的同时测定（平均验证误差分别约为9%和10%）可在单次运行中进行。此外，可以同时获得三种胆红素（游离胆红素、结合胆红素和白蛋白结合胆红素）的半定量形态。(C)2002 Elsevier Science B. V.保留所有权利。
The chemical derivatization of bilirubin oxidase (Box) with a fluorescein derivative (FS) yields a chemically modified enzyme (BOx-FS), with excitation and emission maxima at 487 and 520 nm, respectively. During the oxygen oxidation reaction of bilirubins, in the presence of the modified enzyme, the change in the fluorescence of the modified enzyme depends on the concentration and type of bilirubin. This effect can be used for analytical purposes. Firstly, a theoretical-experimental study of the analytical system was carried out. The mechanism responsible for the fluorescence variation was clarified, a mathematical model developed and the variables affecting the fluorescence changes optimized. The concentration ranges in which the model can be applied (up to 12 mg bilirubin 1(-1)), and the precision of the measurement (about 4%) were established for the three bilirubins. The application of the methodology to the simultaneous determination of direct and total bilirubins were studied by applying multivariate calibration methods to the whole kinetic profiles. A reduced calibration matrix (derived from a 53 base matrix) is proposed for calibration and different numerical methods were tested: Principal Components Regression (PCR), Partial Least Squares Regression (PLS) and Artificial Neural Networks (ANN). The simultaneous determination of direct and total bilirubin (average validation errors of about 9 and 10%, respectively) can be carried out from a single run. Furthermore, a semi-quantitative speciation of the three bilirubins (free, conjugated and albumin-bonded bilirubin) may be simultaneously obtained. (C) 2002 Elsevier Science B.V. All rights reserved.