Sánchez-Martínez M L, Aguilar-Caballos M P, Gómez-Hens A
Department of Analytical Chemistry, University of Córdoba, Campus of Rabanales, Córdoba, Spain.
Talanta. 2009 Apr 15;78(1):305-9. doi: 10.1016/j.talanta.2008.11.014. Epub 2008 Nov 21.
A simple and rapid homogeneous enzyme immunoassay involving the use of the malic dehydrogenase enzyme and a long-wavelength fluorophor, the oxazine Cresyl Violet, is proposed for the determination of the antibiotic amikacin in water samples. An enzymatic tracer has been synthesized by covalent binding of amikacin to malic dehydrogenase via a carbodiimide derivative. Free tracer catalyses the reaction between Cresyl Violet and malic acid giving rise to a decrease in the fluorescence of the fluorophor. Kinetic curves for this reaction have been monitored at lambda(ex) 585 and lambda(em) 624 nm using the stopped-flow mixing technique, being the initial rate measured in only 2-3s. The dynamic range of the method is 1-15 ng mL(-1) and the detection limit is 0.3 ng mL(-1), using aqueous standard solutions or water samples. The precision, obtained at 1 and 5 ng mL(-1) and expressed as relative standard deviation, was 6.0 and 9.6%, respectively. The method has been applied to the analysis of drinking, river and wastewater samples. The sample pre-treatment involved a solid-phase extraction step for the clean-up of the samples. A recovery study was carried out to validate the method, being the values obtained in the range 80-114%, with a mean value of 96.7%.
本文提出了一种简单快速的均相酶免疫分析法，该方法使用苹果酸脱氢酶和长波长荧光团恶嗪甲酚紫来测定水样中的抗生素阿米卡星。通过碳二亚胺衍生物将阿米卡星与苹果酸脱氢酶共价结合，合成了一种酶标记物。游离的标记物催化恶嗪甲酚紫与苹果酸之间的反应，导致荧光团的荧光强度降低。使用停流混合技术，在激发波长585nm和发射波长624nm处监测该反应的动力学曲线，初始速率仅在2 - 3秒内即可测定。使用水溶液标准溶液或水样时，该方法的动态范围为1 - 15 ng mL(-1)，检测限为0.3 ng mL(-1)。在1和5 ng mL(-1)水平下获得的精密度，以相对标准偏差表示，分别为6.0%和9.6%。该方法已应用于饮用水、河水和废水样品的分析。样品预处理包括固相萃取步骤以净化样品。进行了回收率研究以验证该方法，回收率在80 - 114%范围内，平均值为96.7%。
