Long-Wavelength Fluorescence Polarization Immunoassay: Determination of Amikacin on Solid Surface and Gliadins in Solution
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- 作者：Marí ; a Lourdes Sá ; nchez-Martí ; nez ; Marí ; a Paz Aguilar-Caballos ; Agustina Gó ; mez-Hens
- 刊名：Analytical Chemistry
- 出版年：2007
- 出版时间：October 1, 2007
- 年：2007
- 卷：79
- 期：19
- 页码：7424 - 7430
- 全文大小：111K
- 年卷期：v.79,no.19(October 1, 2007)
- ISSN：1520-6882
文摘
The versatility of the fluorescence polarization immunoassay (FPIA) is increased by using two long-wavelengthlabels, Nile Blue and a ruthenium(II) chelate. The firstlabel has been used to study the potential of FPIA on asolid surface using dry reagent technology. The aminoglycoside antibiotic amikacin has been used as an analytemodel, and the method has been applied to the analysisof serum samples. The second label has been used toshow the practical application of FPIA to the determination of macromolecules, using gliadins as an analytemodel, which have been determined in gluten-free food.Very low amounts of anti-amikacin antibodies and amikacin-Nile Blue tracer were immobilized onto nitrocellulose membranes, for the development of the amikacinmethod, and the consumption of reagents is lower thanin conventional FPIA. Only the addition of the standardor sample extract at an adequate pH is required at theanalysis time. The analyte displaces the tracer from thetracer-antibody immunocomplex, obtaining a decreasein the fluorescence polarization proportional to the analyteconcentration. The gliadin-Ru(II) chelate tracer shows arelatively long lifetime, which allows the observation ofdifferences in fluorescence polarization values betweenthe tracer-antibody complex and the tracer alone. Thedynamic range of the calibration graphs for both analytesis 0.5-10 g mL-1 and the detection limits are 0.1 and0.09 g mL-1 for amikacin and gliadins, respectively. Thestudy of the precision gave values of relative standarddeviations lower than 5 and 1.5% for the amikacin andgliadin methods, respectively. Amikacin was determinedin human serum samples using a previous deproteinization step with acetonitrile, obtaining recovery values inthe range 83.4-122.8%. The gliadin method was appliedto the analysis of gluten-free food samples by using aprevious extraction step. The recovery study gave valuesbetween 94.3 and 105.0%.