Long-wavelength fluorimetry as an indirect detection system in immunoaffinity chromatography: application to environmental analysis

Sánchez-Martínez, M. L.; Aguilar-Caballos, M. P.; Eremin, S. A.; Gómez-Hens, A.
December 2006
Analytical & Bioanalytical Chemistry;Dec2006, Vol. 386 Issue 5, p1489
Academic Journal
The potential of long-wavelength fluorimetry when used as the detection system in immunoaffinity chromatography is assessed for the first time by applying this approach to the analysis of water and sludge samples. Nile blue (NB) was used to synthesize a long-wavelength fluorescent tracer for linear alkylbenzenesulfonates (LASs) using the carbodiimide method, in which the amino group of NB is covalently coupled to the activated carboxylic acid group of a LAS mimic with N-hydroxysuccinimide and dicyclohexylcarbodiimide. The method consists of the injection of a pre-incubated mixture containing linear sodium 4-dodecylbenzenesulfonate (LDS; used as the LAS model), anti-LAS antibodies, and the long-wavelength tracer into a commercial Protein G column. Free and bound tracer fractions are separated in the column, and the peak height of the immunochromatogram (corresponding to the free tracer) is directly measured at 626 nm ( λ ex) and 674 nm ( λ em), and then correlated to the analyte concentration. It is not necessary to perform an elution step immediately after every sample application. The dynamic range of the method is 0.05–2.5 μg ml−1 LDS, and the detection limit is 15 ng ml−1. The precision, expressed as the relative standard deviation, is 4.8–6.4%. Other surfactants (sodium dodecylsulfate and Triton X-100) do not cause interference. The recoveries obtained by applying the method to the analysis of water (ground- and wastewater) and sludge (primary and activated) samples ranged from 86.0 to 111.3%. Water sample analysis included an initial solid-phase extraction step, which cleaned up the samples and improved the detection limit fivefold.


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