Automatic evaluation of peroxidase activity using different substrates under a micro sequential injection analysis/lab-on-valve (μSIA-LOV) format

Abstract

In the present work, an automatic methodology, allowing the evaluation of peroxidase activity, and the comparison of the effect of two substrates in this activity, was developed. Automation was carried out using a miniaturized micro-sequential injection analysis system, usually called μSIA-LOV (Lab-on-valve). This kind of system allows downscaling of current flow injection and sequential injection systems. For the automatic evaluation of peroxidase activity it was selected two different reactions using different substrates - 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate (ABTS) and a mixture of 4-aminoantipyrine and phenol, that were implemented in a μSIA-LOV manifold. The experimental parameters such as reagent ratios and volumes, pH, temperature and reaction time, were optimized and kept constant for the two reactions. The use of these two reactions allowed the applicability of the system in two different ranges of concentrations (up to 0.20U mL−1 when it was used ABTS and up to 0.80U mL−1 for the reaction with 4-AAP and phenol) with different sensitivities and detection limits of 1.9×10−4U mL−1 and 9.2×10−3U mL−1 (using ABTS and 4-AAP with phenol, respectively). Horseradish, ginger and avocado samples were analyzed proving that the developed methodology constitutes a robust, versatile, rapid and simple alternative for the evaluation of peroxidase activity, adapted to the actual demands of reagents economy and reduced residue production, being in good agreement with the actual recommendations of green chemistry.