A fluorometric enzyme-linked immunosensing system using 4-(p-hydroxystyryl)pyridine as a new substrate of horseradish peroxidase for Brucella melitensis antibody assay

A novel substrate, 4-(p-hydroxystyryl)pyridine (pHSP) was synthesized and firstly used in a horseradish peroxidase (HRP) based fluorometric enzyme-linked immunosensing system. The results of the property investigation of pHSP demonstrate the advantages in stability and reactivity with HRP over conventional substrates such as p-hydroxyphenylacetic acid (p-HPA), Amplex red and chavicol. In a pH 5.8 Britton-Robinson buffer solution, HRP-antibody conjugate (HRP-IgG) could catalyze the oxidation reaction of pHSP by H2O2, and the pHSP was converted to significantly fluorescent dimers. The increase of. the fluorescence intensity (excitation: 300 nm, emission: 437 nm) of the HRP enzymatic product is proportional to the concentration of HRP-IgG binding to the Brucella melitensis antigen modified matrix. An enzyme-linked immunosensing method based on this principle was developed. The linear range of determination is 6.3 x 10(-11) similar to 1 x 10(-8) mol(.)L(-1) with the relative standard deviation of 4.1%. The detection limit is 6.3 x 10(-11) mol(.)L(-1). The another obvious advantage of the proposed procedure is the sensitivity deriving from the solution of the insoluble substrate determination.