Rapid determination of hydrogen peroxide produced by Lactobacillus using enzyme coupled rhodamine isocyanide/calcium...

A sensitive method for detecting hydrogen peroxide (H2O2) using rhodamine isocyanide incorporated calcium phosphate nanoparticles (Rho/CaP) was developed. The synthesized nanoparticles were characterized based on transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier transformed infrared spectroscopy (FTIR), Raman spectroscopy, energy-dispersive X-ray spectroscopy (EDX) and X-ray diffraction patterns (XRD). To study the application, the nanoparticles were functionalized with horse radish peroxidase (HRP) based on aminopropyl triethoxy silane (APTES) and used as tools to detect H2O2. The detection strategy was based on fluorescence quenching or colorimetric detection. The enzyme immobilized nanoparticles were titrated with different concentrations of H2O2 and a fixed concentration of O-phenylenediamine (OPD). The HRP conjugated Rho/CaP strongly catalyzed H2O2 oxidation of OPD that caused fluorescence quenching at 575 nm. For colorimetric detection, the OPD product was read at 492 nm. In the fluorescence quenching assay, the minimum detectable concentration was ~1 pmol in contrast to ~5 nmol in the colorimetric assay. The minimum detectable concentration by visual detection was ~500 nmol. The specificity of the developed assay method was examined with different interferences which did not produce any significant response. This assay was applied, along with a commercially available kit to compare the H2O2 production capacities of different Lactobacillus strains. The results indicated that the developed assay and commercially available kit methods were highly correlated. The fluorescence quenching kinetics is also discussed.