Sensitive ratiometric fluorescence assay for detecting xanthine in serum based on the inner filter effect of enzyme-catalyzed oxidation products to silicon nanoparticles.

A new type of fluorescent silicon nanoparticles (SiNPs) were prepared via a facile one-pot hydrothermal method by using N-[3-(trimethoxysilyl)propyl]-ethylenediamine (DAMO) and glucose as reagents, and were subsequently applied to construct a ratiometric fluorescence assay for sensitive and rapid determination of xanthine in human serum. Two catalytic oxidation reactions were employed to induce a fluorescence response of the testing system towards xanthine. Under the catalysis of xanthine oxidase (XOD), xanthine in serum samples was oxidized and produced hydrogen peroxide (H2O2). By utilizing o-phenylenediamine (OPD) as the substrate for horseradish peroxidase (HRP) in the presence of H2O2, fluorescent 2,3-diaminophenazine (DAP) was finally generated. A ratiometric fluorescence assay for xanthine was established by determining the ratio of the green-yellow fluorescence emission of DAP and the blue fluorescence emitted from SiNPs under the inner filter effect (IFE) of DAP. Instead of traditional multi-step procedures for adding reacting reagents to the testing solution, all the reaction reagents were mixed with serum samples in a single step for this assay to shorten the total reaction time. This assay demonstrates superiority over a solo DAP fluorescence-based assay as well as other reported methods, with excellent sensitivity and reduced testing time. The strategies proposed in this work for both synthesis and application of fluorescent SiNPs can be used in future fabrication of novel fluorescent probes, especially for sensing biological metabolites involved in H2O2-generation or consumption reactions.