ИСТИНА

Nowadays nanozymes – inorganic nanoparticles mimicking enzymatic activity – are an important object of research. For instance, using peroxidase-mimicking nanozymes as catalytic labels for immunoassay may allow substituting costly and unstable horseradish peroxidase (HDP), the most widely used label at the moment. Such nanoparticles must possess enzymatic specificity and retain high activity in physiological pH (7.0-7.5). Among known nanozymes1 both characteristics are intrinsic only to “catalytically synthesized” Prussian Blue based nanozymes. Their peroxidase-like activity exceeds even that of HDP2, however, for immunoassay applications nanozymes must be functionalized to conjugate them with an antibody. In the present work we suggest a method of the nanozymes’ functionalization by using monomers (aniline and 3,4-(2-(azidomethyl)ethylenedioxy)thiophene) of conductive polymers as reductants. While reducing the Fe[Fe(CN)6] complex into Prussian Blue, monomers undergo oxidative polymerization and form conductive polymers. This allows functionalizing the nanoparticles on the stage of their synthesis and controlling their diameter (32-59 nm) by varying the concentrations of reagents. The nanoparticles formed in this reaction are indeed composite ones, which was confirmed by Raman spectra containing characteristic signals of both Prussian Blue and corresponding polymers. Moreover, their colloidal stability is significantly increased compared to those obtained earlier, which can be illustrated by measurements of zeta potential, which is up to three times more (-33.3 mV). In addition, X-ray powder diffraction pattern confirms that obtained nanozymes are of Prussian Blue crystalline structure. Kinetics of H2O2 reduction catalyzed by obtained nanoparticles in the presence of 3,3’,5,5’-tetramethylbenzydine, most common HDP substrate were investigated spectrophotometrically by accumulation of TMB2+. Dependency of initial reaction rate on TMB concentration obeys Michaelis-Menten equation. Calculated kcat are volume dependent, thus, H2O2 penetrates the bulk of nanoparticles. For 46 nm diameter particles kcat is 460 s-1, which is more than two times more than kcat for HDP in identical conditions. Moreover, in the absence of H2O2 no TMB oxidation was registered, indicating that obtained nanozymes do not show oxidase activity. Modification of nanoparticles with azide and amino functional groups will allow linking them with antibodies. Together with their high stability and specificity of the obtained nanoparticles it may allow for substitution of natural and recombinant peroxidases in immunoassays.