ИСТИНА

There is an increasing interest in mimicking peroxidase activity. However, real analytical applications became possible only after the discovery of nanoparticles with peroxidase-like activity (catalysis of hydrogen peroxide reduction), later referred to as nanozymes. The main disadvantages of the reported metal oxide and noble metal based nanozymes are their low specificity in addition to their oxidase-like and catalase-like activities. Moreover, in physiological solutions (pH 7.0 – 7.5) commonly used for bioanalytical applications such nanozymes are catalytically inactive. Prussian Blue (PB) is recognized as the most advantageous electrocatalyst for hydrogen peroxide reduction in neutral media over all known systems [1, 2] and, thus, is favorable for bioanalytical applications. In the present study we synthesized Prussian Blue nanoparticles in course of reduction of ferricyanide, [Fe(CN)6]3−, and ferric ions, Fe3+ mixture either by hydrogen peroxide, or by conducting polymer forming organic molecules. In this case the precipitation is caused by the same reaction that occurs upon electrocatalysis, which results in formation of Prussian Blue nanoparticles with the highest catalytic activity [3]. Kinetic properties of the catalytically synthesized PB nanoparticles allow to conclude that they defeat natural enzyme peroxidase. Peroxidase activity of nanoparticles and the enzyme has been investigated oxidizing 3,3’,5,5’-tetramethylbenzidine (TMB), apparently the fastest substrate of the enzyme peroxidase. The initial reaction rate of hydrogen peroxide reduction, catalyzed by PB nanoparticles, is linearly dependent on H2O2 concentration, which has never been observed either for peroxidase-like nanozymes, or for natural peroxidase. This indicates that substrate (H2O2) activation by PB nanoparticles occurs much faster even compared to the natural enzyme. Moreover, importantly, in the absence of hydrogen peroxide no oxidation of TMB has been registered in its entire concentration range. Hence, the obtained PB nanoparticles do not display oxidase-like activity (reduction of molecular oxygen). Catalytic constants in H2O2 reduction for catalytically synthesized PB nanoparticles are nearly volume dependent (the slope of size dependence in double logarithmic plots exceeds 2.5) indicating that hydrogen peroxide penetrates the bulk of nanoparticles. Most importantly, for all sizes of PB nanoparticles the catalytic rate constants are higher, than for natural peroxidase (for PB nanoparticles 200 nm in diameter the turnover number is 300 times higher). Except for the activity defeating even the natural enzyme catalytically synthesized PB nanoparticles are highly active even at pH 7.4, and kcat evaluated for TMB as a substrate at pH 7.4 is less than 1.5 times lower than in pH 5.0 used for kinetic investigations. Drop-casting of the nanoparticles suspension with subsequent drying allows to modify the electrodes. The latter, similarly to conventional Prussian Blue film modified electrodes, display the two sets of peaks corresponding to Prussian Blue|Prusssian White and Berlin Green|Prussian Blue redox transitions in their cyclic voltammograms. Analytical properties of PB nanoparticles modified electrodes were investigated at constant potential (E = 0.00 V) providing hydrogen peroxide reduction. Linear calibration range is prolonged over more than 3 orders of magnitude of H2O2 concentrations. Sensitivity evaluated as a slope of the calibration graph is of 0.85 A·M·cm-2 (30% higher compared to it for similar three-electrode structure modified with PB film). The improved analytical performance characteristics of the PB nanoparticles modified electrodes are most probably due to their increased roughness providing more efficient mass transfer compared to conventional PB films. Financial support through Russian Foundation for Basic Research grant # 18-33-00392 is greatly acknowledged. References: 1. Karyakin A.A. Advances of Prussian blue and its analogues in (bio)sensors // Current Opinion in Electrochemistry, 2017, 5(1), p. 92. 2. Sitnikova N.A., Komkova M.A., … A.A. Karyakin, Transition Metal Hexacyanoferrates in Electrocatalysis of H2O2 Reduction: An Exclusive Property of Prussian Blue // Analytical Chemistry, 2014, 86(9), pp. 4131-4134. 3. Komkova M.A., Karyakina E.E., Karyakin A.A. et al. Catalytically Synthesized Prussian Blue Nanoparticles Defeating Natural Enzyme Peroxidase // JACS, 2018, 140, pp. 11302-11307.