ИСТИНА

Inspired by nature and aiming to overcome the high cost and low stability of enzymes, nanozymes – nanomaterials mimicking enzymatic activity – have emerged as a distinct branch of biomimetics [1]. Nanozymes with peroxidase-like activity are of particular interest as horseradish peroxidase (HRP) is the most widely used enzyme in both biotechnology and medical analysis. Unfortunately, two essential criteria to be eligible for either application, catalytic selectivity and high activity in pH 7.0-7.4, aren’t met by an overwhelming majority of known nanozymes. However, Prussian Blue nanoparticles (PBNPs) obtained through catalytic synthesis (by reducing Fe[Fe(CN)6] with H2O2) meet both criteria, surpassing turnover numbers of natural HRP by up to 4 orders [2]. Electrochemical properties of catalytically synthesized PB based nanozymes are notable. Simple drop-casting of their colloidal solution followed by annealing at 100 °C results in a ready-to-use H2O2 sensor. Its sensitivity, which reaches 0.85 A⋅M⋅cm-2, exceeding that of PB film by 30%, allows detecting sub-micromolar concentrations of H2O2 [3]. This value of sensitivity apparently corresponds to the formation of a continuous monolayer of PBNPs: while being linearly dependent on the deposited amount of PBNPs at low concentrations, sensitivity plateaus as the concentration of PBNPs increases. Charge transfer resistance, which was calculated from electrochemical impedance spectra, linearly decays at low PBNPs concentration, also reaching its lower limit in the similar concentration region. Achieved sensitivity of 0.85 A⋅M⋅cm-2 can be further increased with carbon black nanoparticles (CBNPs), resulting in record sensitivity of 1.15 A⋅M⋅cm-2, almost doubling the sensitivity of PB film based sensors. Both glucose and lactate oxidases were co-immobilized with PBNPs-CBNPs mixture. The aforementioned drop-casting approach results in biosensors advantageous over conventional sensors produced upon layer-by-layer immobilization in terms of two times higher sensitivity and three times extended operation time. Functionalization of catalytically synthesized PBNPs would allow using them as catalytic labels, significantly extending their potential applications. However, modifying the surface of PBNPs drastically decreases their catalytic activity, as the diffusion of substrates to their surface is disrupted. This problem is solved with a modification of catalytic synthesis of PBNPs, which allows their functionalization at the stage of synthesis while retaining their ultrahigh activity. This is achieved by swapping the reducing agent (originally H2O2) for monomers of conductive polymers. This modification of the “catalytic synthesis” protocol allows functionalization of PBNPs with amine, azide, boronate, sulphonate and carboxylate functional groups. Furthermore, drop-casting of polymer-modified PBNPs results in considerately more operationally stable sensors. Such sensors retain 95% of their initial signal twice longer than those based on unmodified PB NPs. Azide-modified PBNPs were successfully bioconjugated with alkene-modified HULC gene fragments through copper(I)-mediated 1,3-dipolar cycloaddition. Practical possibility of DNA hybridization detection, and thus, possibility of applying the noted PBNPs as electrocatalytic labels, was shown. The detection limit of oligonucleotides in model systems does not exceed 100 pM. Financial support through Russian Science Foundation grant # 19-73-00166 is greatly acknowledged.