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. 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 meet both criteria, which makes them prominent candidates for analyses in biological matrices. The procedure noted as catalytic synthesis consists in controlled reduction of Fe[Fe(CN)6]. Using H2O2 as the reductant, on the one hand, forms pure PBNPs with minimal impurities as oxygen, the product of H2O2 oxidation, leaves the reaction mixture. This protocol yields PBNPs with record catalytic activity, beating HRP in terms of turnover number by up to 4 orders of magnitude [1]. On the other hand, monomers of conductive polymers are also applicable as reductants. Undergoing oxidative polymerization, they reduce Prussian Green to Prussian Blue, thus forming composite PB/conductive polymer NPs. By using monomers with different functional groups, PBNPs can be functionalized at the stage of synthesis. Moreover, diameter of noted PBNPs can also be controlled by simply varying the concentration of reactants. Both catalytic and electrocatalytic activity of noted PBNPs are notable. PBNPs possess record catalytic activity with size-dependent catalytic constants, which are comparable and exceeding that of HRP by up to four orders of magnitude. Simple drop-casting of PBNPs colloidal solution followed by annealing at 100 °C results in ready-to-use H2O2 electrochemical sensor. Sensitivity of noted sensors (0.85 A‧M-1‧cm-2) is higher than that of PB film based sensors by 30% and allows detecting submocromolar H2O2 concentrations. Notably, modifying PBNPs with conductive polymers barely decreases their (electro)catalytic activity while significantly improving operational stability. Electrochemical H2O2 sensors based on PB/p-(3-aminophenylboronic acid) NPs retain 90% of their initial signal twice as long as sensors based on non-functionalized PBNPs [2]. Achieved sensitivity of 0.85 A⋅M-1⋅cm-2 can be further increased with carbon black nanoparticles (CBNPs), resulting in record sensitivity of 1.15 A⋅M-1⋅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 one order of magnitude higher sensitivity and three times extended operation time [3]. Azide-modified PBNPs were successfully bioconjugated with alkene-modified oligonucleotide fragments through copper(I)-mediated 1,3-dipolar cycloaddition. Obtained conjugates were used in a prototype of electrochemical DNA sensor. Practical possibility of detection of conjugates’ hybridization with immobilized DNA probes was shown, thus allowing development of universal DNA/RNA sensors. The detection limit of oligonucleotides in model systems does not exceed 100 pM. Amine- and carboxy-modified PBNPs were linked to rabbit and donkey antibodies by means of N,N’-dicyclocarbodiimide for direct electrochemical immunoassay trials. Antibodies forming affine complexes seemingly separate the electrode surface and PBNPs, which dramatically decreases the electrocatalytic current of H2O2 reduction in noted immunoassay trials. However, this problem was solved by using a redox mediator, catechol. In presence of catechol, at concentrations of conjugates around 100-200 pmol/cm2 the electrochemical signal is an order of magnitude higher than that of control experiments. This shows the potential of PBNPs as potential catalytic labels in various immunoassays. References [1] Komkova M.A., Karyakina E.E., Karyakin A.A., J. Am. Chem. Soc 2018, 140, 11302–11307 [2] Komkova M.A., Zarochintsev A.A., Karyakina E.E., Karyakin A.A., J. Electroanal. Chem. 2020, SI(872), 114048 [3] Komkova M.A., Andreeva K.D., Zarochintsev A.A., Karyakin A.A. ChemElectroChem 2021, 8, 1117-1122 Acknowledgements. This work was supported by the Russian Science Foundation (project No 21-73-10123).