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The pathway from the advanced electrocatalyst to nanozymes defeating natural enzyme is reviewed. Prussian blue, being the most advantageous electrocatalyst for hydrogen peroxide reduction, is obviously the best candidate for mimicking peroxidase activity. Indeed, catalytically synthesized Prussian blue nanoparticles are characterized by the catalytic rate constants, which are significantly (up to 4 orders of magnitude) higher than for enzyme peroxidase. Displaying in addition the enzymatic specificity in terms of an absence of oxidase-like activity, catalytically synthesized Prussian blue nanoparticles can be referred to as nanozymes. The latter provide the most versatile method for surface covering with the electrocatalyst, allowing to modify non-traditional materials like boron-doped diamond. For stabilization, Prussian blue core can be covered with nickel hexacyanoferrate shell; the resulting core-shell nanozymes still defeat natural enzyme in terms of activity. Discovering the catalytic pathway of nanozymes "artificial peroxidase" action, we have found the novel advantage of nanozymes over the corresponding biological catalysts: their dramatically (100 times) improved bimolecular rate constants., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

We report on the nanoparticles composed of the catalytically synthesized Prussian Blue (PB) core stabilized with the nickel hexacyanoferrate (NiHCF) shell. Catalyzing hydrogen peroxide reduction, the resulting nanozymes (ø = 66 nm) display catalytic rate constants, which for pyrogallol or ferrocyanide are, respectively, 25 and 35 times higher than those for peroxidase enzyme. After more than half a year of storage at a room temperature, the core-shell PB-NiHCF nanozymes retain both their size and physicochemical properties; such stability is unreachable for the enzymes. Being immobilized, core-shell PB-NiHCF nanozymes (ø = 45 nm) result in a hydrogen peroxide sensor with a sensitivity similar to that of the sensor based on sole PB nanoparticles. However, whereas the latter response in hard inactivating conditions (25 min in 1 mM H 2 O 2 ) drops down to 7.5%, the PB-NiHCF nanozymes-based sensor retains >75% of initial sensitivity. Application of the core-shell PB-NiHCF nanozymes "artificial peroxidase" would obviously open new horizons in elaboration of anti-inflammatory drugs and (bio)sensors.

We report on the kinetic mechanism of the catalytically synthesized Prussian Blue nanoparticles denoted as "artificial peroxidase". In contrast to the enzyme, whose active site first interacts with hydrogen peroxide forming the so-called Compound I, in the case of the nanozymes, H 2 O 2 oxidizes their complex with reducing substrate. Slow release of the product (oxidized form of the latter) from the nanozymes has been registered. The interaction of substrates with the nanozymes is 100 times faster than with enzyme peroxidases, and the rate-limiting constant for the nanozymes is also 2 orders of magnitude greater: for pyrogallol k 2 = 1.3 ± 0.1 × 10 8 M -1 s -1 and for ferrocyanide k 2 = 1.9 ± 0.1 × 10 8 M -1 s -1 . Thus, the discovered novel advantage of nanozymes over the corresponding enzymes is the 100-fold greater bimolecular rate constants, resulting, most probably, from their uniformly accessible surface, avoiding the effect of rotation on the diffusion-controlled rate.

In contrast to bienzyme biosensors, we propose the nanozyme-enzyme based ones substituting the enzyme peroxidase with the more active and stable nanoparticles "artificial peroxidase". The use of catalytically synthesized Prussian Blue based nanozymes simplifies assembling of hydrogen peroxide transducer providing its higher sensitivity. For immobilization of lactate oxidase the composite alkoxysilane - perfluorosulfonated ionomer (PFSI) membranes are proposed achieving the significantly improved operating stability. The resulting nanozyme-enzyme lactate biosensor displays twice higher sensitivity (>0.2 A M -1  cm -2 ) compared to the Prussian Blue film based one. Nanozymes "artificial peroxidase" are expected to find wide use in elaboration of oxidase based biosensors., (Copyright © 2019 Elsevier B.V. All rights reserved.)

We synthesized Prussian Blue (PB) nanoparticles through catalytic reaction involving hydrogen peroxide (H 2 O 2 ) activation. The resulting nanoparticles display the size-dependent catalytic rate constants in H 2 O 2 reduction, which are significantly improved compared to natural enzyme peroxidase: for PB nanoparticles 200 nm in diameter, the turnover number is 300 times higher; for 570 nm diameter nanoparticles, it is 4 orders of magnitude higher. Comparing to the known peroxidase-like nanozymes, the advantages of the reported PB nanoparticles are their true enzymatic properties: (1) enzymatic specificity (an absence of oxidase-like activity) and (2) an ability to operate in physiological solutions. The ultrahigh activity and enzymatic specificity of the catalytically synthesized PB nanoparticles together with high stability and low cost, obviously peculiar to noble metal free inorganic materials, would allow the substitution of natural and recombinant peroxidases in biotechnology and analytical sciences.

We report on fully electrochemical flow-through synthesis of Prussian Blue based nanozymes defeating peroxidase in terms of more than 200 times higher catalytic rate constant (kcatapp = 6 x 104 s-1). Being reagentless, reproducible, simple and scalable, the proposed approach blazes new trails for the electrosynthesis of functional conductive and electroactive nanomaterials. [ABSTRACT FROM AUTHOR]

We report the single‐step fabrication of advanced electrochemical biosensors upon drop‐casting of "artificial peroxidase", that is, oxidase‐containing mixtures. Compared to bienzyme biosensors, we substitute peroxidase with more active and stable nanozymes based on catalytically synthesized Prussian Blue. The electroactivity of nanozymes in the sensing membrane was facilitated with carbon black nanoparticles, which resulted in record sensitivity of the H2O2 transducer exceeding 1.1 A M−1 ⋅ cm−2. Both glucose and lactate oxidases were co‐immobilized with nanozymes in alkoxysilane or perfluorosulfonated ionomer membranes. The proposed drop‐casting approach results in biosensors that are advantageous over those conventionally produced upon layer‐by‐layer immobilization in terms of i) almost an order of magnitude increased sensitivity, ii) unsurpassed biosensor‐to‐transducer sensitivity ratio reaching the value of 0.3, and iii) three times extended operational lifetime. [ABSTRACT FROM AUTHOR]

• Prussian Blue based nanozymes catalyze reduction of organic peroxides. • Nanozymes are significantly more active than horseradish peroxidase. • Selectivity of nanozymes to H 2 O 2 is higher as compared to peroxidase. Prussian Blue based nanozymes are able to catalyze reduction of organic peroxides similarly to the enzyme peroxidase. For the whole range of peroxides under study the catalytic rate constant for 3,3′,5,5′-tetramethylbenzidine in case of nanozymes is significantly higher as compared to horseradish peroxidase. Moreover, the selectivity of nanozymes towards hydrogen peroxide relatively organic peroxides reduction is higher than that for the enzyme, which can be explained in terms of different ability of peroxides to penetrate to the bulk of catalytically synthesized Prussian Blue nanoparticles. On the contrary, selectivity of Prussian Blue film modified electrodes is much lower and even negligible among hydrogen, urea and methyl ethyl ketone peroxides. An ability to tune the selectivity would allow individual detection of peroxides in their complex mixtures with Prussian Blue nanostructures. [ABSTRACT FROM AUTHOR]

Simple drop-casting of the catalytically synthesized Prussian Blue nanoparticles with subsequent annealing results in modified electrodes with high electrochemical and electrocatalytic activity. The electroactive fraction of the deposited Prussian Blue being independent of the surface concentration, is decreased with an increase of the nanoparticles size reaching its lower saturation at approximately 25%. Sensitivity of the corresonding hydrogen peroxide sensor is increased linearly with the amount of Prussian Blue reaching its upper saturation together with formation of continuative film, indicating most probably, a nanoparticles monolayer. Due to their performance characteristics the Prussian Blue nanozymes modified electrodes would be highly attractive for corresponding sensors and biosensors. [ABSTRACT FROM AUTHOR]