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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]

We propose catalytically synthesized nanozymes based on Prussian Blue (PB) and azidomethyl-substituted poly (3,4-ethylenedioxythiophene) (azidomethyl-PEDOT) as novel electrocatalytic labels for DNA/RNA sensors. Catalytic approach allowed to synthesize highly redox and electrocatalytically active Prussian Blue nanoparticles functionalized with azide groups that enable 'click' conjugation with alkyne-modified oligonucleotides. Both competitive and sandwich-type schemes were realized. As the sensor response the direct (mediator-free) electrocatalytic current of H 2 O 2 reduction can be measured, which is proportional to the concentration of the hybridized labeled sequences. The current of H 2 O 2 electrocatalytic reduction is only 3–8 times increased in the presence of the freely diffusing mediator catechol, which indicates high efficiency of direct electrocatalysis with the elaborated labels. Electrocatalytic amplification of the signal allows robust detection of (63–70)-base target sequences with concentrations below 0.2 nM in blood serum within an hour. We believe, the use of advanced Prussian Blue based electrocatalytic labels sets new avenues for point-of-care DNA/RNA sensing. [Display omitted] • Catalytically synthesized functionalized nanozymes possess high catalytic activity. • Nanozyme functionalization with azide groups enables 'click' conjugation. • Efficient direct electrocatalysis with the labels provides mediator-free operation. • Electrocatalytic signal amplification allows detecting down to 0.01 nM of DNA. [ABSTRACT FROM AUTHOR]

The ability of Prussian Blue, ferric hexacyanoferrate (FeHCF), to sensitively and selectively detect hydrogen peroxide by its reduction in the presence of oxygen is of high importance for analytical chemistry. Success with Prussian Blue (PB) provided an appearance of contradictory reports concerning electrocatalysis of the other transition metal hexacyanoferrates (HCFs) in H2O2 reduction. Investigating thermodynamics of the catalyzed reactions as well as electrochemical properties of the hexacyanoferrates, we are able to conclude that the noniron hexacyanoferrates themselves are completely electrocataryrically inactive, except for a minor electrocatalysis in the opposite reaction, hydrogen peroxide oxidation, registered for NiHCF. Concerning the most important reaction, H2O2 reduction, the observed electrocatalytic activity (by the way, 100 times decreased compared to PB) is due to the presence of FeHCF (Prussian Blue) as defects in the structure of noniron hexacyanoferrates. This finding, considering other unique properties of transition metal HCFs, will provide a systematic search for sensing materials with improved analytical performance characteristics. [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]