**1. Introduction**

Artificial enzymes are stable and low-cost mimetics of natural enzymes. The search for effective novel artificial enzymes, especially nanozymes, and the development of simple methods for their synthesis and characterization, as well as the selection of novel branches for their application, are currently challenging problems in different fields of biotechnology, industry, and medicine [1–9].

Peroxidase (PO) mimetics are the most frequently investigated artificial enzymes [10–12]. One of the well-known effective PO-like artificial enzymes is Prussian blue (PB) or iron(III) hexacyanoferrate (FeHCF). PB is a member of a well-documented family of synthetized coordination compounds with an extensive 300-year history [13–16]. PB and its analogs (PBAs) are cheap and easy to synthesize, environmentally friendly, and have potential applications for basic research and industrial purposes [12–18] in a large variety of fields,

**Citation:** Gayda, G.Z.; Demkiv, O.M.; Gurianov, Y.; Serkiz, R.Y..; Klepach, H.M.; Gonchar, M.V.; Nisnevitch, M. "Green" Prussian Blue Analogues as Peroxidase Mimetics for Amperometric Sensing and Biosensing . *Biosensors* **2021**, *11*, 193. https://doi.org/10.3390/bios11060193

Received: 30 April 2021 Accepted: 9 June 2021 Published: 10 June 2021

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particularly in medicine [13,19–23]. Despite their multifunctionality, PBAs have complicated compositions, which are largely dependent on the synthesis methods and storage conditions [14–16]. Insoluble PB can be described by the formula Fe4[Fe(CN)6]3, while KFe[Fe(CN)6] corresponds to a colloidal solution of PB. The general formula of hexacyanoferrate (HCF) is Mk[Fe(CN)6] × H2O, where M is a transition metal [14,15].

Due to their capability to insert various ions as counter-ions during the redox process, PB and PBAs have attracted increasing interest as electrode materials for energy storage in fuel cells [15,24,25]. Having remarkable super-magnetic properties, redox and PO-like activities, PBAs are widely applied in bioreactors for detoxification of dangerous chemicals [13,17,26], in molecular magnets, and in optical and electrochemical biosensors [12–16,24,27].

The first report of electrochemical reduction of H2O2 on PB-modified electrodes was published by Itaya in 1984 [28]. In 2000, Karyakin named PB as an "artificial PO" and published numerous reports concerning PB-based amperometric biosensors (ABSs) [24,29–33]. Numerous other scientific groups, especially from China, have also worked diligently on this problem [18–23,34–37].

PBAs are usually obtained via various techniques, including chemical [12–16] and biological methods [38–40]. The biosynthesis of materials using plants, microorganisms and their metabolites as biosurfactants can be related to "green synthesis (GS) [41–44]. Purified enzymes were also shown to be capable of reducing metal ions to obtain metallic nanoparticles [40,45,46].

The application of green-synthesized PBAs (gPBAs or gHCFs) for the construction of ABSs is not ye<sup>t</sup> well documented. The main advantages of green-synthesized nanomaterials (gNMs) are the low energy cost of their synthesis, lack of toxic chemicals, simplicity of procedure, high adaptability of the synthesized gNM, and the presence of functional groups on their surface. The latter is promising for simple immobilization of bioorganic molecules, including enzymes, during biosensor construction [40,47]. If the gNM has additional catalytic properties, it plays a dual role in biosensors, simultaneously serving as the carrier of bio-elements and as the enzyme mimetic (nanozyme).

In our previous research, we reported obtaining gHCFs of transition metals using the purified yeas<sup>t</sup> enzyme flavocytochrome *b*2 (Fc*b*2; L-lactate: ferricytochrome c oxidoreductase, EC 1.1.2.3). The structure, size, composition, electro-catalytic properties, electro-mediator activity, and PO-like properties of the obtained gHCFs, which were synthesized via an enzyme and incorporated with it, were characterized. A more detailed study was performed on copper hexacyanoferrate (gCuHCF or gCuPBA), which was found to be the most effective PO mimetic. When immobilized on a GE, the gCuHCF under special pH conditions and working potential gave the intrinsic amperometric response to hydrogen peroxide. We demonstrated that the synthesized gCuHCF may be successfully used as an artificial PO for sensor analysis of hydrogen peroxide in a real disinfectant sample [40].

In the current work, we describe in more detail the synthesis and characteristics of new gHCFs of transition and noble metals with PO-like activity, an additional structural study of the most effective gCuHCF, development of an improved and highly sensitive ABS using glucose oxidase (GO) and gCuHCF, and testing of the constructed GO/gCuHCF ABS for glucose analysis in real samples of fruit juices.

#### **2. Materials and Methods**
