**4. Conclusions**

In the current research, we report the development of reagentless amperometric H2O2- sensitive sensors with artificial peroxidases (PO). As PO mimetics, "green" hexacyanoferrates (gHCFs) of transition and noble metals were used, which were synthesized via the oxidoreductase Fc*b*2. The gCuHCF was identified as the most effective PO mimetic and was characterized in detail concerning its structural, catalytic and electrochemical properties.

SEM analysis demonstrated that the gCuHCF formed a flower-like micro/nano superstructure. Thus, it may be used not only as a H2O2-sensitive platform for the development of oxidase-based biosensors but also as a carrier for enzyme concentration, immobilization and stabilization.

An amperometric glucose-oxidase-based biosensor with gCuHCF as the PO mimetic was developed. It exhibited high sensitivity (710 A <sup>M</sup>−1m−2), a broad linear range and good selectivity. The practical feasibility of the constructed biosensor was demonstrated on samples of fruit juices.

The obtained results indicated that the gCuHCF and other gHCFs may have a potential for use as PO-like composites for the construction of amperometric biosensors with any oxidase.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/bios11060193/s1, Figure S1. Amperometric characteristics of the several modified electrodes: chronoamperograms (left), dependence of the current response on increasing concentrations of H2O2 (middle), and calibration graphs (right); H2O2-sensing films are the following: gPdHCF (a); gCeHCF (b); gYHCF (c); gCoHCF (d); gMnHCF (e); gZnHCF (f); gNdHCF (g); gCdHCF (h) and chCuHCF (i). Conditions: working potential −50 mV vs. Ag/AgCl (reference electrode), 50 mM NaOAc buffer, pH 4.5 at 23 ◦C. Figure S2. FTIR spectrum of the gCuHCF. Figure S3. DLS plots of particle hydrodynamic diameter of the sample at various concentrations: the green line represents a 1.3 × 10<sup>8</sup> mL−1, the yellow line 6.6 × 10<sup>7</sup> mL−1, and the red line 3.3 × 10<sup>7</sup> mL−<sup>1</sup> particle concentration. Figure S4. X-ray diffraction analysis of the gCuHCF's synthesized particles. Figure S5. Effect of the PO-mimetic activity on the efficiency of H2O2 sensing: current response to increasing concentrations of H2O2 (a–e); and calibration graphs (f) for the GEs modified with different quantities of gCuHCF: (a) 1 mU, (b) 2 mU, (c) 5 mU, (d) 10 mU, (e,f); combined graph lines (1–4) correspond to graphs (a–d), respectively. Conditions: working potential −50 mV, Ag/AgCl (reference electrode) in 50 mM NaOAc, pH 4.5. Figure S6. Effect of PO-mimetic activity and working potential on analytical characteristics of the gCuHCF/GE: current responses to increasing concentrations of H2O2 (a,c); and the corresponding calibration graphs (b,d) for the GE modified with different quantities of gCuHCF: (1)—0.07 mU, (2)—0.15 mU, (3)—0.40 mU. Conditions: working potential −50 mV (a,b) and −200 mV (c,d), Ag/AgCl (reference electrode) in 50 mM phosphate buffer, pH 6.0.

**Author Contributions:** Conceptualization: G.Z.G. and M.N.; methodology: O.M.D., G.Z.G., Y.G.; investigation: O.M.D., R.Y.S., Y.G. and H.M.K.; resources: M.V.G.; data curation: G.Z.G. and M.N.; writing—original draft preparation: G.Z.G. and O.M.D.; writing—review and editing: G.Z.G. and M.N.; supervision: M.V.G.; project administration: G.Z.G.; funding acquisition: M.V.G. and M.N. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was partially funded by NAS of Ukraine (the program "Smart sensor devices of a new generation based on modern materials and technologies", project 0118U006260), by the Ministry of Education and Science of Ukraine (Ukrainian-Lithuanian R&D, project 0120U103398), by the National Research Foundation of Ukraine (project 0100/02.2020 "Development of new nanozymes as

catalytic elements for enzymatic kits and chemo/biosensors") and by the Research Authority of the Ariel University, Israel.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article: Gayda, G.; Demkiv, O.; Gurianov, Y.; Serkiz, R.; Gonchar, M.; Nisnevitch, M. 2020. "Green" nanozymes: synthesis, characterization and application in amperometric (bio)sensors. Proceedings 60(1), 58; doi.org/10.3390/IECB2020-07072 and Supplementary Material section.

**Acknowledgments:** We acknowledge Oksana M. Zakalska (Institute of Cell Biology, Lviv, Ukraine) for technical support and experimental assistance. The authors would like to thank Alexey Kossenko (Ariel University) for his help with X-ray crystallography analyses.

**Conflicts of Interest:** The authors declare no conflict of interest.
