# Peroxidase-Like Metal-Based Nanozymes: Synthesis, Catalytic Properties, and Analytical Application
## 3 Results and Discussion
### Development and Characterization of the NZs-Modified Electrodes  
Our next task was to select the most PO-active NPs immobilized on the electrode surface. It is known that modification of an electrode with some metallic NPs can improve the efficiency of an electron transfer, due to an increase in the electrochemically accessible electrode surface area. Numerous NZs/GEs were screened for their ability to decompose hydrogen peroxide. The electrocatalytic activities of the synthesized NZs, while immobilized on the surface of GEs, were tested by CV and chronoamperometry, as described in Section 2.5.2 (Figure 1 and Figure S2). In our research, the amperometric responses of different NZs/GEs to added portions of H\({}_{2}\)O\({}_{2}\) were compared under mild conditions at the working potential of \(-\)50 mV. This rather low potential was chosen from the CV profiles (Figure 1), since the main drawback  
Figure 1: Cyclic voltammograms (CV) of the graphite electrodes modified with Cu/Ce\({}^{S}\) (**a**), Fe/Ce (**b**), Au/Cu (**c**), and Ag/Ce (**d**). CV profiles as outputs on addition of H\({}_{2}\)O\({}_{2}\) in concentration: (**a**)\(-\)0 mM (blue), 0.2 mM (cyan), 0.5 mM (pink), 1 mM (red); (**b**–**d**)\(-\)0 mM (black), 1 mM (red), 3 mM (green). Conditions: scan rate 50 mV\(\cdot\)s\({}^{-1}\); Ag/AgCl (reference electrode) in 50 mM NaOAc buffer, pH 4.5. The surfaces of the graphite electrode (GE) were modified with 1 \(\upmu\)g (**a**) or 5 \(\upmu\)g NZs (**b**–**d**) and covered with Nafion. of many H\({}_{2}\)O\({}_{2}\)-sensitive NZ-based electrochemical sensors is their nonselectivity under high positive (or negative) working potentials. H\({}_{2}\)O\({}_{2}\) is able to direct the auto-oxidation on electroactive surfaces at a working potential above +0.4 V, or auto-reduction at \(-0.4\) V or less vs. Ag/AgCl. Moreover, the real samples containing organic compounds may be easily co-oxidized/co-reduced at extreme potentials, which may result in overestimation of the target analytes in the presence of oxygen. To avoid these problems during sensor analysis, it was important to screen new NZs while working at operating potentials close to zero (0) V vs. Ag/AgCl [12; 43; 44; 45]. This requirement is especially relevant for the construction of biosensors and their exploitation for analysis of real samples (food products, biological liquids, etc.). Our previous experiments with natural PO as a biorecognition element of amperometric biosensors were also carried out at \(-50\) mV [41; 42; 43; 44]. Using the chronoamperograms, calibration curves were plotted for H\({}_{2}\)O\({}_{2}\) determination by the developed electrodes (Figure S2). The analytical characteristics of the modified GEs, as deduced from the graphs in comparison with natural PO [42; 43; 44], are summarized in Table 3. The linear ranges, limits of detection (LOD), and sensitivities of the electrodes modified with nanoPOs were calculated. The limit of blank values (LOB) for NZ was calculated as 0.3 \(\times\) LOD. The sensitivity of each NZ-modified electrode was measured for an electrode area of 7.30 mM\({}^{2}\). Figure 2 summarizes the data concerning the PO-like properties of the selected NZs, namely, Cu/Ce\({}^{5}\), Au/Cu, Ag/Ce, Fe/Ce, Ag/Zn, Pd/Ce, and Pt/Cu. The results in Table 3 demonstrate that Cu/Ce\({}^{5}\)/GE exhibited the highest sensitivity (1890 A\(\cdot\)M\({}^{-1}\cdot\)m\({}^{-2}\)) and current response (786 \(\mu\)A), the lowest LOD (0.42 \(\mu\)M), and a broad linear range (1.5-20,000 \(\mu\)M) upon H\({}_{2}\)O\({}_{2}\) addition. This structure was therefore studied in more detail as the most effective artificial PO immobilized on the electrode.