## 3 Results and Discussion
### Evaluation and Optimization of the Arg-Sensitive Bioelectrodes  
The aim of our work was to construct ArgO-based ABSs for Arg determination using NZs as PO-mimetics. To form an enzymatic layer of the bio-electrode, NZs were coupled with ArgO on a GE and covered with Nafion, as described in the experimental part (see Section 2.5.2). The general principle of Arg assay by the developed ABSs is based on the detection of H\({}_{2}\)O\({}_{2}\) generated as a result of Arg hydrolysis under ArgO catalysis (Scheme 1). The efficiency of freely defusing electron transfers from the oxidized ArgO to the electrode via PO-like NZs (as mediator) was evaluated using CV (Figure 2a or Figure 3a). As it was shown there, the potential \(-150\) mV vs. Ag/AgCl was chosen as the optimal working potential for ArgO reduction in our system. The obtained ABSs (namely, ArgO/nCeCu/GE, ArgO/nNiPtPd/GE, and ArgO/gCuHCF/GE) were used for the investigation of the analytical parameters. Figures 3-5 demonstrate the amperometric properties of the developed ABSs. Current responses of the ABSs to the stepwise addition of a standard Arg solution were tested. Figure 4: Amperometric characteristics of ArgO/nNiPtPd/GE: (**a**) CV profiles as current responses upon addition of Arg up to final concentrations: (1) 0 mM (black), (2) 0.6 mM (red); (3) 1.2 mM (blue), scan rate 50 mV\(\cdot\)s\({}^{-1}\); (**b**) dependence of amperometric signal on concentration of Arg; (**c**) calibration graph for Arg determination. Conditions: working potential \(-150\) mV vs. Ag/AgCl/3 M KCl in 50 mM phosphate buffer, pH 7.0. Figure 3: Amperometric characteristics of ArgO/nCeCu/GE: (**a**) CV profiles as current responses upon addition of Arg up to final concentrations: (1) 0 mM (drop-black), (2) 2.4 mM (red); (3) 6.5 mM (blue); (4) 13 mM (green); (**b**) dependence of amperometric signal on concentration of Arg; (**c**) calibration graph for Arg determination. Conditions: Ag/AgCl (reference electrode) in 50 mM phosphate buffer, pH 7.0; scan rate 50 mV\(\cdot\)s\({}^{-1}\) (**a**); working potential \(-150\) mV (**b**,**c**). The prepared ABSs in this research, based on ArgO and NZs, demonstrated several advantages compared to the previously reported ones (Table 2). Our ABSs are highly selective, rapid, extremely sensitive, easy to use, reliable and portable. Additionally, the developed ABSs are cost-effective because they require only one enzyme (ArgO) and rather inexpensive functional nanomaterials as mimetics of the costly enzyme (PO). The values of \(K_{M}{}^{app}\) for Arg estimated with ArgO/nCeCu/GE and ArgO/nNiPtPd/GE are 0.32 \(\pm\) 0.05 mM and 0.35 \(\pm\) 0.03 mM, respectively. These values are practically identical with that for ADI in solution (0.35 mM) [28]. The application of NZs in a bioselective layer has a positive impact on the ABSs properties, due to their lower LODs for Arg determination and their high sensitivities. The constructed ArgO-based biosensors demonstrate slower amperometric responses (up to 50-60 s) in comparison with the previously reported by us biosensors on Arg [21, 26]. The arginase/urase/PANi/Pt- and ADI/PANi/Nafion/Pt-SPE-based ABSs demonstrated the response times 10 s [21] and 15 s [26], respectively. This phenomenon may be explained by different contents of enzymes in sensing films as well as by different origins and areas of working electrodes. In our present work, only (3-7) mu ArgO were contained in the biorecognition film on the surface of each ArgO/NZ-modified electrodes. Previously, ADI was immobilized in PANi/Nafion film on the surface of Pt-SPE at much higher amount (540 mU) [26]. The stabilities of the developed ArgO-based ABSs were explored by monitoring their responses to the injection of a 0.2 mM Arg standard solution over a period of seven days. The ArgO/nCeCu/GE and ArgO/nNiPtPd/GE preserved around 50% stability after five