###### Abstract  
There are limited data on amperometric biosensors (ABSs) for L-arginine (Arg) determination based on oxidases that produce hydrogen peroxide (H\({}_{2}\)O\({}_{2}\)) as a byproduct of enzymatic reaction, and artificial peroxididases (POs) for decomposition of H\({}_{2}\)O\({}_{2}\). The most frequently proposed Arg-sensitive oxidase-based ABSs contain at least two enzymes in the bioselective layer; this complicates the procedure and increases the cost of analysis. Therefore, the construction of a one-enzyme ABS for Arg analysis is a practical problem. In the current work, fabrication, and characterization of three ABS types for the direct measurement of Arg were proposed. L-arginine oxidase (ArgO) isolated from the mushroom _Amantia phalloides_ was co-immobilized with PO-like nanozymes (NZs) on the surface of graphite electrodes. As PO mimetics, chemically synthesized NZs of CeCu (nCeCU) and NiPtPd (nNiPtPd), as well as green-synthesized hexacyanoferrate of copper (gCuHCF), were used. The novel ABSs exhibited high sensitivity and selectivity to Arg, broad linear ranges and good storage stabilities. Two ABSs were tested on real samples of products containing Arg, including the pharmaceutical preparation \"Tivortine\", juices, and wine. A high correlation (R = 0.995) was demonstrated between the results of testing \"Tivortine\" and juice using nCeCU/GE and nNiPtPd/GE. It is worth mentioning that only a slight difference (less than 1%) was observed for \"Tivortin\" between the experimentally determined content of Arg and its value declared by the producer. The proposed ArgO-NZ-based ABSs may be promising for Arg analysis in different branches of science, medicine, and industry. L-arginine analysis; amperometric biosensors; L-arginine oxidase; hydrogen peroxide; artificial peroxidase 20 June 2021  
27 July 2021  
Multi-Step Analysis]Amperometric Biosensors for L-Arginine Determination Based on L-Arginine Oxidase and Peroxidase-Like Nanozymes  
1  
[1][PERSON], A., O.  
may be utilized. However, the necessity of several enzymes in the biosective layer complicates the process of biosensor fabrication, increases the cost of analysis, and causes losses in bioelectrode stability and sensitivity. Thus, using only one enzyme may be a more promising method for the fabrication of biosensors. Alternately, many efforts have been devoted in recent years to the development of non-enzymatic sensors. This approach is based on functional materials of different compositions, including nanomaterials. A limitation of this method is insufficient selectivity to the target analyte [34; 35; 36; 37; 38]. Nanomaterials with intrinsic enzyme-like activities or nanozymes (NZs) are promising alternatives to natural enzymes in biosensor construction. Artificial POs are the most urgently needed type of NZs for this goal. A limited number of amperometric biosensors (ABSs), especially mono-enzyme ABSs, have been developed to date to measure Arg concentrations [13; 27]. Yet a simple construction of stable ABSs based on one oxidase, namely ArgO, co-immobilized with H\({}_{2}\)O\({}_{2}\)-sensitive NZs in a biosective layer, remains a real problem. ArgO (L-arginine: oxygen oxidoreductase, EC 1.4.3.25) belongs to a class of oxidases of L-amino acids, that are accumulated in the tissues of some mammals, marine mollusks, and fish, in fungi, algae, bacteria, in the venoms of insects and snakes in the fruiting bodies of poisonous mushrooms [39; 40; 41; 42; 43; 44]. Oxidases of L-amino acids are promising analytical tools for quantifying the concentrations of the corresponding L-amino acids in different samples [30; 31]. ArgO is a poorly studied enzyme to date, as evidenced by a limited list of publications on the problem [29; 30; 39]. ArgO is a FAD-containing enzyme that catalyzes the conversion of Arg to 5-guanidino-2-oxopentanoate, ammonia and hydrogen peroxide. The enzymatic conversion of Arg is presented in Scheme 1.