The current review is devoted to nanozymes, i.e., nanostructured artificial enzymes which mimic the catalytic properties of natural enzymes. Use of the term “nanozyme” in the literature as indicating an enzyme is not always justified. For example, it is used inappropriately for nanomaterials bound with electrodes that possess catalytic activity only when applying an electric potential. If the enzyme-like activity of such a material is not proven in solution (without applying the potential), such a catalyst should be named an “electronanocatalyst”, not a nanozyme. This paper presents a review of the classification of the nanozymes, their advantages vs. natural enzymes, and potential practical applications. Special attention is paid to nanozyme synthesis methods (hydrothermal and solvothermal, chemical reduction, sol-gel method, co-precipitation, polymerization/polycondensation, electrochemical deposition). The catalytic performance of nanozymes is characterized, a critical point of view on catalytic parameters of nanozymes described in scientific papers is presented and typical mistakes are analyzed. The central part of the review relates to characterization of nanozymes which mimic natural enzymes with analytical importance (“nanoperoxidase”, “nanooxidases”, “nanolaccase”) and their use in the construction of electro-chemical (bio)sensors (“nanosensors”).

Nanozymes (NZs) are nanostructured artificial enzymes that mimic catalytic properties of natural enzymes. The NZs have essential advantages over natural enzymes, namely low preparation costs, stability, high surface area, self-assembling capability, size and composition-dependent activities, broad possibility for modification, and biocompatibility. NZs have wide potential practical applications as catalysts in biosensorics, fuel-cell technology, environmental biotechnology, and medicine. Most known NZs are mimetics of oxidoreductases or hydrolases. The present work aimed to obtain effective artificial peroxidase (PO)-like NZs (nanoPOs), to characterize them, and to estimate the prospects of their analytical application. NanoPOs were synthesized using a number of nanoparticles (NPs) of transition and noble metals and were screened for their catalytic activity in solution and on electrodes. The most effective nanoPOs were chosen as NZs and characterized by their catalytic activity. Kinetic parameters, size, and structure of the best nanoPOs (Cu/Ce^S) were determined. Cu/Ce^S-based sensor for H₂O₂ determination showed high sensitivity (1890 A·M⁻¹·m⁻²) and broad linear range (1.5–20,000 µM). The possibility to apply Cu/Ce^S-NZ as a selective layer in an amperometric sensor for hydrogen-peroxide analysis of commercial disinfectant samples was demonstrated.

There are limited data on amperometric biosensors (ABSs) for L-arginine (Arg) determination based on oxidases that produce hydrogen peroxide (H₂O₂) as a byproduct of enzymatic reaction, and artificial peroxidases (POs) for decomposition of H₂O₂. 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 Amanita 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.

Amperometric biosensors, due to their high sensitivity, fast response time, low cost, simple control, miniaturization capabilities, and other advantages, are receiving significant attention in the field of medical diagnostics, especially in monitoring blood glucose levels in diabetic patients. In this study, an amperometric glucose biosensor based on immobilized enzyme glucose oxidase (GOx) and bimetallic platinum cobalt (PtCo) nanoparticles was developed. The PtCo nanoparticles, deposited on a graphite rod electrode, exhibited peroxidase-like catalytic properties and were able to electrocatalyze the reduction of H₂O₂. After immobilization of the GOx, an amperometric signal generated by the biosensor was directly proportional to the glucose concentration in the range of 0.04–2.18 mM. The biosensor demonstrated a sensitivity of 19.38 μA mM⁻¹ cm⁻², with a detection limit of 0.021 mM and a quantification limit of 0.064 mM. In addition to this analytical performance, the biosensor exhibited excellent repeatability (relative standard deviation (RSD) was 4.90%); operational and storage stability, retaining 98.93% and 95.33% of its initial response after 26 cycles of glucose detection and over a 14-day period, respectively; and anti-interference ability against electroactive species, as well as exceptional selectivity for glucose and satisfactory reproducibility (RSD 8.90%). Additionally, the biosensor was able to detect glucose levels in blood serum with a high accuracy (RSD 5.89%), indicating potential suitability for glucose determination in real samples.

L-Lactate is an indicator of food quality, so its monitoring is essential. Enzymes of L-Lactate metabolism are promising tools for this aim. We describe here some highly sensitive biosensors for L-Lactate determination which were developed using flavocytochrome b₂ (Fcb₂) as a bio-recognition element, and electroactive nanoparticles (NPs) for enzyme immobilization. The enzyme was isolated from cells of the thermotolerant yeast Ogataea polymorpha. The possibility of direct electron transfer from the reduced form of Fcb₂ to graphite electrodes has been confirmed, and the amplification of the electrochemical communication between the immobilized Fcb₂ and the electrode surface was demonstrated to be achieved using redox nanomediators, both bound and freely diffusing. The fabricated biosensors exhibited high sensitivity (up to 1436 A·M⁻¹·m⁻²), fast responses, and low limits of detection. One of the most effective biosensors, which contained co-immobilized Fcb₂ and the hexacyanoferrate of gold, having a sensitivity of 253 A·M⁻¹·m⁻² without freely diffusing redox mediators, was used for L-Lactate analysis in samples of yogurts. A high correlation was observed between the values of analyte content determined using the biosensor and referenced enzymatic-chemical photometric methods. The developed biosensors based on Fcb₂-mediated electroactive nanoparticles can be promising for applications in laboratories of food control.

Metallic nanoparticles potentially have wide practical applications in various fields of science and industry. In biosensorics, they usually act as catalysts or nanozymes (NZs) and as mediators of electron transfer. We describe here the development of amperometric biosensors (ABSs) based on purified oxidases, synthesized nanoparticles of CuCe (nCuCe), and micro/nanoporous gold (pAu), which were electro-deposited on a graphite electrode (GE). As an effective peroxidase (PO)-like NZ, nCuCe was used here as a hydrogen-peroxide-sensing platform in ABSs that were based on glucose oxidase, alcohol oxidase, methylamine oxidase, and L-arginine oxidase. At the same time, nCuCe is an electroactive mediator and has been used in laccase-based ABSs. As a result, the ABSs we constructed and characterized were based on glucose, methanol, methyl amine, L-arginine, and catechol, respectively. The developed nCuCe-based ABSs exhibited improved analytical characteristics in comparison with the corresponding PO-based ABSs. Additionally, the presence of pAu, with its extremely advanced chemo-sensing surface layer, was shown to significantly increase the sensitivities of all constructed ABSs. As an example, the bioelectrodes containing laccase/GE, laccase/nCuCe/GE, and laccase/nCuCe/pAu/GE exhibited sensitivities to catechol at 2300, 5055, and 9280 A·M⁻¹·m⁻², respectively. We demonstrate here that pAu is an effective carrier of electroactive nanomaterials coupled with oxidases, which may be promising in biosensors.