Biosensor Based on Peroxidase-Mimetic Nanozyme and Lactate Oxidase for Accurate L-Lactate Analysis in Beverages.

Precision analysis of the key biological metabolites such as L-lactate has great practical importance for many technological processes in food technology, including beverage production. Here we describe a new, highly selective, and sensitive biosensor for accurate L-lactate assay based on a combination of peroxidase-mimetic nanozymes with microbial lactate oxidase (LOx) immobilized onto the surface of a graphite-rod electrode (GE). The peroxidase-like nanozymes were synthesized using the debris of carbon microfibers (CFs) functionalized with hemin (H) and modified with gold nanoparticles (AuNPs) or platinum microparticles (PtMPs). The nanozyme formed with PtMPs as well as corresponding bioelectrodes based on it (LOx-CF-H-PtMPs/GE) is characterized by preferable catalytic and operational characteristics, so it was selected for the analysis of L-lactate content in real samples of grape must and red wine. The results of the L-lactate analysis obtained by the developed biosensors are highly correlated with a very selective spectrophotometric approach used as a reference. The developed biosensor, due to its high selectivity and sensitivity, is very prospective not only for the beverage industry and food technology, but also for clinical diagnostics and medicine, as well as in other applications where the accurate analysis of L-lactate is highly important.

New micro/nanocomposite with peroxidase-like activity in construction of oxidases-based amperometric biosensors for ethanol and glucose analysis.

Development of artificial enzymes, including nanozymes as an alternative for non-stable and expensive natural enzymes, is a booming field of modern Biosensorics and Biofuel Technology. In this study, we describe fabrication and characterization of sensitive biosensors for the detection of ethanol and glucose based on new micro/nanocomposite electrodes with peroxidase-like activity (nanozyme) coupled with microbial oxidases: alcohol oxidase (AOX) and glucose oxidase (GOX). The nanozyme was synthesized by modification of carbon microfibers (CF) by hemin (H) and gold (Au) nanoparticles. The formation of gold nanoparticles on the surface of hemin-modified carbon microfibers has been confirmed by the UV-Vis and X-ray spectroscopy as well by the SEM analysis. Compared to hemin-only modified electrodes, the resulting micro/nanocomposite CF-H-Au electrodes exhibit a higher specific catalytic activity and a better affinity for H2O2 in solution. The H2O2-sensitive CF-H-Au-modified electrodes showed a higher sensitivity (1.3-2.6-fold) compared with the nearest carbon-derived analogs and were used for the construction of highly sensitive ethanol and glucose biosensors. To eliminate diffusion limitation for substrates, AOX or GOX were fixed on the CF-H-Au-modified electrodes using a highly porous Nafion membrane. The main biosensors' characteristics have been investigated. The developed biosensors were tested for ethanol and glucose analysis in the real samples of both grape must and wine. The results are in good agreement with the results obtained using enzymatic kits as reference approaches.

Synthesis, Catalytic Properties and Application in Biosensorics of Nanozymes and Electronanocatalysts: A Review.

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").

D-lactate-selective amperometric biosensor based on the mitochondrial fraction of Ogataea polymorpha recombinant cells.

During the recent decades, a lot of data about the significance of D-lactate determination in food technology and quality control have been accumulated. Nowadays, the development of new methods for the determination of D-lactate is very relevant, especially with regard to biosensors. To construct a D-lactate-selective biosensor, we suggest using the mitochondria of recombinant yeast cells of Ogataea (Hansenula) polymorpha "tr6" (gcr1 catX/Δcyb2, prAOX_DLDH) overproducing D-lactate: cytochrome c-oxidoreductase (DLDH, EC 1.1.2.4) and lacking an L-lactate-specific enzyme (flavocytochrome b2 , E.C. 1.1.2.3). The usage of the pure enzyme is problematic due to the complexity of its isolation and stabilization because of the intramembranous localization of DLDH. The enzyme catalyzes D-lactate oxidation to pyruvate coupled with ferricytochrome c reduction to ferrocytochrome c. The constructed biosensor is characterized by high sensitivity (18.5 Ð·Ðœ-1 ·m-2 ), a low detection limit (3 µM of D-lactate), wide linear ranges, good selectivity, and sufficient stability. The real samples' analysis of D-lactate in dairy products was performed, and high correlation of the obtained results with the reference approach (0.7 < r < 1) and literature data was demonstrated.

Mutations and environmental factors affecting regulation of riboflavin synthesis and iron assimilation also cause oxidative stress in the yeast Pichia guilliermondii.

Iron deficiency causes oversynthesis of riboflavin in several yeast species, known as flavinogenic yeasts. However, the mechanisms of such regulation are not known. We found that mutations causing riboflavin overproduction and iron hyperaccumulation (rib80, rib81 and hit1), as well as cobalt excess or iron deficiency all provoke oxidative stress in the Pichia guilliermondii yeast. Iron content in the cells, production both of riboflavin and malondialdehyde by P. guilliermondii wild type and hit1 mutant strains depend on a type of carbon source used in cultivation media. The data suggest that the regulation of riboflavin biosynthesis and iron assimilation in P. guilliermondii are linked with cellular oxidative state.