Flavocytochrome b2-Mediated Electroactive Nanoparticles for Developing Amperometric L-Lactate Biosensors.

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.

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.

Reusable alcohol oxidase-nPtCu/alginate beads for highly sensitive ethanol assay in beverages.

Nanozymes (NZs) are nanoparticles that mimic the catalytic properties of natural enzymes. The present work aimed to obtain effective peroxidase mimetics (PO-like NZs), to characterize their morphological properties, estimate the kinetic parameters of NZs and evaluate the prospects of their application in analysis of ethanol. Herein, we have proposed a convenient spectrophotometric method for ethanol assay using reusable alginate beads enriched with alcohol oxidase (AO) and nanoparticles of PtCu (nPtCu) as PO-like NZs, and 3,3',5,5'-tetramethylbenzidine (TMB) as a chromogen. The linear range for the proposed nPtCu-AO/alginate beads/TMB-based method is from 0.01 mM to 0.15 mM with a limit of detection of 3.3 µM ethanol. The method is used for the quantitative determination of ethanol in alcoholic beverages. The obtained results proved to be in a good correlation with the enzymatic reference method. These results highlight the potential of the nPtCu with PO-like activity in bioanalytical applications. The proposed method, being sensitive, economical and suitable for routine and micro-volume formats, can be used in clinical diagnostics for the detection of ethanol.

"Green" Prussian Blue Analogues as Peroxidase Mimetics for Amperometric Sensing and Biosensing.

Prussian blue analogs (PBAs) are well-known artificial enzymes with peroxidase (PO)-like activity. PBAs have a high potential for applications in scientific investigations, industry, ecology and medicine. Being stable and both catalytically and electrochemically active, PBAs are promising in the construction of biosensors and biofuel cells. The "green" synthesis of PO-like PBAs using oxido-reductase flavocytochrome b2 is described in this study. When immobilized on graphite electrodes (GEs), the obtained green-synthesized PBAs or hexacyanoferrates (gHCFs) of transition and noble metals produced amperometric signals in response to H2O2. HCFs of copper, iron, palladium and other metals were synthesized and characterized by structure, size, catalytic properties and electro-mediator activities. The gCuHCF, as the most effective PO mimetic with a flower-like micro/nano superstructure, was used as an H2O2-sensitive platform for the development of a glucose oxidase (GO)-based biosensor. The GO/gCuHCF/GE biosensor exhibited high sensitivity (710 A M-1m-2), a broad linear range and good selectivity when tested on real samples of fruit juices. We propose that the gCuHCF and other gHCFs synthesized via enzymes may be used as artificial POs in amperometric oxidase-based (bio)sensors.

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.

Amperometric biosensors based on oxidases and PtRu nanoparticles as artificial peroxidase.

Catalytically active nanomaterials have several advantages over their natural analogues when used as artificial enzymes (nanozymes), namely, higher stability and lower cost. Nanozymes with metallic nanocomposites are promising catalysts for biosensing applications. The aim of the current research is to construct oxidase-based bioelectrodes for food analysis using nanozymes as peroxidase mimetics. Bimetallic PtRu nanoparticles (nPtRu) coupled with alcohol oxidase (AO) and methylamine oxidase (AMO) were chosen to construct amperometric biosensors (ABSs) for primary alcohols and methylamine (MA). Both ABSs show high sensitivities (336 A·M-1·m-2 for the AO-ABS and 284 A·M-1·m-2 for the AMO-ABS), broad linear ranges (25-200 µM ethanol and 20-600 µM MA) and satisfactory storage stabilities. Practical feasibility of the constructed ABSs was demonstrated on food samples. High correlation between contents of MA and ethanol in foods determined by the ABSs and reference methods was observed.

'Cold' crystallization in nanostructurized 80GeSe2-20Ga2Se3 glass.

'Cold' crystallization in 80GeSe2-20Ga2Se3 chalcogenide glass nanostructurized due to thermal annealing at 380°C for 10, 25, 50, 80, and 100 h are probed with X-ray diffraction, atomic force, and scanning electron microscopy, as well as positron annihilation spectroscopy performed in positron annihilation lifetime and Doppler broadening of annihilation line modes. It is shown that changes in defect-related component in the fit of experimental positron lifetime spectra for nanocrystallized glasses testify in favor of structural fragmentation of larger free-volume entities into smaller ones. Nanocrystallites of Ga2Se3 and/or GeGa4Se8 phases and prevalent GeSe2 phase extracted mainly at the surface of thermally treated samples with preceding nucleation and void agglomeration in the initial stage of annealing are characteristic features of cold crystallization.