Highly sensitive amperometric sensors based on laccase-mimetic nanozymes for the detection of dopamine.

The current research presents novel sensors based on laccase-like mimetics for the detection of dopamine (DA). The synthesized laccase-like nanozymes (nAuCu, nPtCu, nCuMnCo, and nCoCuCe) were prepared by a simple hydrothermal method and exhibited an attractive catalytic activity toward DA. The developed amperometric sensors based on laccase nanozymes (nAuCu and nPtCu) are more stable, selective, and revealed a higher sensitivity (6.5-fold than the biosensor based on the natural fungal laccase from Trametes zonata). The amperometric sensors were obtained by modification of the glassy carbon electrodes (GCEs) with AuPt nanoparticles. Functionalization of the electrode surface by AuPt NPs resulted in increased catalytic activity of the laccase-like layer and higher sensitivity. Among studied configurations, the sensor containing nAuCu and nAuPt possesses a wide linear range for dopamine detection (10-170 µM), the lowest limit of detection (20 nM), and the highest sensitivity (10 650 ± 8.3 A M-1 m-2) at a low applied potential (+0.2 V versus Ag/AgCl). The proposed simple and cost-effective sensor electrode was used for the determination of DA in pharmaceuticals.

Highly Porous 3D Gold Enhances Sensitivity of Amperometric Biosensors Based on Oxidases and CuCe Nanoparticles.

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.

"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.

Alcohol Oxidase from the Methylotrophic Yeast Ogataea polymorpha: Isolation, Purification, and Bioanalytical Application.

Alcohol oxidase (EC 1.1.3.13; AOX) is a flavoprotein that catalyzes the oxidation of primary short-chain alcohols to corresponding carbonyl compounds with a concomitant release of hydrogen peroxide. It is a key enzyme of methanol metabolism in methylotrophic yeasts, catalyzing the first step of methanol oxidation to formaldehyde.Here we describe the isolation and purification of AOX from the thermotolerant methylotrophic yeast Ogataea (Hansenula) polymorpha, and using this enzyme in enzymatic assay of ethanol, simultaneous analysis of methanol and formaldehyde, and in construction of amperometric biosensors selective to primary alcohols and formaldehyde.

Alcohol oxidase- and formaldehyde dehydrogenase-based enzymatic methods for formaldehyde assay in fish food products.

For Gadoid fishes, formaldehyde can be generated in tissues in huge amounts during endogenous enzymatic degradation of natural osmoprotectant trimethylamine-N-oxide. This paper describes two enzymatic methods for assay of formaldehyde in fish food products using alcohol oxidase (AOX) and formaldehyde dehydrogenase (FdDH) isolated from the thermotolerant methylotrophic yeast Hansenula polymorpha. AOX-based method exploits an ability of the enzyme to oxidise a hydrated form of formaldehyde to formic acid and hydrogen peroxide monitored in peroxidase-catalysed colorimetric reaction. In FdDH-based method, a monitored coloured formazane is formed from nitrotetrazolium salt during reduction by NADH, produced in formaldehyde-dependent reaction. It was demonstrated an applicability of both methods for assay of formaldehyde in fish products. The optimal protocols for analysis procedures have been elaborated and analytical parameters of both enzymatic methods have been established. The both methods were demonstrated that some fish products (hake and cod) contain high formaldehyde concentrations (up to 100mg/kg wet weight).