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.

Ammonium nanochelators in conjunction with arginine-specific enzymes in amperometric biosensors for arginine assay.

Amino acid L-arginine (Arg), usually presented in food products and biological liquids, can serve both as a useful indicator of food quality and an important biomarker in medicine. The biosensors based on Arg-selective enzymes are the most promising devices for Arg assay. In this research, three types of amperometric biosensors have been fabricated. They exploit arginine oxidase (ArgO), recombinant arginase I (ARG)/urease, and arginine deiminase (ADI) coupled with the ammonium-chelating redox-active nanoparticles. Cadmium-copper nanoparticles (nCdCu) as the most effective nanochelators were used for the development of ammonium chemosensors and enzyme-coupled Arg biosensors. The fabricated enzyme/nCdCu-containing bioelectrodes show wide linear ranges (up to 200 µM), satisfactory storage stabilities (14 days), and high sensitivities (A⋅M-1⋅m-2) to Arg: 1650, 1700, and 4500 for ADI-, ArgO- and ARG/urease-based sensors, respectively. All biosensors have been exploited to estimate Arg content in commercial juices. The obtained data correlate well with the values obtained by the reference method. A hypothetic scheme for mechanism of action of ammonium nanochelators in electron transfer reaction on the arginine-sensing electrodes has been proposed.

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.

The Peroxidase-like Nanocomposites as Hydrogen Peroxide-Sensitive Elements in Cholesterol Oxidase-Based Biosensors for Cholesterol Assay.

Catalytically active nanomaterials, in particular, nanozymes, are promising candidates for applications in biosensors due to their excellent catalytic activity, stability and cost-effective preparation. Nanozymes with peroxidase-like activities are prospective candidates for applications in biosensors. The purpose of the current work is to develop cholesterol oxidase-based amperometric bionanosensors using novel nanocomposites as peroxidase (HRP) mimetics. To select the most electroactive chemosensor on hydrogen peroxide, a wide range of nanomaterials were synthesized and characterized using cyclic voltammetry (CV) and chronoamperometry. Pt NPs were deposited on the surface of a glassy carbon electrode (GCE) in order to improve the conductivity and sensitivity of the nanocomposites. The most HRP-like active bi-metallic CuFe nanoparticles (nCuFe) were placed on a previously nano-platinized electrode, followed by conjugation of cholesterol oxidase (ChOx) in a cross-linking film formed by cysteamine and glutaraldehyde. The constructed nanostructured bioelectrode ChOx/nCuFe/nPt/GCE was characterized by CV and chronoamperometry in the presence of cholesterol. The bionanosensor (ChOx/nCuFe/nPt/GCE) shows a high sensitivity (3960 A·M-1·m-2) for cholesterol, a wide linear range (2-50 µM) and good storage stability at a low working potential (-0.25 V vs. Ag/AgCl/3 M KCl). The constructed bionanosensor was tested on a real serum sample. A detailed comparative analysis of the bioanalytical characteristics of the developed cholesterol bionanosensor and the known analogs is presented.

Amperometric biosensors based on alcohol oxidase and peroxidase-like nanozymes for ethanol determination.

The aim of the current research is to design alcohol oxidase-based amperometric biosensors (ABSs) using hybrid metallic nanoparticles as artificial peroxidases (PO) or PO-like nanozymes (NZs). A lot of metallic PO-like NZs were synthesized and tested with respect to their ability to substitute natural PO in solution and on amperometric electrode. The most effective PO mimetics were coupled with alcohol oxidase (AOX) on graphite electrodes (GEs) and characterized. Two types of modified GEs, namely, the AOX/nAuCePt/GE and the AOX/nFePtAu/GE show the highest sensitivities to ethanol (2600 A⋅M-1⋅m-2 and 1250 A⋅M-1⋅m-2, respectively), low limits of detection (1.5 µM and 2.2 µM), broad linear ranges (5 - 100 µM and 12 - 120 µM), as well as satisfactory storage stabilities. The most sensitive bioelectrode AOX/nAuCePt/GE was used as ABS for ethanol determination in real samples. The practical feasibility of the constructed ABS was demonstrated by determination of ethanol in beverages, human blood and saliva.

Nanomaterials as Redox Mediators in Laccase-Based Amperometric Biosensors for Catechol Assay.

Laccase is a copper-containing enzyme that does not require hydrogen peroxide as a co-substrate or additional cofactors for an enzymatic reaction. Nanomaterials of various chemical structures are usually applied to the construction of enzyme-based biosensors. Metals, metal oxides, semiconductors, and composite NPs perform various functions in electrochemical transformation schemes as a platform for the enzyme immobilization, a mediator of an electron transfer, and a signal amplifier. We describe here the development of amperometric biosensors (ABSs) based on laccase and redox-active micro/nanoparticles (hereafter-NPs), which were immobilized on a graphite electrode (GE). For this purpose, we isolated a highly purified enzyme from the fungus Trametes zonatus, and then synthesized bi- and trimetallic NPs of noble and transition metals, as well as hexacyanoferrates (HCF) of noble metals; these were layered onto the surfaces of GEs. The electroactivity of many of the NPs immobilized on the GEs was characterized by cyclic voltammetry (CV) experiments. The most effective mediators of electron transfer were selected as the platform for the development of laccase-based ABSs. As a result, a number of catechol-sensitive ABSs were constructed and characterized. The laccase/CuCo/GE was demonstrated to possess the highest sensitivity to catechol (4523 A·M-1·m-2) among the tested ABSs. The proposed ABSs may be promising for the analysis of phenolic derivatives in real samples of drinking water, wastewater, and food products.

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.

Improvement of laccase biosensor characteristics using sulfur-doped TiO2 nanoparticles.

The search for new nanoscale materials with predictable properties to target the timely and fast detection of toxic components in wastewater is one of the most promising directions of modern biosensorics. We have shown that TiO2 nanoparticles modified with sulfur significantly improve the main operational parameters of laccase-based electrodes when compared with controls. The nanoparticle samples were labeled as TiO2S(0.75), TiO2S(1.5), and TiO2S(3.0), in which the numbers in parentheses refer to the quantity of H2SO4 (mL) used in the synthesis. The nanoparticles and enzyme were immobilized by means of Nafion film formed on a carbon rod electrode. It was shown that the modification of Nafion film by TiO2 or TiO2S(1.5) nanoparticles does not affect the size of the nanocavities and defect structure of the main polymer matrix as revealed by positron annihilation spectroscopy. It testifies that the structural-morphological difference between the film samples is rather small, and the improving of the sensor operational parameters for TiO2S(1.5)-based laccase electrodes is connected only with the impact of sulfur doping, but not the difference in membrane properties. The developed bioelectrodes were tested for phenol analysis in real communal wastewater samples spiked with these analytes, demonstrating the high accuracy of the assay.

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.

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").

Microporous carbon fibers as electroconductive immobilization matrixes: Effect of their structure on operational parameters of laccase-based amperometric biosensor.

In this study, we describe the fabrication of sensitive biosensor for the detection of phenolic substrates using laccase immobilized onto two types of microporous carbon fibers (CFs). The main characteristics of microporous CFs used for preparation of biosensors are given. Two CFs were characterized by different specific surface area, CFA (<1 m2·g-1) and CFB (1448 m2·g-1), but with comparable size of the micropores estimated by positron annihilation lifetime spectroscopy. The structural analysis was shown that CFA is formed by thin interwoven fibers forming a highly porous structure, as well as CFB - by granular formations with uneven edges that shape a cellulose membrane of lower porosity. The results of amperometric analysis revealed that the laccase-bound CFs possesses better electrochemical behavior for laccase than non-modified rod carbon electrodes (control). Using chronoamperometric analysis, the operational parameters of the CFs-modified bioelectrodes were compared to control bioelectrodes. The bioelectrodes based on CFs have demonstrated 2.4-2.7 folds enhanced maximal current at substrate saturation (Imax) values, 1.2-1.4 folds increased sensitivity and twice wide linearity compared with control bioelectrodes. The sensitivity of the developed CFs-based bioelectrodes was improved compared with the laccase-bound electrodes, described in literature. The developed biosensor was tested for catechol analysis in the real communal wastewater sample.

A Reagentless Amperometric Formaldehyde-Selective Chemosensor Based on Platinized Gold Electrodes.

Fabrication and characterization of a new amperometric chemosensor for accurate formaldehyde analysis based on platinized gold electrodes is described. The platinization process was performed electrochemically on the surface of 4 mm gold planar electrodes by both electrolysis and cyclic voltamperometry. The produced electrodes were characterized using scanning electron microscopy and X-ray spectral analysis. Using a low working potential (0.0 V vs. Ag/AgCl) enabled an essential increase in the chemosensor's selectivity for the target analyte. The sensitivity of the best chemosensor prototype to formaldehyde is uniquely high (28180 A·M-1·m-2) with a detection limit of 0.05 mM. The chemosensor remained stable over a one-year storage period. The formaldehye-selective chemosensor was tested on samples of commercial preparations. A high correlation was demonstrated between the results obtained by the proposed chemosensor, chemical and enzymatic methods (R = 0.998). The developed formaldehyde-selective amperometric chemosensor is very promising for use in industry and research, as well as for environmental control.

Detection of Waterborne and Airborne Formaldehyde: From Amperometric Chemosensing to a Visual Biosensor Based on Alcohol Oxidase.

A laboratory prototype of a microcomputer-based analyzer was developed for quantitative determination of formaldehyde in liquid samples, based on catalytic chemosensing elements. It was shown that selectivity for the target analyte could be increased by modulating the working electrode potential. Analytical parameters of three variants of the amperometric analyzer that differed in the chemical structure/configuration of the working electrode were studied. The constructed analyzer was tested on wastewater solutions that contained formaldehyde. A simple low-cost biosensor was developed for semi-quantitative detection of airborne formaldehyde in concentrations exceeding the threshold level. This biosensor is based on a change in the color of a solution that contains a mixture of alcohol oxidase from the yeast Hansenula polymorpha, horseradish peroxidase and a chromogen, following exposure to airborne formaldehyde. The solution is enclosed within a membrane device, which is permeable to formaldehyde vapors. The most efficient and sensitive biosensor for detecting formaldehyde was the one that contained alcohol oxidase with an activity of 1.2 U·mL-1. The biosensor requires no special instrumentation and enables rapid visual detection of airborne formaldehyde at concentrations, which are hazardous to human health.

Immobilized formaldehyde-metabolizing enzymes from Hansenula polymorpha for removal and control of airborne formaldehyde.

Formaldehyde (FA)-containing indoor air has a negative effect on human health and should be removed by intensive ventilation or by catalytic conversion to non-toxic products. FA can be oxidized by alcohol oxidase (AOX) taking part in methanol metabolism of methylotrophic yeasts. In the present work, AOX isolated from a Hansenula polymorpha C-105 mutant (gcr1 catX) overproducing this enzyme in glucose medium, was tested for its ability to oxidize airborne FA. A continuous fluidized bed bioreactor (FBBR) was designed to enable an effective bioconversion of airborne FA by AOX or by permeabilized mutant H. polymorpha C-105 cells immobilized in calcium alginate beads. The immobilized AOX having a specific activity of 6-8 U mg⁻¹ protein was shown to preserve 85-90% of the initial activity. The catalytic parameters of the immobilized enzyme were practically the same as for the free enzyme (k(cat)/K(m) was 2.35×10³ M⁻¹ s⁻¹ vs 2.89×10³ M⁻¹ s⁻¹, respectively). The results showed that upon bubbling of air containing from 0.3 up to 18.5 ppm FA through immobilized AOX in the range of 1.3-26.6 U g⁻¹ of the gel resulted in essential decrease of FA concentration in the outlet gas phase (less than 0.02-0.03 ppm, i.e. 10-fold less than the threshold limit value). It was also demonstrated that a FBBR with immobilized permeabilized C-105 cells provided more than 90% elimination of airborne FA. The process was monitored by a specially constructed enzymatic amperometric biosensor based on FA oxidation by NAD+ and glutathione-dependent formaldehyde dehydrogenase from the recombinant H. polymorpha Tf 11-6 strain.

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).

Reagentless amperometric formaldehyde-selective biosensors based on the recombinant yeast formaldehyde dehydrogenase.

Novel formaldehyde-selective amperometric biosensors were developed based on NAD(+)- and glutathione-dependent formaldehyde dehydrogenase isolated from a gene-engineered strain of the methylotrophic yeast Hansenula polymorpha. Electron transfer between the immobilized enzyme and a platinized graphite electrode was established using a number of different low-molecular free-diffusing redox mediators or positively charged cathodic electrodeposition paints modified with Os-bis-N,N-(2,2'-bipyridil)-chloride ([Os(bpy)(2)Cl]) complexes. Among five tested Os-containing redox polymers of different chemical structure and properties, complexes of osmium-modified poly(4-vinylpyridine) with molecular mass of about 60 kDa containing diaminopropyl groups were selected. The positively charged cathodic paint exhibited the best electron-transfer characteristics. Moreover, the polymer layers simultaneously served as a matrix for keeping the negatively charged low-molecular cofactors, glutathione and NAD(+), in the bioactive layer. Additionally, covering the enzyme/polymer layer with a negatively charged Nafion membrane significantly decreased cofactors leakage and simultaneously enhanced the sensor' stability. The developed sensors revealed a high selectivity to formaldehyde (FA) and a low cross-sensitivity to other substances (such as, e.g. butyraldehyde, propionaldehyde, acetaldehyde, methylglyoxal). The maximum current value was 34.2+/-0.72 microA/mm(2) (3.05 mm diameter electrode) and the apparent Michaelis-Menten constant (K(M)(app)) derived from the FA calibration curves was 120+/-5mM with a linear detection range for FA up to 20mM. The best observed sensitivity for reagentless sensor was 1.8 nA microM(-1) (358 Am(-2)M(-1)). The developed sensors had a good operational and storage stability. The laboratory prototype of the sensor was applied for FA testing in some real samples of pharmaceutical (formidron), disinfectant (descoton forte) and industrial product (formalin). A good correlation was revealed between the concentration values measured using the developed FdDH-based sensor, an enzymatic method and standard chemical methods of FA determination.

Formaldehyde dehydrogenase from the recombinant yeast Hansenula polymorpha: isolation and bioanalytic application.

A recombinant yeast clone, a derivative of the recipient Hansenula polymorpha strain NCYC 495, was chosen as an NAD and glutathione-dependent formaldehyde dehydrogenase overproducer. Optimal cultivation conditions for the highest yield of enzyme were established. A simple scheme for the isolation of formaldehyde dehydrogenase from the recombinant strain was proposed, and some characteristics of the purified enzyme were studied. An enzymatic method for formaldehyde assay based on formaldehyde dehydrogenase was developed and used for testing real samples.

Intact and permeabilized cells of the yeast Hansenula polymorpha as bioselective elements for amperometric assay of formaldehyde.

Intact and permeabilized yeast cells were tested as the biorecognition elements for amperometric assay of formaldehyde (FA). For this aim, the mutant C-105 (gcr1 catX) of the methylotrophic yeast Hansenula polymorpha with a high activity of AOX was chosen. Different approaches were used for monitoring FA-dependent cell response including analysis of their oxygen consumption rate by the use of a Clark electrode, as well as assay of oxidation of redox mediator at a screen-printed platinum electrode covered by cells entrapped in Ca-alginate gel. It was shown that oxygen consumption rate of permeabilized cells reached its saturation at 4mM of FA (23 degrees C). The detection limit was found to be 0.27mM. In the presence of redox mediator 2,6-dichlorophenolindophenol (DCIP), the screen-printed platinum band electrode covered by permeabilized cells did not show any current output to FA. In contrast, well-pronounced amperometric response to FA was observed in the case of intact yeast cells in the presence of DCIP. It was shown that current output reached its maximum at 7mM concentration of FA. The detection limit was found to be 0.74mM. Obviously, it is necessary to perform a directed genetic engineering of the yeast cells to improve their bioanalytical characteristics in the corresponding biosensors.