Towards a Self-Powered Amperometric Glucose Biosensor Based on a Single-Enzyme Biofuel Cell.

This paper describes the study of an amperometric glucose biosensor based on an enzymatic biofuel cell consisting of a bioanode and a biocathode modified with the same enzyme-glucose oxidase (GOx). A graphite rod electrode (GRE) was electrochemically modified with a layer of Prussian blue (PB) nanoparticles embedded in a poly(pyrrole-2-carboxylic acid) (PPCA) shell, and an additional layer of PPCA and was used as the cathode. A GRE modified with a nanocomposite composed of poly(1,10-phenanthroline-5,6-dione) (PPD) and gold nanoparticles (AuNPs) entrapped in a PPCA shell was used as an anode. Both electrodes were modified with GOx by covalently bonding the enzyme to the carboxyl groups of PPCA. The developed biosensor exhibited a wide linear range of 0.15-124.00 mM with an R2 of 0.9998 and a sensitivity of 0.16 µA/mM. The limit of detection (LOD) and quantification (LOQ) were found to be 0.07 and 0.23 mM, respectively. The biosensor demonstrated exceptional selectivity to glucose and operational stability throughout 35 days, as well as good reproducibility, repeatability, and anti-interference ability towards common interfering substances. The studies on human serum demonstrate the ability of the newly designed biosensor to determine glucose in complex real samples at clinically relevant concentrations.

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.

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.

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.

Nanozymes with reductase-like activities: antioxidant properties and electrochemical behavior.

Nanozymes (NZs) as stable cost-effective mimics of natural enzymes may be promising catalysts in food and environmental biotechnology, biosensors, alternative energy and medicine. The majority of known NZs are mimetics of oxidoreductases, although there are only limited data regarding mimetics of reductases. In the present research, a number of metal-based NZs were synthesized via chemical methods and screened for their antioxidant ability in solution. The most effective reductase-like Zn/Cd/Cu NZ was characterized in detail. Its antioxidant properties in comparison with several food products and Trolox, as well as substrate specificity, size and composition were studied. Zn/Cd/Cu NZ was shown to mimic preferentially selenite reductase. The amperometric sensor was constructed possessing a high sensitivity (1700 A M-1 m-2) and a broad linear range (16-1000 µM) for selenite ions. The possibility to apply the fabricated sensor for selenite determination in commercial mineral water has been demonstrated.

"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.

Extracellular laccase from Monilinia fructicola: isolation, primary characterization and application.

Laccases are enzymes belonging to the family of blue copper oxidases. Due to their broad substrate specificity, they are widely used in many industrial processes and environmental bioremediations for removal of a large number of pollutants. During last decades, laccases attracted scientific interest also as highly promising enzymes to be used in bioanalytics. The aim of this study is to obtain a highly purified laccase from an efficient fungal producer and to demonstrate the applicability of this enzyme for analytics and bioremediation. To select the best microbial source of laccase, a screening of fungal strains was carried out and the fungus Monilinia fructicola was chosen as a producer of an extracellular enzyme. Optimal cultivation conditions for the highest yield of laccase were established; the enzyme was purified by a column chromatography and partially characterized. Molecular mass of the laccase subunit was determined to be near 35 kDa; the optimal pH ranges for the highest activity and stability are 4.5-5.0 and 3.0-5.0, respectively; the optimal temperature for laccase activity is 30°C. Laccase preparation was successfully used as a biocatalyst in the amperometric biosensor for bisphenol A assay and in the bioreactor for bioremediation of some xenobiotics.

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").

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.

Highly selective apo-arginase based method for sensitive enzymatic assay of manganese (II) and cobalt (II) ions.

A novel enzymatic method of manganese (II) and cobalt (II) ions assay, based on using apo-enzyme of Mn2+-dependent recombinant arginase I (arginase) and 2,3-butanedione monoxime (DMO) as a chemical reagent is proposed. The principle of the method is the evaluation of the activity of L-arginine-hydrolyzing of arginase holoenzyme after the specific binding of Mn2+ or Co2+ with apo-arginase. Urea, which is the product of enzymatic hydrolysis of L-arginine (Arg), reacts with DMO and the resulted compound is detected by both fluorometry and visual spectrophotometry. Thus, the content of metal ions in the tested samples can be determined by measuring the level of urea generated after enzymatic hydrolysis of Arg by reconstructed arginase holoenzyme in the presence of tested metal ions. The linearity range of the fluorometric apo-arginase-DMO method in the case of Mn2+ assay is from 4pM to 1.10nM with a limit of detection of 1pM Mn2+, whereas the linearity range of the present method in the case of Co2+ assay is from 8pM to 45nM with a limit of detection of 2.5pM Co2+. The proposed method being highly sensitive, selective, valid and low-cost, may be useful to monitor Mn2+ and Co2+ content in clinical laboratories, food industry and environmental control service.

Fluorometric enzymatic assay of l-arginine.

The enzymes of l-arginine (further - Arg) metabolism are promising tools for elaboration of selective methods for quantitative Arg analysis. In our study we propose an enzymatic method for Arg assay based on fluorometric monitoring of ammonia, a final product of Arg splitting by human liver arginase I (further - arginase), isolated from the recombinant yeast strain, and commercial urease. The selective analysis of ammonia (at 415nm under excitation at 360nm) is based on reaction with o-phthalaldehyde (OPA) in the presence of sulfite in alkali medium: these conditions permit to avoid the reaction of OPA with any amino acid. A linearity range of the fluorometric arginase-urease-OPA method is from 100nM to 6µÐœ with a limit of detection of 34nM Arg. The method was used for the quantitative determination of Arg in the pooled sample of blood serum. The obtained results proved to be in a good correlation with the reference enzymatic method and literature data. The proposed arginase-urease-OPA method being sensitive, economical, selective and suitable for both routine and micro-volume formats, can be used in clinical diagnostics for the simultaneous determination of Arg as well as urea and ammonia in serum samples.

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.

Bi-enzyme L-arginine-selective amperometric biosensor based on ammonium-sensing polyaniline-modified electrode.

A novel L-arginine-selective amperometric bi-enzyme biosensor based on recombinant human arginase I isolated from the gene-engineered strain of methylotrophic yeast Hansenula polymorpha and commercial urease is described. The biosensing layer was placed onto a polyaniline-Nafion composite platinum electrode and covered with a calcium alginate gel. The developed sensor revealed a good selectivity to L-arginine. The sensitivity of the biosensor was 110 ± 1.3 nA/(mM mm(2)) with the apparent Michaelis-Menten constant (K(M)(app)) derived from an L-arginine (L-Arg) calibration curve of 1.27 ± 0.29 mM. A linear concentration range was observed from 0.07 to 0.6mM, a limit of detection being 0.038 mM and a response time - 10s. The developed biosensor demonstrated good storage stability. A laboratory prototype of the proposed amperometric biosensor was applied to the samples of three commercial pharmaceuticals ("Tivortin", "Cytrarginine", "Aminoplazmal 10% E") for L-Arg testing. The obtained L-Arg-content values correlated well with those declared by producers.

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.

The chromate resistance phenotype of some yeast mutants correlates with a lower level of Cr(V)-species generated in the extra-cellular medium.

The paper describes the selection of chromate-resistant mutants of the yeast Pichia guilliermondii with a higher chromate-reducing activity and reports the EPR-study of Cr(V)-generation in the extra-cellular medium during the reduction of chromate by the yeast culture. It is shown that the reduction of chromate to Cr(III) species runs through the extra-cellular generation of Cr(V)-intermediate(s), thus supporting the assumption about the existence of an extra-cellular pathway of Cr(VI)-reduction. Furthermore, it is demonstrated that the chromate-resistance phenotype of tested mutants correlates with a lower stationary level of Cr(V)-species in the medium. It is thus suggested that isolated mutants can be used as sources of Cr(III)-biocomplexes due to their ability to effectively reduce chromate to Cr(III)-chelates with potential pharmacological applications.