Search Results (128)

This study discloses the noncovalent immobilization of a bienzyme cascade composed of glucose oxidase (GOx) and horseradish peroxidase (HRP) onto magnetically responsive polyamide microparticles (PA MPs). Porous PA6, PA4, and PA12 MPs containing iron fillers were synthesized via activated anionic ring-opening polymerization in suspension, alongside neat PA6 MPs used as a reference. Four hybrid catalytic systems (GOx/HRP@PA) were prepared through sequential adsorption of HRP and GOx onto the various PA MP supports. The initial morphologies of the supports and the hybrid biocatalysts were characterized by SEM, followed by evaluation of the catalytic performance using a two-step glucose oxidation cascade process. Among all systems, the GOx/HRP@PA4-Fe complex exhibited the highest activity, being approximately 1.5 times greater than the native enzyme dyad, followed by the PA6-supported system with slightly inferior performance. All systems obeyed Michaelis–Menten kinetics, with the immobilized cascades displaying higher Kₘ and Vₘₐₓ values than the non-immobilized enzyme pair while maintaining comparable catalytic efficiencies, CE (CE = k_cat/Kₘ). Subsequently, the immobilized and native enzyme systems were employed for the polymerization of aniline. According to UV–VIS, complete monomer conversion was achieved within 24 h for selected catalysts, and FTIR analysis confirmed the formation of polyaniline in the emeraldine base form without the use of template molecules. These findings highlight the potential of Fe-containing polyamide microparticles as efficient supports for the sustainable, enzyme-mediated synthesis of intrinsically conductive aromatic polymers. Full article

Heavy metal contamination in livestock and poultry farm wastewater poses significant risks to both the environment and human health, so it is critical to accurately and rapidly quantify heavy metal ion concentrations in water. This research develops a bioelectric active hydrogel sensor for detecting heavy metal ions in livestock wastewater. The sensor integrates microbial surface display technology with graphene hydrogel, displaying glucose oxidase (GOx) on the surface of yeast cells, and covalently incorporating it into the graphene hydrogel through the bio-reduction activity of metal-reducing bacteria, enhancing its electrochemical performance. The sensor demonstrates excellent sensitivity and stability in detecting Cu²⁺, with a detection limit for Cu²⁺ of 17.0 µM. This sensor is also applicable for detecting Zn²⁺ in wastewater. When various heavy metal ions coexist in the solution, they exert a more pronounced inhibitory effect on enzyme activity. Consequently, the sensor can be employed to assess the overall heavy metal content in water samples. In the detection of Cu²⁺ in real livestock and poultry wastewater, the recovery rate of the graphene hydrogel electrode ranged from 88% to 106.5%, indicating that the sensor holds significant potential for application in actual sample analysis. Full article

Glucose (Glu) detection, as a fundamental analytical technique, has applications in medical diagnostics, clinical testing, bioanalysis and environmental monitoring. In this work, a solid-phase electrochemiluminescence (ECL) enzyme sensor was developed by immobilizing the ECL emitter in a stable manner within bipolar silica nanochannel array film (bp-SNA), enabling sensitive glucose detection. The sensor was constructed using an electrochemical-assisted self-assembly (EASA) method with various siloxane precursors to quickly modify the surface of indium tin oxide (ITO) electrodes with a bilayer SNA of different charge properties. The inner layer, including negatively charged SNA (n-SNA), attracted the positively charged ECL emitter tris(2,2′-bipyridyl)ruthenium(II) (Ru(bpy)₃²⁺) via electrostatic interaction, while the outer layer, including positively charged SNA (p-SNA), repelled it, forming a barrier that efficiently concentrated the Ru(bpy)₃²⁺ emitter in a stable manner. After modifying the amine groups on the p-SNA surface with aldehyde groups, glucose oxidase (GOx) was covalently immobilized, forming the enzyme electrode. In the presence of glucose, GOx catalyzed the conversion of glucose to hydrogen peroxide (H₂O₂), which acted as a quencher for the Ru(bpy)₃²⁺/triethanolamine (TPA) system, reducing the ECL signal and enabling quantitative glucose analysis. The sensor exhibited a wide linear range from 10 μM to 7.0 mM and a limit of detection (LOD) of 1 μM (S/N = 3). Glucose detection in fetal bovine serum was realized. By replacing the enzyme type on the electrode surface, this sensing strategy holds the potential to provide a universal platform for the detection of different metabolites. Full article

A biosensor for the determination of glucose, lactate, ethanol and starch in beverages has been developed using enzymes immobilized by a redox-active gel on a screen-printed electrode. A significant improvement proposed for multichannel biosensors, overcoming stability and sensitivity issues by covalently binding phenazine mediators to a biocompatible protein hydrogel, enhancing the packaging of the enzyme. Glucose oxidase (GOx), alcohol oxidase (AOx) and lactate oxidase (LOx) were used as biological materials, as well as a mixture of GOx with γ-amylase (Am). Redox gels were synthesized from bovine serum albumin (BSA) and phenazine derivatives. It was shown that a neutral red-based redox gel combined with single-walled carbon nanotubes is more promising than other substrates for enzyme immobilization. The lower limit of quantification for glucose, ethanol, lactate and starch using these systems is 0.035 mM, 2.3 mM, 15 mM and 2 mg/L, respectively. Biosensors were used to analyze the content of these substances in alcoholic, kvass and fermentation mass. Statistical analysis of the results showed that the values of glucose, ethanol, lactic acid and starch determined using biosensors and obtained by reference methods differ insignificantly. A set of biosensors developed on the basis of specifically selected enzymes is effective for controlling biotechnological processes and can be used as an alternative to classical analytical methods. Full article

The need for sustainable and efficient water decontamination methods has led to the increasing use of redox enzymes such as glucose oxidase (GOx). GOx immobilization on textile supports provides a promising alternative for catalyzing pollutant degradation in bio-Fenton (BF) and bio-electro-Fenton (BEF) systems. However, challenges related to enzyme stability, reusability, and environmental impact remain a concern. This communication paper outlines innovative strategies developed to address these challenges, notably the use of ecotechnologies to achieve efficient GOx immobilization while maintaining biocatalytic activity. Plasma ecoprocesses, amino-bearing biopolymer-chitosan, as well as a bio-crosslinker genipin have been used efficiently on conductive carbon and non-conductive polyester-PET nonwovens. In certain cases, immobilized GOx can retain high catalytic activity after multiple cycles, making them an effective biocatalyst for organic dye degradation (Crystal Violet and Remazol Blue) via bio-Fenton reactions, including total heterogeneous bio-Fention system. Moreover, the conductive carbon felt-based bioelectrodes successfully supported simultaneous pollutant degradation and energy generation in a BEF system. This work highlights the potential of textile-based enzyme immobilization for sustainable wastewater treatment, bio-electrochemical energy conversion, and also for bacterial deactivation. Future research will focus on optimizing enzyme stability and enhancing BEF efficiency for large-scale applications. Full article

Postoperative peritoneal adhesion and high recurrence rates are critical challenges in the clinical treatment of colorectal cancer. In this study, based on amyloid-like protein self-assembly technology, a novel Janus protein film was developed. The protein film encapsulates glucose oxidase (GOx) and catalase (CAT), which is named PTL@GC. Through a one-step method involving cysteine-reduced lysozyme-induced amyloid-like self-assembly, the film was co-loaded with GOx and CAT to achieve synergistic anti-adhesion and anti-tumor recurrence effects. The Janus film features a hydrophobic side that stably adheres to the intestinal surface without exogenous chemical modification and a hydrophilic side that prevents adhesion. The loaded GOx selectively induces disulfidptosis in SLC7A11-overexpressing tumor cells, while CAT degrades H₂O₂ to alleviate hypoxia and inhibit oxidative stress, significantly reducing adhesion-related fibrosis. The experimental results demonstrate that PTL@GC exhibited excellent mechanical properties, high enzyme activity retention (>90%), and controllable degradability (complete metabolism within 50 days). In animal models, PTL@GC reduced postoperative adhesion area by 22.77%, decreased local tumor burden to 28.42% of the control group, and achieved an inhibition rate of 58.49%, without inducing systemic toxicity. This study presents a biologically safe and functionally synergistic approach to addressing dual complications following colorectal cancer surgery, offering potential insights for future research on multifunctional Janus materials. Full article

Glucose oxidase (GOX) is widely used in bakery applications to improve dough rheology and bread quality. However, its direct addition to formulations limits its functionality due to premature enzymatic activity. This study used electrospraying to encapsulate GOX using chia mucilage and sodium alginate as biopolymeric wall materials. Three drying methods—critical point drying (CPD), Lyophilization/freeze-drying (LC), and oven drying (OD)—were compared to evaluate their impact on encapsulation efficiency (EE), enzymatic activity retention, and microstructural integrity. Our findings reveal that CPD preserved the porous structure of the microcapsules, minimizing enzymatic leakage and yielding the highest EE (70%). In contrast, LC induced ice crystal formation, disrupting the polymer network and leading to a moderate EE (27.43%), whereas OD resulted in extensive capsule shrinkage, causing significant enzyme loss (57.1%). The release kinetics of GOX during mixing were best described by the Korsmeyer–Peppas model (R² = 0.999), indicating a non-Fickian diffusion mechanism influenced by polymer relaxation. These results demonstrate that drying technique selection plays a crucial role in encapsulated enzymes’ stability and release behavior, providing new insights for optimizing enzyme delivery in bakery applications. Full article

A cutting-edge biosensor has been developed to monitor blood glucose levels, which is particularly vital for people with diabetes. This advanced technology uses a miniaturized and membraneless enzymatic fuel cell (EFC) as a compact electrical reader for rapid on-site diabetes diagnosis. Using disposable screen-printed gold electrodes (Au-SPE) modified with the enzyme glucose oxidase (GOx), the biosensor enables the oxidation of glucose at both the anode (counter electrode) and cathode (working electrode) of the EFC. The cathode contains graphene oxide/Prussian blue nanocubes (GO/PBNCs), while the anode uses a biographene layer. Both electrodes were modified with GOx by electrostatic/hydrogen bonding the enzyme to the modified electrodes surface. Individual evaluations of each electrode system emphasized their effectiveness. The integration of both electrodes resulted in an EFC that can generate an output power of approximately 1.8 μW/cm² at a glucose concentration of 5 mmol/L, which is very close to physiological conditions (3.8 to 6.9 mmol/L). This technology represents a significant advance and promises fully autonomous diagnostic devices suitable for a wide range of analytes. It paves the way for diagnostics everywhere and marks a fundamental shift in point-of-care (PoC) diagnostics. Full article

Rising temperatures have caused a major shift in wine chemistry, including increased sugar and pH along with decreased acidity. Wines produced from such grapes tend to be microbiologically unstable and are often described as unpalatable. This study looks at treatments to lower pH and enhance sensory characteristics of wines produced from grapes grown at higher temperatures. The four acidification treatments included the following: tartaric acid; verjus—an acidic juice made from unripe grapes; glucose oxidase with catalase enzyme (GOx), which converts glucose to gluconic acid; and ion exchange. All treatments were able to reduce pH to the target pH of 3.6. Sensory analysis was conducted using the Hierarchical Rate-All-That-Apply (HRATA) method and preference testing. Analysis of the HRATA and GCMS data using Principal Component Analysis (PCA) accounted for 78.69% and 70% of the variance observed, respectively. Wines treated with GOx and verjus exhibited the most distinct sensory profiles when compared to each other, the other treatments, and the control group. GOx-treated wines were associated with positive flavor descriptors including caramel, hazelnut, lemon, and fruity which correlated well with the aromatic compounds determined by GCMS. There were no significant differences in consumer preferences of treatments. This study shows how different acidifiers can be utilized by winemakers to affect not just the pH and acidity but also the aromatic and flavor profile of the wine. Full article

Listeria monocytogenes represents one of the main risks for food safety worldwide. Two enzyme-based antimicrobials (enzybiotics) have been combined in a novel treatment against this pathogenic bacterium, resulting in a powerful synergistic effect. One of the enzymes is an endolysin from Listeria phage vB_LmoS_188 with amidase activity (henceforth A10), and the other is an engineered version of glucose oxidase from Aspergillus niger (GOX). Both enzymes, assayed separately against Listeria innocua, showed antibacterial activity at the appropriate doses. The combination of the two enzybiotics resulted in a synergistic effect with a log reduction in viable cells (log N₀/N) of 4, whereas, taken separately, the same dose of A10 and GOX caused only 1.2 and 0.2 log reductions, respectively. Flow cytometry and microscopy analyses revealed that A10 treatment alone induced the aggregation of dead cells. L. monocytogenes showed higher resistance to single treatment with GOX or A10 than L. innocua. However, the synergic combination of A10 and GOX resulted in a high lethality of L. monocytogenes with a log N₀/N higher than 5 (below the detection limit in our analysis). Altogether, these results represent a novel efficient and eco-friendly antimicrobial treatment against the most lethal food-borne pathogen. Full article

With the continuous advancement of contemporary medical technology, an increasing number of individuals are inclined towards self-monitoring their physiological health information, specifically focusing on monitoring blood glucose levels. However, as an emerging flexible sensing technique, continuous and non-invasive monitoring of glucose in sweat offers a promising alternative to conventional invasive blood tests for measuring blood glucose levels, reducing the risk of infection associated with blood testing. In this study, we fabricated a flexible and wearable electrochemical enzyme sensor based on a two-dimensional Ti₃C₂T_x MXene nanosheets and coral-like polyaniline (PANI) binary nanocomposite (denoted as Ti₃C₂T_x/PANI) for continuous, non-invasive, real-time monitoring of sweat glucose. The exceptional conductivity of Ti₃C₂T_x MXene nanosheets, in conjunction with the mutual doping effect facilitated by coral-like PANI, significantly enhances electrical conductivity and specific surface areas of Ti₃C₂T_x/PANI. Consequently, the fabricated sensor exhibits remarkable sensitivity (25.16 μA·mM⁻¹·cm⁻²), a low detection limit of glucose (26 μM), and an extensive detection range (0.05 mM ~ 1.0 mM) in sweat. Due to the dense coral-like structure of Ti₃C₂T_x/PANI binary nanocomposite, a larger effective area is obtained to offer more active sites for enzyme immobilization and enhancing enzymatic catalytic activity. Moreover, the sensor demonstrates exceptional mechanical performance, enabling a 60° bend in practical applications, thus satisfying the rigorous demands of human sweat detection applications. The results obtained from continuous 60 min in vitro monitoring of sweat glucose levels demonstrate a robust correlation with the data of blood glucose levels collected by a commercial glucose meter. Furthermore, the fabricated Ti₃C₂T_x/PANI/GOx sensor demonstrated agreement with HPLC findings regarding the actual concentration of added glucose. This study presents an efficient and practical approach for the development of a highly reliable MXene glucose biosensor, enabling stable and long-term monitoring of glucose levels in human sweat. Full article

Bioremediation, the degradation of environmental pollutants by living organisms, has immense potential to lead to a greener planet. Bioinformatics analysis can contribute to the identification of novel microorganisms, which biodegrade contaminants, or of participating proteins and enzymes, and the elucidation of the complex metabolic pathways involved. In this study, we focus on C-P lyase and glyphosate oxidoreductase (Gox), two enzymes which degrade glyphosate, a widely used pesticide. Amino acid sequences of the two enzymes were collected from a broad range of microorganisms using the KEGG database and BLAST. Based on this, we identified additional lineages, with putative glyphosate-degrading activity, for which no glyphosate-degrading species have been reported yet. The conserved residues in each enzyme were identified via multiple alignments and mapped onto the 3D structures of the enzymes, using PyMOL, leading to novel insights into their function. As the experimental structure of Gox is still unknown, we created structural models based on three different programs and compared the results. This approach can be used to yield insights into the characteristics of potential glyphosate-degrading enzymes. Given the limited information available, such a step is important to gain further knowledge about them, which can contribute to their application in bioremediation in the future. Full article

Graphene and its variants exhibit excellent electrical properties for the construction of enzymatic interfaces. In particular, the direct electron transfer of glucose oxidase on the electrode surface is a very important issue in the development of enzyme-based bioelectrodes. However, the number of studies conducted to assess how pristine graphene forms different interfaces with other carbon materials is insufficient. Enzyme-based electrodes (formed using carbon materials) have been extensively applied because of their low manufacturing costs and easy production techniques. In this study, the characteristics of a single-walled carbon nanotube/graphene-combined enzyme interface are analyzed at the atomic level using molecular dynamics simulations. The morphology of the enzyme was visualized using an elastic network model by performing normal-mode analysis based on electrochemical and microscopic experiments. Single-carbon electrodes exhibited poorer electrical characteristics than those prepared as composites with enzymes. Furthermore, the composite interface exhibited 4.61- and 2.45-fold higher direct electron efficiencies than GOx synthesized with single-carbon nanotubes and graphene, respectively. Based on this study, we propose that pristine graphene has the potential to develop glucose oxidase interfaces and carbon-nanotube–graphene composites for easy fabrication, low cost, and efficient electrode structures for enzyme-based biofuel cells. Full article

Nowadays, biosensors are gaining increasing interest in foods’ and beverages’ quality control, owing to their economic production, enhanced sensitivity, specificity, and faster analysis. In particular, colorimetric biosensors can be combined with color recognition applications on smartphones for the detection of analytes, rendering the whole procedure more applicable in everyday life. Herein, chitosan (CS) films were prepared with the deep eutectic solvent (DES) choline chloride/urea/glycerol (ChCl:U:Gly). Glucose oxidase (GOx), a widely utilized enzyme in quality control, was immobilized within CS films through glutaraldehyde (GA), leading to the formation of CS/GOx films. The optimized GOx concentration and DES content were determined for the films. Moreover, the effect of the pH and temperature of the glucose oxidation reaction on the enzymatic activity of GOx was studied. The structure, stability, and specificity of the CS/GOx films as well as the Km values of free and immobilized GOx were also determined. Finally, the analytical performance of the films was studied by using both a spectrophotometer and a color recognition application on a smartphone. The results demonstrated that the films were highly accurate, specific to glucose, and stable when stored at 4 °C for 4 weeks and when reused 10 times, without evident activity loss. Furthermore, the films displayed a good linear response range (0.1–0.8 mM) and a good limit of detection (LOD, 33 μM), thus being appropriate for the estimation of glucose concentration in real samples through a smartphone application. Full article

We describe the creation of a conductive microcavity based on the assembly of two pieces of carbon nanotube buckypaper for the entrapment of two enzymes, horseradish peroxidase (HRP) and glucose oxidase (GOx), as well as a redox mediator: 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt (ABTS). The hollow electrode, employing GOx, HRP, and the mediator, as an electrochemical enzyme cascade model, is utilized for glucose sensing at a potential of 50 mV vs. Ag/AgCl. This bienzyme electrode demonstrates the ability to oxidize glucose by GOx and subsequently convert H₂O₂ to water via the electrical wiring of HRP by ABTS. Different redox mediators (ABTS, potassium hexacyanoferrate (III), and hydroquinone) are tested for HRP wiring, with ABTS being the best candidate for the electroenzymatic reduction of H₂O₂. To demonstrate the possibility to optimize the enzyme cascade configuration, the enzyme ratio is studied with 1 mg HRP combined with variable amounts of GOx (1–4 mg) and 2 mg GOx combined with variable amounts of HRP (0.5–2 mg). The bienzyme electrode shows continuous operational stability for over a week and an excellent storage stability in phosphate buffer, with a decay of catalytic current by only 29% for 1 mM glucose after 100 days. Full article