Immobilization Of Glucose Oxidase Research Articles

Magnetic Nanoparticle Support with an Ultra-Thin Chitosan Layer Preserves the Catalytic Activity of the Immobilized Glucose Oxidase.

Here, we developed magnetically recoverable biocatalysts based on magnetite nanoparticles coated with an ultra-thin layer (about 0.9 nm) of chitosan (CS) ionically cross-linked by sodium tripolyphosphate (TPP). Excessive CS amounts were removed by multiple washings combined with magnetic separation. Glucose oxidase (GOx) was attached to the magnetic support via the interaction with N-hydroxysuccinimide (NHS) in the presence of carbodiimide (EDC) leading to a covalent amide bond. These steps result in the formation of the biocatalyst for D-glucose oxidation to D-gluconic acid to be used in the preparation of pharmaceuticals due to the benign character of the biocatalyst components. To choose the catalyst with the best catalytic performance, the amounts of CS, TPP, NHS, EDC, and GOx were varied. The optimal biocatalyst allowed for 100% relative catalytic activity. The immobilization of GOx and the magnetic character of the support prevents GOx and biocatalyst loss and allows for repeated use.

Intensification of a biocatalytic oxidation under fine bubble aeration in a rotating bed reactor

The utilization of oxidases suffers from insufficient oxygen availability due to its low solubility in gas-liquid multiphase systems. To overcome this limitation, oxygen is continuously supplied to the reaction medium during the course of the reaction, however, it causes significant gas consumption. In this study, a novel experimental set-up was established by installing a sintered frit porous sparger in the SpinChem® rotating bed reactor. Covalently immobilized glucose oxidase on epoxy-functionalized carriers was used in the rotating bed reactor with an activity yield of the immobilization of over 98%. For fine bubble aeration, the volumetric mass transfer coefficient kLa (67.3 h−1) tripled compared to macrobubble aeration (22.5 h−1), while the volume-specific aeration rate remained constant. A maximum reaction yield of 96.5% was reached. The reaction rate was improved by a factor of 12.9 for the fine bubble aeration compared to macrobubble aeration under identical conditions. With fine bubble aeration, the oxygen consumption was reduced by 87.5% over macrobubble aeration, establishing the same enzyme-specific reaction rates at a comparable kLa. Validation was carried out in repetitive batches, achieving the same glucose oxidase specific activity. This study demonstrates the advantages of fine bubble aeration and presents promising results for addressing the sustainable biotransformation processes.

Design and Synthesis of an Integrated Nanozyme by Immobilizing Glucose Oxidase on an Iron-Based Metal-Organic Framework for the Development of a Glucose Assay

Design and Synthesis of an Integrated Nanozyme by Immobilizing Glucose Oxidase on an Iron-Based Metal-Organic Framework for the Development of a Glucose Assay

A Smartphone-Enabled Colorimetric Platform Based on Enzyme Cascade Amplification Strategy for Detection of Staphylococcus aureus in Milk

Staphylococcus aureus (S. aureus) is a pathogenic bacterium-contaminating milk and dairy foods causing food poisoning and foodborne pathogens. In this work, a smartphone-enabled enzyme cascade-triggered colorimetric platform was constructed using cascade bio-nanozyme formed by immobilized glucose oxidase (GOx) on the Fe3O4@Ag for rapid detection of S. aureus. Benefiting from reasonable experimental design, a bio-nanozyme cascade-triggered reaction was achieved through H2O2 produced by GOx oxidation of glucose, followed by in situ catalysis of 3,3',5,5'-tetramethylbenzidine (TMB) by the inherent peroxidase-like activity of Fe3O4@Ag to produce color signals. S. aureus detection could be performed through naked-eye observation and smartphone measurement, the developed assay can achieve quantitative and qualitative detection of S. aureus. The on-site nanoplatform had satisfactory specificity and sensitivity with a low detection limit of 6.9 cfu·mL-1 in 50 min. Moreover, the nanoplatform has good practicality in the detection of S. aureus in milk samples. Therefore, the assay has potential application prospects in food safety inspection.

Organosilicon Material in Combination with Structure-Controlling Agents as a Basis for Immobilization of the Enzyme Glucose Oxidase

Organosilicon Material in Combination with Structure-Controlling Agents as a Basis for Immobilization of the Enzyme Glucose Oxidase

Fabrication of flexible glucose sensor based on heterostructure ZnO nanosheets decorated PU/Chitosan-PANI hybrid nanofiber

Designing and fabrication of immensely accurate, affordable, sensitive, and portable glucose sensors is rapidly increasing in clinical applications. However, commercialization of wearable sensors has been impeded by inadequate conductivity, insufficient durability, poor flexibility, and enzyme immobilization. Herein, a scalable approach for fabricating a nanofiber-based highly electroactive, flexible, and disposable (PUC-PANI-ZnO@GOx) sensor that can assess glucose stimuli with sufficient liquid permeability by anchoring ZnO nanosheets over polyaniline (PANI) coated polyurethane-chitosan (PU-Chitosan; PUC) fibrous mat followed by glucose oxidase (GOx) immobilization is described. Regarding the electrocatalytic performance, PUC-PANI-ZnO@GOx sensor showed a high sensitivity (∼998.72 µA mM−1 cm−2) and a low detection limit (∼7.31 µM) for a broad linear range of 0.01 – 9.48 mM with acceptable stability and reproducibility. The fabricated sensor was highly selective towards glucose in the presence of exogenous and endogenous interfering species. To the best of our knowledge, this novel polymer-based nanocomposite adorned on nanofiber substrate was utilized for the first time for the fabrication of a flexible glucose sensor. It can be used for producing a large-scale, cost-effective, and high-performing sensor. Hence, this sustainable and eco-friendly approach can lead to new techniques for designing next-generation wearable and implantable glucose sensors.

Synthesis of a Multifunctional Glyco-Block Copolymer through Reversible Addition-Fragmentation Chain Transfer Polymerization and Click Chemistry for Enzyme and Drug Loading into MDA-MB-231 Cells.

Reversible addition-fragmentation chain transfer polymerization has been used in various applications such as preparing nanoparticles, stimulus-responsive polymers, and hydrogels. In this study, the combination of this polymerization method and Cu(I)-catalyzed azide-alkyne cycloaddition click chemistry was used to prepare the multifunctional glyco-diblock copolymer P(PEG-co-AM)-b-PF, which is composed of mannosides for cell targeting, poly(ethylene glycol) (PEG) for biocompatibility, and aryl-aldehyde moieties for enzyme immobilization. The alkyne group in the polymer structure enables the alternation for other azide-conjugated monomers. The stepwise synthesis of the polymers was fully characterized. P(PEG-co-AM)-b-PF was self-assembled into polymeric nanoparticles (BDOX-GOx@NPs) for glucose oxidase immobilization through Schiff base formation and for encapsulating the prodrug of arylboronate-linked doxorubicin (BA-DOX) under optimal conditions. Glucose oxidase in BDOX-GOx@NPs catalyzes glucose oxidation to produce gluconic acid and H2O2, which cause oxidative stress. Glucose oxidase also consumes glucose, causing starvation in cancer cells. The produced H2O2 can selectively activate the anticancer prodrug BA-DOX for chemotherapy. In vitro data indicate that GOx and the prodrug BA-DOX present inside BDOX-GOx@NPs exhibit higher stability than free glucose oxidase with a favorable active DOX release profile. MDA-MB-231 cells, which express mannose receptors, were used to establish a model in this study. The bioactivity of the nanoplatform in the two- and three-dimensional models of MDA-MB-231 cancer cells was investigated to ascertain its antitumor efficacy.

Dual Stimulus-Responsive Enzyme@Metal-Organic Framework-Polymer Composites toward Enhanced Catalytic Performance for Visual Detection of Glucose.

Enzyme immobilization on a metal-organic framework (enzyme@MOF) has been proven to be a promising strategy for boosting catalysis and biosensing applications. However, promoting the catalytic performance of polymer-modified enzyme@MOF composites remains an ongoing challenge. Herein, a protocol for enzyme immobilization was designed by using a smart polymer-modified MOF (UiO-66-NH2, UN) as the support. Through in situ polymerization, the dual stimulus-responsive poly(N-2-dimethylamino ethyl methacrylate) (PDM) was prepared. The PDM as a "soft cage" protected the immobilized glucose oxidase (GOx)-horseradish peroxidase (HRP) on the surface of the rigid UN. The confinement effect was generated by varying the temperature and pH, thereby improving the catalytic activity of the GOx-HRP@UN-PDM composites. In comparison with free enzymes, the fabricated composites exhibited an 8.9-fold enhancement in catalytic performance (Vmax) at pH 5.0 and 49 °C. Furthermore, relying on a cascade reaction generated in the composites, an assay was developed for the visual detection of glucose in rat serum. This study introduces a groundbreaking approach for the construction of smart enzyme@MOF-polymer composites with high catalytic activity for sensitive monitoring of biomolecules.

Fe3O4 NPs-encapsulated metal-organic framework/enzyme hybrid nanoreactor for drug-resistant bacterial elimination via enhanced chemodynamictherapy

As a potential tool for combating bacterial infections, chemodynamic therapy (CDT), which utilizes the highly toxic hydroxyl radical (•OH), has shown tremendous promise. Its antibacterial efficacy, however, is compromised by insufficient H2O2 levels and a near-neutral pH at infection sites. Herein, a glucose-fueled and H2O2 self-supplying •OH nanogenerator (GOx-Fe3O4@MIL) based on cascade catalytic reactions is successfully constructed by immobilizing glucose oxidase (GOx) on the surface of Fe3O4 NPs-encapsulated MIL88B–NH2. MIL88B–NH2 (MIL NPs), a peroxidase-mimicking enzyme, immobilizes and protects GOx while synergistically enhancing the CDT effect of Fe3O4 NPs. GOx can continuously convert glucose into gluconic acid and H2O2. The former reduces the pH value to approximately 4, at which point Fe3O4@ MIL NPs exhibit the highest reaction activity. The continually produced H2O2 is used for subsequent catalysis of activated Fe3O4 @MIL NPs to generate highly toxic •OH for antibacterial applications, avoiding the direct use of relatively high concentrated and toxic H2O2. In vitro and in vivo results indicate that the designed self-activated nanoreactor can significantly inhibit methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Acinetobacter baumannii (MDR-AB) growth with negligible biotoxicity.

Highly sensitive and stable glucose sensing using N-type conducting polymer based organic electrochemical transistor

Glucose sensors have emerged as indispensable tools in monitoring glucose concentrations, enabling individuals to take proactive measures in managing their health effectively. Organic electrochemical transistors (OECTs) have emerged as promising platforms for glucose sensing, offering inherent advantages, such as stability in aqueous environments, low operating voltage requirements, and remarkable sensitivity. In this study, we present the development of an OECT-based glucose sensor using a N-type conducting polymer poly(benzimidazobenzophenanthroline) (BBL) as the active layer. BBL exhibited high performance and stability in an aqueous electrolyte, making it an ideal candidate for glucose sensing applications. By incorporating ferrocene (FC) as an electron mediator and immobilizing glucose oxidase (GOx) on the gate electrode, we achieved remarkable sensitivity and linearity within the glucose concentration range relevant to typical blood sugar levels. Stability tests revealed consistent performance over multiple cycles and continuous stressing, indicating the sensor's long-term stability and reliability. Additionally, we investigated the role of FC through electrochemical absorption and Raman shift spectra, providing insights into the glucose sensing mechanism. Moreover, the glucose sensor demonstrated promising performance on a flexible PET substrate, highlighting its versatility for practical applications in glucose sensing and beyond.

DNA flexible chain modified MOFs as a versatile platform for chemoenzymatic cascade reactions in glucose catalysis

Glucose oxidase (GOD) is widely used in the pharmaceutical industry, fermentation products and glucose biosensors for its essential role in catalyzing the conversion of glucose to gluconic acid and hydrogen peroxide (H2O2). As H2O2 is the by-product and will have a toxic effect on glucose oxidase, so introducing another enzyme that could consume H2O2 to form an enzymatic cascade reaction is a practical solution. However, this decision will lead to extra expenses and complex condition optimization such as the specific mass ratio, temperature and pH to improve the activity, stability and recyclability. Herein, we describe a mild and versatile strategy by anchoring GOD on carboxyl-activated MOF (Cu-TCPP(Fe)) through DNA-directed immobilization (DDI) technology. Robust MOF nanosheets were utilized as not only the carrier for the immobilization of GOD, but also a peroxidase-like catalyst for the decomposition of H2O2 to reduce its harmful impacts. In this work, the immobilized GOD retained 55.78% of its initial activity after being used for 7 times. More than 60% of the immobilized enzyme's catalytic activity was still maintained after 96 h of being stored at 50 ℃. This study provides a new idea for preparing immobilized enzymes with enhanced stability, fast diffusion and high activity, which can be used in fields such as biocatalysis and biotechnology.

Immobilizing glucose oxidase on AuCu hydrogels for enhanced electrochromic biosensing

Development of highly sensitive and accurate biosensors still faces a great challenge. Herein, glucose oxidase (GOx) is efficiently immobilized on the AuCu hydrogels owing to their porous structure and interfacial interaction, demonstrating enhanced catalytic activity, satisfactory stability and recyclability. Besides, by integration of AuCu@GOx and electrochromic material of Prussian blue, a sensitive and stable biosensing platform based on the excellent electrochromic property of Prussian blue and the enhanced enzyme activity of AuCu@GOx is developed, which enables the electrochemical and visual dual-mode detection of glucose. The as-constructed biosensing platform possesses a wide linear range, and good selectivity for glucose detection with a limit of detection of 0.82 μM in visual mode and 0.84 μM in electrochemical mode. This easy-to-operate biosensing platform opens a door for the practical application of the multi-mode strategy for glucose detection.

The Development of Reagentless Amperometric Glucose Biosensor Based on Gold Nanostructures, Prussian Blue and Glucose Oxidase.

Precise blood glucose detection plays a crucial role in diagnosing and medicating diabetes, in addition to aiding diabetic patients in effectively managing their condition. In this research, a first-generation reagentless amperometric glucose biosensor was developed by combining the graphite rod (GR) electrode modification by gold nanostructures (AuNS) and Prussian blue (PB) with glucose oxidase (GOx)-an enzyme that can oxidize glucose and produce H2O2. Firstly, AuNS was electrochemically deposited on the GR electrode (AuNS/GR), and then PB was electrochemically synthesized on the AuNS/GR electrode (PB/AuNS/GR). Finally, GOx was immobilized over the PB/AuNS nanocomposite with the assistance of Nafion (Nf) (Nf-GOx/PB/AuNS/GR). An application of PB in the design of a glucose biosensor enables an easy electrochemical reduction and, thus, the determination of the H2O2 produced during the GOx-catalyzed oxidation of glucose in the sample at a low operation potential of -0.05 V vs. Ag/AgCl/KCl3 mol L-1. In addition, AuNS increased the electrochemically active surface area, improved the GOx immobilization and ensured a higher analytical signal. The developed glucose biosensor based on the Nf-GOx/PB/AuNS/GR electrode exhibited a wide linear range, from 0.025 to 1 mmol L-1 of glucose, with a 0.0088 mmol L-1 limit of detection, good repeatability and high selectivity over electroactive interfering substances. The developed biosensor is convenient for the determination of glucose in the physiological environment.

Response surface methodology use in construction of polianiline-coated carbon paste electrode-based biosensor: Modification and characterization.

In this study, the effect of amperometric glucose biosensor construction and using conditions on the current response was investigated in detail applying experimental design. Polyaniline (PANI) was synthesized on the carbon paste electrode (CPE) surface using the cyclic voltammetry technique in sodium oxalate (NaOx ) electrolyte medium, and an amperometric biosensor was constructed by immobilizing glucose oxidase (GOD). Biosensor preparation (aniline, GOD and NaOx concentrations, and scan rate) and operating conditions (pH and applied potential) were optimized by Box-Behnken and optimal designs, respectively, via State Ease Design Expert 7.0.1.1 software. ANOVA analyses showed that among the biosensor preparation parameters, the NaOx concentration has the highest effect on the current measured in the presence of glucose, whereas in the optimization of pH and potential parameters applied in current measurement studies, it has been revealed that pH has a very high effect on the measured current. Several compounds, such as MWCNT, two different ionic liquids and two different organic molecules were added to carbon paste, and, among them, 2-cyanoethylpyrrole (CPy) enhanced the efficacy highly, most probably due to its polymerization in the paste and increasing the electron transfer rate of the CPE. Sucrose- and lactose-sensitive biosensors were also constructed by co-immobilizing GOD with invertase (INV) or β-galactosidase, respectively, onto modified CPE, and sensitivities to their substrates were shown by cyclic voltammetry and impedance analysis. CPy modification caused an increase in the current values, and also Imax /KM values increased approximately 11.8, 7.83, and 2.56 times for glucose-, sucrose-, and lactose-sensitive CPEs, respectively.

Laser induced graphene (LIG) biosensors derived from chitosan: Towards sustainable and green electronics

Modern biosensors can provide real-time monitoring of individuals' health status, revolutionizing traditional healthcare diagnostics. As demand for these devices is continuously growing, the development of novel green materials and processes is mandatory to ensure sustainability. In this work, a simple laser direct writing approach was utilized to fabricate a novel green Laser Induced Graphene (LIG) glucose biosensor, whereby chitosan-based biofilms were used as writing feedstock material. Careful optimization of the biofilm composition was carried out in conjunction with the optimization of laser irradiation parameters to obtain bio-LIG structures with low sheet resistance and spectral characteristics of graphene-like materials. The surface of bio-LIG electrodes was modified with Prussian Blue nanoparticles and the electrocatalytic performance of bio-LIG sensors towards H2O2 was investigated using voltammetric techniques. The response of H2O2 was linear in the range 3 μM - 1 mM (sensitivity, 103.4 μA mM−1 cm−2 and the limit of detection (LOD), 1.9 μM). Following immobilization of glucose oxidase, the bio-LIG sensors showed a linear response of glucose in phosphate buffer (PBS) in the relevant physiological range of 25–300 μM (sensitivity, 457 nA mM−1 cm−2). The LOD was calculated as 9.6 μM. Additionally, the biosensor showed comparable results in artificial sweat.

Glucose Oxidase Loading in Ordered Porous Aluminosilicates: Exploring the Potential of Surface Modification for Electrochemical Glucose Sensing.

Enzymatic electrochemical sensors have become the leading glucose detection technology due to their rapid response, affordability, portability, selectivity, and sensitivity. However, the performance of these sensors is highly dependent on the surface properties of the electrode material used to store glucose oxidase and its ability to retain enzymatic activity under variable environmental conditions. Mesoporous thin films have recently attracted considerable attention as promising candidates for enzyme storage and activity preservation due to their well-defined nanoarchitecture and tunable surface properties. Herein, we systematically compare pathways for the immobilization of glucose oxidase (GOx) and their effectiveness in electrochemical glucose sensing, following modification protocols that lead to the electrostatic attraction (amino functionalization), covalent bonding (aldehyde functionalization), and electrostatic repulsion (oxygen plasma treatment) of the ordered porous aluminosilicate-coated electrodes. By direct comparison using a quartz crystal microbalance, we demonstrate that glucose oxidase can be loaded in a nanoarchitecture with a pore size of ∼50 nm and pore interconnections of ∼35 nm using the native aluminosilicate surface, as well as after amino or aldehyde surface modification, while oxygen plasma exposure of the native surface inhibits glucose oxidase loading. Despite a variety of routes for enzyme loading, quantitative electrochemical glucose sensing between 0 and 20 mM was only possible when the porous surface was functionalized with amino groups, which we relate to the role of surface chemistry in accessing the underlying substrate. Our results highlight the impact of rational surface modification on electrochemical biosensing performance and demonstrate the potential of tailoring porous nanoarchitecture surfaces for biosensing applications.

Glucose Sensor Using Sol-Gel Coating Layer Deposited on PMMA Optical Fiber: An Enzyme Activity Measurement System.

In the present work, a biocatalytic glucose optical sensor produced by immobilizing glucose oxidase (GOD) as a recognition molecule over a PMMA (polymethylmethacrylate) optical fiber is introduced. An enzymatic encapsulation process was carried out using the sol-gel method, depositing a TEOS-based coating by immersion at the end of an optical fiber; the biosensor was characterized using different glucose levels. Finally, the best way to encapsulate the enzyme and prevent it from degrading is to perform the process at room temperature, and later implement the deposition of the coating on the fiber. The drying process was optimal below 8 °C.

Electrochemical Fine-Tuning of the Chemoresponsiveness of Langmuir-Blodgett Graphene Oxide Films.

Graphene oxide has been widely deployed in electrical sensors for monitoring physical, chemical, and biological processes. The presence of abundant oxygen functional groups makes it an ideal substrate for integrating biological functional units to assemblies. However, the introduction of this type of defects on the surface of graphene has a deleterious effect on its electrical properties. Therefore, adjusting the surface chemistry of graphene oxide is of utmost relevance for addressing the immobilization of biomolecules, while preserving its electrochemical integrity. Herein, we describe the direct immobilization of glucose oxidase onto graphene oxide-based electrodes prepared by Langmuir-Blodgett assembly. Electrochemical reduction of graphene oxide allowed to control its surface chemistry and, by this, regulate the nature and density of binding sites for the enzyme and the overall responsiveness of the Langmuir-Blodgett biofilm. X-ray photoelectron spectroscopy, surface plasmon resonance, and electrochemical measurements were used to characterize the compositional and functional features of these biointerfaces. Covalent binding between amine groups on glucose oxidase and epoxy and carbonyl groups on the surface of graphene oxide was successfully used to build up stable and active enzymatic assemblies. This approach constitutes a simple, quick, and efficient route to locally address functional proteins at interfaces without the need for additives or complex modifiers to direct the adsorption process.

A novel effective immobilization of glucose oxidase on Ni0.25Zn0.25Cu0.25Co0.25La0.06Fe1.94O4 – Chitosan nanocomposite as an enzymatic glucose biosensor

An effectual enzymatic glucose biosensor has drawn significant attention in the natural world due to its continuous glucose monitoring systems on human beings. A need for accurate and dependable glucose biosensors is needed and has notably augmented the keen interest to synthesize new non-invasive glucose monitoring systems in the recent phase. A novel Ni0.25Zn0.25Cu0.25Co0.25La0.06Fe1.94O4 nanocomposite has been synthesized via the combustion method to develop an appreciable glucose biosensor. The glucose biosensor was fabricated by immobilization of glucose oxidase (GOx) onto chitosan (CH)-Ni0.25Zn0.25Cu0.25Co0.25La0.06Fe1.94O4 heterojunction nanocomposite on FTO glass substrate. The performance of the as-prepared enzymatic glucose biosensor was estimated using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The electrochemical studies revealed an enhanced diffusion of molecules on the electrode surface, superior charge transfer rate, high sensitivity, and fast response time. The Ni0.25Zn0.25Cu0.25Co0.25La0.06Fe1.94O4-CH bi-junction conjoining with GOx exhibits a higher sensitivity of 52.76 µAmM-1cm-2 in a comprehensive undeviating range. The catalytic properties of the electrode in the H2O2 solution were studied using cyclic voltammetry, which showed a good linear response with an increase in scan rate and peak current resulting in enriched electrostatic interaction. In addition, the fabricated biosensor with a low Michaelis-Menten constant contributes a better affinity of the electrode surface towards glucose oxide.

Electrochemical Properties Analysis of Immobilized Glucose Oxidase on Multi-Walled Carbon Nanotubes and Hydroxy-Fullerene Nanocomposites

The development of a glucose sensor that can monitor blood glucose levels is particularly important with the increasing number of diabetes patients. Here, a glucose biosensor based on Nafion (NF), glucose oxidase (GOx), multi-walled carbon nanotubes (MWCNTs), hydroxy fullerene (HFs) composite modified glassy carbon electrode (GCE) is presented. The transmission electron microscopy (TEM), field emission scanning electron microscope (FESEM), UV–visible spectroscopy (UV–vis), cyclic voltammetry (CV), square wave voltammetry (SWV), and electrochemical impedance spectroscopy (EIS) methods are used to study the characteristics of the modification materials and to evaluate electrochemical properties of the modified electrodes. The heterogeneous electron transfer constant (ks) is calculated to be 4.49 s−1. The response to glucose is linear in the range of 0.02–0.25 mM and 0.25–4.0 mM with a sensitivity of 80.59 μA mM−1 cm−2 and detection limit (LOD) of 9.93 μM. The NF/MWCNTs-HFs-GOx/GCE biosensor demonstrate high sensitivity to glucose with excellent storage stability and anti-interference capability.