Immobilization Of Glucose Oxidase Research Articles

Synthesis and comparison of the performance of two different water-soluble phthalocyanine based electrochemical biosensor

Herein, a comparative study between novel water-soluble phthalocyanine-based biosensors was performed for the application of glucose sensing. For this purpose, two different copper (II) and manganese (III) phthalocyanines and their water-soluble derivatives were synthesized, and then their role as a supporting material for enzyme immobilization was evaluated by comparing their sensor performances. Two different phthalocyanine (AP-OH2-MnQ (MnPc) and AP-OH2-CuQ (CuPc)) were tested using electrochemical biosensor with immobilized glucose oxidase (GOx). To the best of our knowledge, the related water-soluble phthalocyanine-based glucose biosensors were attempted for the first time, and the developed approach resulted in improved biosensor characteristics. The constructed biosensors GE/MnPc/GOx and GE/CuPc/GOx showed good linearity between 0.003–1.0 mM and 0.05–0.4 mM, respectively. The limit of detection was estimated at 0.0026 mM for the GE/MnPc/GOx and 0.019 mM for the GE/CuPc/GOx. KMapp and sensitivity values were also calculated as 0.026 mM and 175.043 µAmM−1 cm−2 for the GE/MnPc/GOx biosensor and 0.178 mM and 117.478 µAmM−1 cm−2 for the GE/CuPc/GOx biosensor. Moreover, the fabricated biosensors were successfully tested to detect glucose levels in beverages with high recovery results. The present study shows that the proposed water-soluble phthalocyanines could be a good alternative for quick and cheap glucose sensing with improved analytical characteristics.

Evaluation of Electrochemical Sensor Using Microfluidic System

This paper presents the characterization and testing of electrochemical sensor using microfluidic system. Various geometric patterns were laser cut into the platinum working electrode of a biosensor. In this work, a microfluidic chamber was designed that allows phosphate buffer solution (PBS) to flow across the sensor, using a peristaltic pump, while varying the concentration of hydrogen peroxide. The amperometric characterization of the electrochemical sensor with 25, 50, 75, and 100[Formula: see text][Formula: see text]m perforation and 75[Formula: see text][Formula: see text]m spacing showed the highest sensitivity. This result was to be expected the purpose of patterning the sensors was to provide a 3-dimensional structure to the planar electrode in order for the enzyme, glucose oxidase, to be immobilized. Future work will include selecting one of the patterns for immobilization of glucose oxidase allowing us to realize a fully functional glucose sensor.

A Mn-porphyrinic metal–organic framework immobilizing glucose oxidase for combined photodynamic/chemodynamic/starvation therapy

A Mn-porphyrinic metal–organic framework immobilizing glucose oxidase for combined photodynamic/chemodynamic/starvation therapy

Enzymes on chemical gardens: Chemobrionics-based electrochemical biosensor

The relationship between biological materials and the immobilization matrix is a critical point in attaching biomolecules onto transducers in the preparation of biosensors. Various types of chemical molecules are used to conjugate biological compounds in the design of biofunctional surfaces. Among them, chemobrionics (CBCs) are good alternatives as an immobilization matrix to improve performance parameters of biosensors such as linear range, limit of detection (LOD), stability, reproducibility, etc. Here, CBCs are used to develop enzymatic biosensors, and glucose oxidase (GOx) is selected as a model enzyme. Firstly, CBC is functionalized using APTES to obtain free amine groups for immobilization of GOx via covalent bonds. The synthesized CBC-APTES is used as a matrix for the conjugation of GOx using glutaraldehyde as a crosslinker. The proposed CBC-APTES/GOx is formed on the glassy carbon electrode, and its performance is checked for glucose detection. The linear range is 0.025–1.25 mM with an LOD of 0.024 mM Glc. After finding no effect of some potential interference on the current response, CBC-APTES/GOx is successfully applied to analyze Glc in artificial samples such as serum, urine, sweat, and saliva. The objective here is to outline the integration of CBC in biosensor preparation, which is successfully carried out for the first time in literature.

A Glucose Oxidase-Curcumin Composite Nanoreactor for Multimodal Synergistic Cancer Therapy.

Glucose oxidase (GOx) selectively oxidizes β-d-glucose into gluconic acid and hydrogen peroxide; thus, it has emerged as a promising anticancer agent by tumor starvation and oxidative therapy. Here, we developed a nanoscale platform or "nanoreactor" that incorporates GOx and the bioactive natural product curcumin (CUR) to achieve a multimodal anticancer nanocomposite. The composite nanoreactor was formed by loading CUR in biodegradable polymeric nanoparticles (NPs) of poly(ethylene glycol)-b-poly(ε-caprolactone) (PEG-PCL). Prime-coating of the NPs with an iron(III)-tannic acid complex enabled facile immobilization of GOx on the NP surface. The NPs were monodisperse with a hydrodynamic diameter of 122 nm and a partially negative surface charge. The NPs were also associated with an excellent CUR loading efficiency and sustained release up to 96 h, which was accelerated by surface-immobilized GOx and followed supercase II transport. Viability assays were conducted on two model cancer cell lines, MCF-7 and MDA-MB-231 cells, as well as human dermal fibroblasts as a representative normal cell line. The assays revealed significantly improved potency of CUR in the composite nanoreactor, with up to 6000- and 1280-fold increase in MCF-7 and MDA-MB-231 cells, respectively, and lower toxicity toward normal cells. The NPs were also able to promote intracellular reactive oxygen species (ROS) generation and dissipation of the mitochondrial membrane potential, providing important clues on the mechanism of action of the nanoreactor. Further investigation of caspase-3 activity revealed that the nanoreactor had no effect or inhibited caspase-3 levels, signifying a caspase-independent mechanism of inducing apoptosis. Our findings present a promising nanocarrier platform that combines therapeutic agents with distinct mechanisms of action acting in synergy for more effective cancer therapy.

Self-propelled enzyme-controlled IR-mesoporous silica Janus nanomotor for smart delivery

Here, we report the preparation of a novel Janus nanoparticle with opposite Ir and mesoporous silica nanoparticles through a partial surface masking with toposelective modification method. This nanomaterial was employed to construct an enzyme-powered nanomachine with self-propulsion properties for on-command delivery. The cargo-loaded nanoparticle was provided with a pH-sensitive gate and unit control at the mesoporous face by first attaching boronic acid residues and further immobilization of glucose oxidase through reversible boronic acid esters with the carbohydrate residues of the glycoenzyme. Addition of glucose leads to the enzymatic production of H2O2 and gluconic acid, being the first compound catalytically decomposed at the Ir nanoparticle face producing O2 and causing the nanomachine propulsion. Gluconic acid leads to a pH reduction at the nanomachine microenvironment causing the disruption of the gating mechanism with the subsequent cargo release. This work demonstrates that enzyme-mediated self-propulsion improved release efficiency being this nanomotor successfully employed for the smart release of Doxorubicin in HeLa cancer cells.

Advancements in Glucose Monitoring: A Thin Film ZnO-Nanoflakes Based Highly Sensitive Wearable Biosensor for Noninvasive Sweat-Based Point-of-Care Monitoring for Diabetes

In the contemporary global landscape, diabetes is a concerning presence that contributes to noteworthy mortality rates. The etiology of this condition involves a subtle interaction between insulin deficiency and elevated blood sugar levels. Against this backdrop, the necessity for prompt, accurate, and continuous noninvasive monitoring of glucose concentrations becomes notably evident. The electrochemical flexible glucose biosensor was fabricated by immobilizing glucose oxidase (GOx) on ZnO nanoflakes, possessing a 20nm thickness, and synthesized on an Au-coated stretchable PET film. The sensitivity of the flexible biosensor was determined by 29.97μA/decade/cm², and the minimum detection limit was 1μM. The coefficient of determination was 97.76%, which is significantly linear.

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.

A smartphone-enabled colorimetric platform based on enzyme cascade amplification strategy for detection of Staphylococcus aureus in milk

Staphylococcus 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 a cascade bio-nanozyme formed by immobilized glucose oxidase (GOx) on 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. Staphylococcus aureus detection could be performed through naked-eye observation and smartphone measurement, and 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.

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

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.