Immobilization Of Glucose Oxidase Enzyme Research Articles

Langmuir-Blodgett Films with Immobilized Glucose Oxidase Enzyme Molecules for Acoustic Glucose Sensor Application.

In this work, a sensitive coating based on Langmuir-Blodgett (LB) films containing monolayers of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) with an immobilized glucose oxidase (GOx) enzyme was created. The immobilization of the enzyme in the LB film occurred during the formation of the monolayer. The effect of the immobilization of GOx enzyme molecules on the surface properties of a Langmuir DPPE monolayer was investigated. The sensory properties of the resulting LB DPPE film with an immobilized GOx enzyme in a glucose solution of various concentrations were studied. It has shown that the immobilization of GOx enzyme molecules into the LB DPPE film leads to a rising LB film conductivity with an increasing glucose concentration. Such an effect made it possible to conclude that acoustic methods can be used to determine the concentration of glucose molecules in an aqueous solution. It was found that for an aqueous glucose solution in the concentration range from 0 to 0.8 mg/mL the phase response of the acoustic mode at a frequency of 42.7 MHz has a linear form, and its maximum change is 55°. The maximum change in the insertion loss for this mode was 18 dB for a glucose concentration in the working solution of 0.4 mg/mL. The range of glucose concentrations measured using this method, from 0 to 0.9 mg/mL, corresponds to the corresponding range in the blood. The possibility of changing the conductivity range of a glucose solution depending on the concentration of the GOx enzyme in the LB film will make it possible to develop glucose sensors for higher concentrations. Such technological sensors would be in demand in the food and pharmaceutical industries. The developed technology can become the basis for creating a new generation of acoustoelectronic biosensors in the case of using other enzymatic reactions.

Biomechanical behaviour of PEDOT:PSS-based hydrogels as an electrode for stent integrated enzyme biofuel cells

The possibility of creating a biofuel cell based on a metal stent was shown in this study. Given the existing stent implantation approaches, the integration of a biofuel cell into a stent naturally entails capacity for biofuel cells to be installed into a human body. As a counter electrode, a hydrogel based on iota-carrageenan, polyvinyl alcohol, and PEDOT:PSS, with an immobilized glucose oxidase enzyme, was proposed. Tension tests demonstrated that the hydrogel mechanical behavior resembles that of a bovine's vein. To obtain an analytical description, the deformation curves were fitted using Gent and Ogden models, prompting the fitting parameters which can be useful in further investigations. During cyclic biaxial studies the samples strength was shown to decreases insignificantly in the first 50 cycles and, further, remains stable up to more than 100 cycles. The biofuel cell was designed with the PEDOT:PSS based material as an anode and a Co–Cr self-expanding stent as a cathode. The maximum biofuel cell power density with a glucose concentration of 5 mM was 7.87 × 10−5 W in phosphate buffer and 3.98 × 10−5 W in blood mimicking buffer. Thus, the biofuel cell integration in the self-expanding stent was demonstrated.

Immobilization of GOx Enzyme on SiO2-Coated Ni-Co Ferrite Nanocomposites as Magnetic Support and Their Antimicrobial and Photocatalytic Activities.

The present study used a sol–gel auto-combustion approach to make silica (SiO2)-coated Ni–Co ferrite nanocomposites that would be used as a platform for enzyme immobilization. Using glutaraldehyde as a coupling agent, glucose oxidase (GOx) was covalently immobilized on this magnetic substrate. X-ray diffraction (XRD), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), high-resolution transmission electron microscopy (HRTEM), and fourier transform infrared spectroscopy (FTIR) was used to determine the structural analysis and morphology of Ni–Co ferrite/SiO2 nanocomposites. FTIR spectra confirmed the binding of GOx to Ni–Co ferrite/SiO2 nanocomposites, with a loading efficiency of around 85%. At alkaline pH and higher temperature, the immobilized GOx enzyme exhibited increased catalytic activity. After 10 times of reuses, it still had 69% catalytic activity. Overall, the immobilized GOx displayed higher operational stability than the free enzyme under severe circumstances and was easily recovered by magnetic separation. With increased doping concentration of the nanocomposites, the photocatalytic activity was assessed using a degradation process in the presence of methylene blue dye under UV light irradiation, which revealed that the surface area of the nanocomposites with increased doping concentration played a significant role in improving photocatalytic activity. The antibacterial activity of Ni–Co ferrite/SiO2 nanocomposites was assessed using the agar well diffusion method against Escherichia coli, a gram-negative bacteria (ATCC 25922). Consequently, it was revealed that doping of Ni2+ and Co2+ in Fe2O4/SiO2 nanocomposites at varied concentrations improved their antibacterial properties.

Genipin-mediated immobilization of glucose oxidase enzyme on carbon felt for use as heterogeneous catalyst in sustainable wastewater treatment

Enzyme immobilization is necessary process to improve the bioactivity and stability of the biocatalyst. In this study, glucose oxidase (GOx) enzyme was immobilized on plasma-treated fibrous carbon felt as a textile carrier to produce a heterogeneous catalyst. Genipin, as a naturally occurring crosslinker, that has less cytotoxicity than conventional crosslinkers, was used in the enzyme immobilization process. UV-Vis and FTIR spectra confirmed the crosslinking reaction between genipin and the primary amines of GOx enzyme, by forming blue-pigmented aggregates. GOx relative activity after crosslinking and immobilization on the carbon felt was maintained up to 40%, with stability in performance up to 6 cycles for the plasma treated carbon, while maintaining their bio-electro-activity as shown from cyclic voltammetry scans (CV). The obtained heterogeneous catalysts have been tested for use in sustainable wastewater treatment of Remazol Blue RR (RB) dyestuff by means of Bio-Fenton (BF) and enzymatic Bio-electro-Fenton (BEF) processes. The produced samples resulted in high color removal efficiency, up to 93% discoloration of (RB) for the first use in (BF) process in 3 h. Meanwhile, enzymatic (BEF) process resulted in up to 34% of COD removal, with simultaneous power density generation up to 0.16 ± 0.01 μW.cm−2 at a current density of around 10 ± 2 μA.cm−2 in 12 h. These results highlight the importance of genipin as a bio-based crosslinker for enzymes, and the potential use in both (BF) and (BEF) as sustainable approaches for wastewater treatment and as a step towards zero-energy degradation of organic matter.

Novel polymeric microspheres: Synthesis, enzyme immobilization, antimutagenic activity, and antimicrobial evaluation against pathogenic microorganisms.

New polymeric microspheres containing azomethine (1a-1c and 2a-2c) were synthesized by condensation to compare the enzymatic properties of the enzyme glucose oxidase (GOx) and to investigate antimutagenic and antimicrobial activities. The polymeric microspheres were characterized by elemental analysis, infrared spectra (FT-IR), proton nuclear magnetic resonance spectra, thermal gravimetric analysis, and scanning electron microscopy analysis. The catalytic activity of the glucose oxidase enzyme follows Michaelis-Menten kinetics. Influence of temperature, reusability, and storage capacity of the free and immobilized glucose oxidase enzyme were investigated. It is determined that immobilized enzymesexhibit good storage stability and reusability. After immobilization of GOx in polymeric supports, the thermal stability of the enzyme increased and the maximum reaction rate (Vmax ) decreased. The activity of the immobilized enzymes was preserved even after 5 months. The antibacterial and antifungal activity of the polymeric microspheres were evaluated by well-diffusion method against some selected pathogenic microorganisms. The antimutagenic properties of all compounds were also examined against sodium azide in human lymphocyte cells by micronuclei and sister chromatid exchange tests.

Immobilization of glucose oxidase enzyme on NiAl-LDHs for application in microfluidic fuel cell and serotonin detection.

In this work, an enzymatic glucose oxidase (GOx) electrode immobilized on double layered hydroxides of NiAl (NiAl-LDHs) was developed for the detection of serotonin neurotransmitters that may be present in human blood samples and carry out the glucose oxidation present in this fluid for energy conversion, and can be applied as self-feeding electrodes in Lab-on-a-Chip devices. The self-feeding electrodes based on fuel cells have a very promising future in lab-on-a-chip devices because they do not occupy an external power source, their manufacturing processes are relatively simple and low cost, and can be developed for different applications such as the determination of biomolecules for diagnosis of various diseases.

Surface modification of polyester fabric using plasma-dendrimer for robust immobilization of glucose oxidase enzyme

Robust immobilization of glucose oxidase (GOx) enzyme was achieved on poly(ethylene terephthalate) nonwoven fabric (PN) after integration of favourable surface functional groups through plasma treatments [atmospheric pressure-AP or cold remote plasma-CRP (N2 + O2)] and/or chemical grafting of hyperbranched dendrimers [poly-(ethylene glycol)-OH or poly-(amidoamine)]. Absorption, stability, catalytic behavior of immobilized enzymes and reusability of resultant fibrous bio-catalysts were comparatively studied. Full characterization of PN before and after respective modifications was carried out by various analytical, instrumental and arithmetic techniques. Results showed that modified polyester having amine terminal functional groups pledged better surface property providing up to 31% enzyme loading, and 81% active immobilized enzymes. The activity of the enzyme was measured in terms of interaction aptitude of GOx in a given time to produce hydrogen peroxide using colorimetric assay. The immobilized GOx retained 50% of its original activity after being reused six (06) times and exhibited improved stability compared with the free enzyme in relation to temperature. The reaction kinetics, loading efficiency, leaching, and reusability analysis of enzyme allowed drawing a parallel to the type of organic moiety integrated during GOx immobilization. In addition, resultant fibrous bio-catalysts showed substantial antibacterial activity against pathogenic bacteria strains (Staphylococcus epidermidis and Escherichia coli) in the presence of oxygen and glucose. These results are of great importance because they provide proof-of-concept for robust immobilization of enzymes on surface-modified fibrous polyester fabric for potential bio-industrial applications.

Novel dendrimers containing redox mediator: Enzyme immobilization and applications

In this study, new dendrimers with a ferrocene core were produced by means of a divergent method for the immobilization of the glucose oxidase enzyme (GOx). Pt(II) and Pt(IV) ions were attached to the terminal groups of dendrimers. Metal-containing dendrimers, [Fc(MGlu)2-Aph-Pt(II)] and [Fc(MGlu)2-Aph-Pt(IV)], were obtained using the “template method” with aminophenol and Pt(II)/Pt(IV) cations. These compounds have been characterized by molar conductivity, magnetic susceptibility, FTIR, UV–Vis, 1H NMR and LC-MS methods. The GOx enzyme was immobilized on the Fc(MGlu)2, [Fc(MGlu)2-Aph-Pt(II)] and [Fc(MGlu)2-Aph-Pt(IV)] dendrimers, and the immobilized enzyme optimization parameters (substrate concentration, temperature, pH, reusability and storage capacity) were determined. Their Km (mM) and Vmax (mM.min−1) values were calculated from the Michaelis–Menten equation. The reusability of the immobilized glucose oxidase enzyme was investigated in an artificial urine medium. The research showed that [Fc(MGlu)2-Aph-Pt(II)] retains more than 61.23% of its initial activity after 10 successive cycles, which is a remarkable result.

Amperometric glucose sensor based on the glucose oxidase enzyme immobilized on graphite rod electrode modified with Fe3O4-CS-Au magnetic nanoparticles

In this report, an amperometric detection method for blood glucose level was developed benefiting from glucose oxidase (GO) enzyme immobilization on nanoparticle modified graphite rod (GR) electrode. Fe3O4-CS-Au magnetic nanoparticles were synthesized, characterized by TEM, UV-Vis, magnetometry, FTIR, and zeta potential measurements and used for the modification of GR electrode. This modified electrode was used for the detection of glucose level amperometrically at 0.6 V. The obtained calibration graph was linear in the range of 5–30 mM glucose concentration with a coefficient of determination (R2) 0.9971. The limit of detection (LOD) and limit of quantification (LOQ) values were calculated as 0.55 and 1.83 mM, respectively. The modified GR electrode showed excellent selectivity in the presence of dopamine, ascorbic acid, and uric acid. The applicability of the developed method was examined in real blood samples by comparing the results obtained from commercial glucose sensor. This novel glucose detection method exhibited fast amperometric response, long storage time, and good selectivity.

Smartphone based non-invasive salivary glucose biosensor

The present work deals with the development of a non-invasive optical glucose biosensor using saliva samples and a smartphone. The sensor was fabricated with a simple methodology by immobilization of Glucose oxidase enzyme along with a pH responsive dye on a filter paper based strip. The strip changes color upon reaction with glucose present in saliva and the color changes were detected using a smartphone camera through RGB profiling. This standalone biosensor showed good sensitivity and low interference while operating within 20 s response time. We used various means for improvements such as the use of slope method instead of differential response; use of a responsive pH indicator and made numerous tweaks in the smartphone app. Calibration with spiked saliva samples with slopes for (R + G + B) pixels revealed an exponentially increasing calibration curve with a linear detection range of 50–540 mg/dL, sensitivity of 0.0012 pixels sec−1/mg dL−1 and LOD of 24.6 mg/dL. The biosensor was clinically validated on both healthy and diabetic subjects divided into several categories based on sex, age, diabetic status etc. and correlation between blood and salivary glucose has been established for better standardization of the sensor. Correlation of 0.44 was obtained between blood and salivary glucose in healthy individuals whereas it was 0.64 and 0.94 in case of prediabetic and diabetic patients respectively. The developed biosensor has the potential to be used for mass diagnosis of diabetes especially in such areas where people remain prohibited from routine analysis due to high healthcare cost. Apart from that, a smartphone would be the only device the user needs for this measurement, along with a disposable low cost test strip.

Preparation of immobilized glucose oxidase wafer enzyme on calcium-bentonite modified by surfactant

Wafer glucose oxidase (GOx) enzymes was produced by addition of PAH (Poly-Allyamine Hydrochloride) polymer into immobilized GOx enzyme on modified-Tetramethylammonium Hydroxide (TMAH) 5%-calsium-bentonite. The use of surfactant molecul (TMAH) is to modify the surface properties and pore size distribution of the Ca-bentonite. These properties are very important to ensure GOx molecules can be bound on the Ca-bentonit surface to be immobilized. The addition of the polymer (PAH) is expected to lead the substrates to be adsorbed onto the enzyme. In this study, wafer enzymes were made in various concentration ratio (Ca-bentonite : PAH) which are 1:0, 1:1, 1:2 and 1:3. The effect of PAH (Poly-Allyamine Hydrochloride) polymer added with various ratios of concentrations can be shown from the capacitance value on LCR meter and enzyme activity using DNS method. The addition of the polymer (PAH) showed effect on the activity of GOx, it can be shown from the decreasing of capacitance value by increasing of PAH concentration.

Process and kinetic characteristics of glucose oxidation catalyzed with immobilized enzyme

The aim of this experiment was to study the efficient immobilization of glucose oxidase enzyme (GOX) on silica gel particles and thereby enhance the conversion of glucose to d-glucono-1,5-lactone using this catalyst. According to a modified method, GOX was covalently immobilized on silica gel particles with the size of 0.063–0.2 mm. Reactions were carried out in a thermostatically controlled laboratory batch reactor at temperatures of 25, 30, 35 and 40 °C, stirrer speed of 0, 150 and 300 min−1, air flow rates of 0, 10, 20 and 30 L/h and at pH values 5, 6, 7 and 8. Glucose conversion was determined by measuring the concentration of unreacted glucose with a spectrophotometer at 500 nm. We examined the effect of main process parameters on the stability and activity of the GOX and on the conversion of glucose oxidation, thus determined the optimal reaction conditions. Kinetic studies of glucose oxidation with immobilized GOX as the catalyst were performed concluding that this reaction follows the kinetics proposed by Michaelis–Menten.

Phospholipid bilayer functionalized membrane pores for enhanced efficiency of immobilized glucose oxidase enzyme

This study integrates the advantages of biomimetic characteristics of phospholipid bilayer and convective flow through microporous membrane within the same configuration and reports the development of novel biofunctionalized membranes by incorporating phospholipid bilayer within membrane pores for efficient enzymatic catalysis. Two different approaches were used for membrane functionalization – (i) direct deposition of lipid bilayer within bare membrane pores, and (ii) immobilization of a polymer cushion prior to lipid bilayer within membrane pores. As a model enzyme, glucose oxidase (GOx) was immobilized into the phospholipid bilayer network within the functionalized membrane pores. In-depth activity study was conducted with different GOx-functionalized membranes while monitoring the production of H2O2 by oxidation of glucose. Biofunctionalized membrane, containing polymer cushion supported phospholipid bilayer and operating under convective mode of flow, exhibited important advancements in the field of immobilized enzymatic catalysis in terms of activity, stability and operating range of pH. Comparison of the results obtained in this study with other literature reported results establish the benefits of the biofunctionalized membrane discussed here. To the best of our knowledge this is the first study that investigates the advantages of incorporation of phospholipid bilayer within membrane pores for immobilized enzymatic catalysis.

Impact of structural features of SBA-15 host particles on activity of immobilized glucose oxidase enzyme and sensitivity of a glucose sensor

Glucose oxidase (GOD) enzyme immobilized mesoporous silica particles (SBA-15) have been developed as an amperometric glucose sensor. SBA-15 particles with fibre, rod and cuboid morphologies of decreasing aspect ratio (12.1, 4.6 and 1.1), and increasing mesopore diameter (6.8, 6.8 and 11.4 nm), respectively, were synthesized, to understand the role of these structural parameters on enzyme activity and sensor performance. Microscopy and gas sorption experiments confirm that, as required, GOD (~7 nm size) immobilizes inside mesopores. Amongst all forms, cuboid SBA-15 showed the highest normalized absolute activity and reusability for at least six cycles. Thus, compared to rod SBA-15, an electrode with GOD immobilized-cuboid SBA-15 showed ten times higher sensitivity of 0.53 µA/cm2/mM, with a linearity range of 1.0–7.7 mM glucose and a short response time (<5 s). Hence, structural features of the host could be directly related to the sensor performance. Therefore, in general, SBA-15 hosts with a smaller particle aspect ratio and a larger internal mesopore diameter (like cuboid), together with appropriate enzyme loading density and presence of ferrocene as an electron mediator, can ensure a superior biosensor performance.

Influence of load on the recycling stability of nanospheres attached platinium ion for determination of glucose

New nanospheres have been prepared with Pt2+ and Pt4+ for compare enzymatic properties of glucose oxidase enzyme (GOx). In this study nanoparticles, (aminomethyl)polystyrene (APS), 2-hydroxy-5-methylbenzaldehyde and Pt2+/Pt4+ have been synthesized by means of template and investigated the enzymatic properties of glucose oxidase enzyme (GOx) immobilized on there. The characteristics of the immobilized glucose oxidase (APS–Pt2+–GOx and APS–Pt4+–GOx) enzyme showed one optimum pH value. The influence of temperature, reusability and storage capacity on the free and immobilized glucose oxidase enzyme have been investigated. It is found that nanosphere including platinium atom exhibits excellent performance as the immobilized supporter of GOx, the immobilized enzyme demonstrates perfected storage and recycling stability. APS–Pt2+–GOx retains more than 30% of the initial activity after 32 successive cycles, which is a remarkable result.

A paper strip based non-invasive glucose biosensor for salivary analysis.

In our present study, we developed an optical biosensor for direct determination of salivary glucose by using immobilized glucose oxidase enzyme on filter paper strip (specific activity 1.4 U/strip) and then reacting it with synthetic glucose samples in presence of co-immobilized color pH indicator. The filter paper changed color based on concentration of glucose in reaction media and hence, by scanning this color change (using RGB profiling) through an office scanner and open source image processing software (GIMP) the concentration of glucose in the reaction medium could be deduced. Once the biosensor was standardized, the synthetic glucose sample was replaced with human saliva from donors. The individual's blood glucose level at the time of obtaining saliva was also measured using an Accuchek(™) active glucometer (Roche Inc.). In this preliminary study, a correlation of nearly 0.64 was found between glucose levels in saliva and blood of healthy individuals and in diabetic patients it was nearly in the order of 0.95, thereby validating the importance of salivary analysis. The RGB profiling method obtained a detection range of 9-1350 mg/dL glucose at a response time of 45 s and LOD of 22.2 mg/dL.

Development and characterization of a novel bioactive polymer with antibacterial and lysozyme‐like activity

The development and characterization of a novel bioactive polymer based on the immobilization of glucose oxidase enzyme (GOx) in a polyvinyl alcohol (PVA) film showing antibacterial activity is presented. The PVA-GOx composite material was extensively characterized by UV-vis, X-ray Photoelectron (XPS) spectroscopy and by Fourier Transform Infrared (FTIR) spectroscopy to verify the preservation of enzyme structural integrity and activity. The antimicrobial activity of this composite material against Escherichia coli and Vibrio alginolyticus was assessed. Furthermore the lysozyme-like activity of PVA-GOx was highlighted by a standard assay on Petri dishes employing Micrococcus lysodeikticus cell walls. The findings from this study have implications for future investigations related to the employment of PVA-GOx system as a composite material of pharmaceutical and technological interest.

Activation of Immobilized Glucose Oxidase Enzyme by Acoustic Wave Resonance Oscillation

Abstract Glucose oxidase enzyme was immobilized on ferroelectric LiNbO3 single crystals that were able to generate acoustic wave resonance oscillations by radio frequency electric power. The generation of the 3.6–14.7 MHz acoustic waves with lattice distortion vertical to the surface caused increases in the enzyme activity for glucose oxidation by decreasing the activation energy of reaction. A mechanism is proposed in which the acoustic waves enhance the interactions between the glucose substrate and the enzyme.

Nanoplatforms attached Schiff bases by condensation method; Investigation of glucose oxidase enzyme as biocatalysts

We report a easy approach for the immobilization of glucose oxidase enzyme (GOx) on nanoparticles. Nanoparticles-modified polystyrene-ANH2 (PSA) with some salicylaldehyde derivatives were synthesized by means of condensation and investigated the enzymatic properties of GOx immobilized on there. Modified polystyrenes were characterized using IR spectra, gel permeation chromatography, and scanning electron microscopy. All the characteristics of the immobilized glucose oxidase (PSA-SalH)-GOx, (PSA-SalCH3)-GOx, and (PSA)-GOx enzyme showed except one of them. Immobilized GOx on to (PSA) showed two optimum pH, other immobilized polymers were one optimum.

Polystyrene attached Pt(IV)-azomethine, synthesis and immmobilization of glucose oxidase enzyme.

Modified polystyrene with Pt(IV)–azomethine (APS–Sch–Pt) was synthesized by means of condensation and demonstrated to be a promising enzyme support by studying the enzymatic properties of glucose oxidase enzyme (GOx) immobilized on it. The characteristics of the immobilized glucose oxidase (APS–Sch–Pt–GOx) enzyme showed two optimum pH values that were pH = 4.0 and pH = 7. The insertion of stable Pt(IV)–azomethine spacers between the polystyrene backbone and the immobilized GOx, (APS–Sch–Pt–GOx), increases the enzymes’ activity and improves their affinity towards the substrate even at pH = 4. The influence of temperature, reusability and storage capacity on the free and immobilized glucose oxidase enzyme was investigated. The storage stability of the immobilized glucose oxidase was shown to be eleven months in dry conditions at +4 °C.