Covalent Immobilization Of Glucose Oxidase Research Articles

Optimizing Covalent Immobilization of Glucose Oxidase and Laccase on PV15 Fluoropolymer-Based Bioelectrodes.

Enzymatic biofuel cells (EBCs) represent a promising technology for biosensors, biodevices, and sustainable green energy applications, thanks to enzymes' high specificity and catalytic efficiency. Nevertheless, drawbacks such as limited output power and short lifetime have to be solved. Nowadays, research is addressed to the use of 3D electrode structures, but the high cost and the industrialization difficulties of such electrodes represent a key issue. The purpose of the paper is thus to describe the use of a low-cost commercial conductive polymer (Sigracell® PV15) as support for the covalent immobilization of glucose oxidase and laccase, for bioanode and biocathode fabrication, respectively. Efficient immobilization protocols were determined for the immobilized enzymes in terms of employed linkers and enzyme concentrations, resulting in significant enzymatic activities for units of area. The analysis focuses specifically on the optimization of the challenging immobilization of laccase and assessing its stability over time. In particular, an optimum activity of 23 mU/cm2 was found by immobilizing 0.18 mg/cm2 of laccase, allowing better performances, as for voltage output and electrochemical stability, and a direct electron transfer mechanism to be revealed for the fabricated biocathode. This study thus poses the basis for the viable development of low-cost functional EBC devices for biomedical applications.

Carboxylic acid-tethered polyaniline as a generic immobilization matrix for electrochemical bioassays.

Polyaniline (PANI) was functionalized by thiol-ene click chemistry to obtain carboxylic acid-tethered polyaniline (PCOOH). The versatility of PCOOH as an immobilization matrix was demonstrated by constructing four different biosensors for detection of metabolites and cancer biomarker. Immobilization efficiency of PCOOH was investigated by surface plasmon resonance and fluorescence microscopic analysis which revealed dense immobilization of biomolecules on PCOOH as compared to conventional PANI. A sandwich electrochemical biosensor was constructed using PCOOH for detection of liver cancer biomarker, α-fetoprotein (AFP). The sensor displayed sensitivity of 15.24 µA (ng mL-1)-1cm-2, with good specificity, reproducibility (RSD 3.4%), wide linear range (0.25-40ngmL-1) at - 0.1V (vs. Ag/AgCl), and a low detection limit of 2pgmL-1. The sensor was validated by estimating AFP in human blood serum samples where the AFP concentrations obtained are consistent with the values estimated using ELISA. Furthermore, utilization of PCOOH for construction of enzymatic biosensor was demonstrated by covalent immobilization of glucose oxidase, uricase, and horseradish peroxidase (HRP) for detection of glucose, uric acid, and H2O2, respectively. The biosensors displayed reasonable sensitivity (50, 148, 127 µA mM-1cm-2), and linear ranges (0.1-5, 0.1-6, 0.1-7mM) with a detection limit of 10, 1, and 8µM for glucose, uric acid, and H2O2, respectively. The present study demonstrates the capability of PCOOH to support and enable oxidation of H2O2 generated by oxidase enzymes as well as HRP enzyme catalyzed reduction of H2O2. Thus, PCOOH offers a great promise as an immobilization matrix for development of high-performance biosensors to quantify a variety of other disease biomarkers. Carboxylic acid-tethered polyaniline synthesized by thiol-ene click chemistry was used as matrix to construct four different electrochemical biosensors for detection of cancer biomarker α-fetoprotein, glucose, uric acid, and H2O2.

In situ growth of TiO2 nanowires on Ti3C2 MXenes nanosheets as highly sensitive luminol electrochemiluminescent nanoplatform for glucose detection in fruits, sweat and serum samples

A sensitive electrogenerated chemiluminescence (ECL) biosensor for glucose was developed based on in situ growth of TiO2 nanowires on Ti3C2 MXenes (TiO2–Ti3C2) as the nanoplatform. Via tuning the alkaline oxidation time, different amount of TiO2 nanowires can be found on MXenes. An ECL biosensor for glucose was constructed by covalent immobilization of glucose oxidase (GODx) on the glycine functional TiO2–Ti3C2 surface, with the ECL signal depending on the in-situ formation of H2O2 via the specifically catalysis of glucose by GODx, resulting in the apparent increase of ECL signal. The TiO2–Ti3C2 can also act as the catalyst for the oxidation of H2O2 into O2 to enhance the ECL of luminol. Based on this strategy, a highly sensitive ECL biosensor for glucose was obtained in wide concentration range of 20 nM-12 mM with a low detection limit of 1.2 nM (S/N = 3). The synergistic effects of large surface area, excellent conductivity, and high catalytic activity of the TiO2–Ti3C2 make the sensor highly sensitive toward glucose; the specific enzyme catalysis reaction promises excellent selectivity of the ECL sensor. The proposed biosensor has been employed to detect glucose in human serum, fruits, and sweat samples with excellent performance, providing a universal approach for glucose in various samples, which shows great prospect in clinical diagnostics and wearable sensors.

Analytical Parameters of a Novel Glucose Biosensor Based on Grafted PFM as a Covalent Immobilization Technique.

Bioanalytical methods, in particular electrochemical biosensors, are increasingly used in different industrial sectors due to their simplicity, low cost, and fast response. However, to be able to reliably use this type of device, it is necessary to undertake in-depth evaluation of their fundamental analytical parameters. In this work, analytical parameters of an amperometric biosensor based on covalent immobilization of glucose oxidase (GOx) were evaluated. GOx was immobilized using plasma-grafted pentafluorophenyl methacrylate (pgPFM) as an anchor onto a tailored HEMA-co-EGDA hydrogel that coats a titanium dioxide nanotubes array (TiO2NTAs). Finally, chitosan was used to protect the enzyme molecules. The biosensor offered outstanding analytical parameters: repeatability (RSD = 1.7%), reproducibility (RSD = 1.3%), accuracy (deviation = 4.8%), and robustness (RSD = 2.4%). In addition, the Ti/TiO2NTAs/ppHEMA-co-EGDA/pgPFM/GOx/Chitosan biosensor showed good long-term stability; after 20 days, it retained 89% of its initial sensitivity. Finally, glucose concentrations of different food samples were measured and compared using an official standard method (HPLC). Deviation was lower than 10% in all measured samples. Therefore, the developed biosensor can be considered to be a reliable analytical tool for quantification measurements.

An ultrasensitive photoelectrochemical biosensor for glucose based on bio-derived nitrogen-doped carbon sheets wrapped titanium dioxide nanoparticles

In this work, an ultra-sensing photoelectrochemical (PEC) glucose biosensor has been constructed from the bio-derived nitrogen-doped carbon sheets (NDC) wrapped titanium dioxide nanoparticles (NDC-TiO2 NPs) followed by the covalent immobilization of glucose oxidase (GODx) on them (designated as a GODx/NDC-TiO2NPs/ITO biosensor). Initially, the TiO2 NPs was synthesized by sol-gel method and then NDC-TiO2 NPs was synthesized utilizing a green source of Prunus persica (peach fruit) through a simple hydrothermal process. The synthesized NDC-TiO2 NPs composite was characterized by FESEM, HRTEM, Raman spectroscopy, XRD, ATR-FTIR spectroscopy and XPS to determine composition and phase purity. These fabricated GODx/NDC-TiO2NPs/ITO biosensor exhibited a good charge separation, highly enhanced and stable photocurrent responses with switching PEC behavior under the light (λ > 400 nm). As a result, GODx/NDC-TiO2NPs/ITO PEC glucose sensor exhibits a good photocurrent response to detection of glucose concentrations (0.05–10 μM) with an ultra-low detection limit of 13 nM under optimized PEC experimental conditions. Also, the PEC glucose sensor revealed a high selectivity, good stability, long time durability, and capability to analyze the glucose levels in real human serum. Also, the further development of this work may provide new insights into preparing other bio-derived carbon nanostructure-based photocatalysts for PEC applications.

Covalent Attachment of Enzymes to Paper Fibers for Paper-Based Analytical Devices.

Due to its unique material properties, paper offers many practical advantages as a viable platform for sensing devices. In view of paper-based microfluidic biosensing applications, the covalent immobilization of enzymes with preserved functional activity is highly desirable and ultimately challenging. In the present manuscript, we report an efficient approach to achieving the covalent attachment of certain enzymes on paper fibers via a surface-bound network of hydrophilic polymers bearing protein-modifiable sites. This tailor-made macromolecular system consisting of polar, highly swellable copolymers is anchored to the paper exterior upon light-induced crosslinking of engineered benzophenone motifs. On the other hand, this framework contains active esters that can be efficiently modified by the nucleophiles of biomolecules. This strategy allowed the covalent immobilization of glucose oxidase and horseradish peroxidase onto cotton linters without sacrificing their bioactivities and performance upon surface binding. As a proof-of-concept application, a microfluidic chromatic paper-based glucose sensor was developed and achieved successful glucose detection in a simple yet efficient cascade reaction.

Covalent Immobilization of Glucose Oxidase onto Electro-synthesized Nanocomposite with PEDOT Derivative for Amperometric Glucose Biosensing

Covalent Immobilization of Glucose Oxidase onto Electro-synthesized Nanocomposite with PEDOT Derivative for Amperometric Glucose Biosensing

Functionalized polypyrrole/sulfonated graphene nanocomposites: Improved biosensing platforms through aryl diazonium electrochemistry

A novel electrochemical approach aimed at developing biosensing platforms based on polypyrrole/sulfonated graphene nanocomposites is reported. Specifically, nanocomposite layers are deposited onto platinum electrodes through the electrochemical polymerization of pyrrole monomer in the presence of reduced graphene oxide bearing phenylsulfonyl groups. Thus, the functionalized graphene nanofiller acts as dopant and balances the positive charges on the polymer chains, leading to an enhancement of the polymer's electrical conductivity and concomitantly increasing the electrode surface area. The polypyrrole/graphene nanocomposite films are further modified with carboxyphenyl groups via electrochemical reduction of 4-carboxyphenyl diazonium tetrafluoroborate. Grafting carboxyphenyl functionalities serves a dual purpose: it permits the covalent immobilization of glucose oxidase via carbodiimide chemistry and also forms an electrode blocking layer which hinders the oxidation of interfering substances. The feasibility of this strategy is demonstrated by the preparation of a glucose biosensor which exhibited an improved performance: wide linear range (0.02–12mM), good sensitivity (0.56μAmM−1cm−2) and adequate selectivity towards common interferents including ascorbic acid, paracetamol, uric acid, and cysteine.

Glucose oxidase immobilization onto Au/poly[anthranilic acid-co-3-carboxy-N-(2-thenylidene)aniline]/PVAc electrospun nanofibers

Au/poly[anthranilic acid-co-3-carboxy-N-(2-thenylidene)aniline/PVAc] [Au/P(ANA-co-CNTA)/PVAc] electrospun nanofibers were fabricated in different electrospinning media including acetone and dimethylformamide (DMF) for covalent immobilization of glucose oxidase (GOx). The surface of copolymer nanofibers was activated by EDC/NHS chemistry, and the presence of Au nanoparticles as tiny conduction centers inside the copolymer matrix enhanced the electrochemical properties. Morphology and composition of enzyme-immobilized nanofibers were characterized by scanning electron microscopy/energy-dispersive X-ray spectroscopy (EM/EDX) and atomic force microscope (AFM). The effective covalent binding of glucose oxidase onto the Au/P(ANA-co-CNTA)/PVAc nanofibers was also confirmed by FTIR-ATR and Raman spectroscopy. EIS measurements revealed that the charge transfer resistances of the enzyme-immobilized nanofibers were decreased with increasing amount of enzyme. The effect of electrospun nanofiber diameter on sensing properties of enzyme-functionalized nanofibers was investigated by EIS. The sensitivities of electrodes calculated from impedance measurement were 7.24 × 106 and 6.67 × 103 Ω mM−1 cm−2 for the Au/P(ANA-co-CNTA)/PVAc-GOX (DMF) and Au/P(ANA-co-CNTA)/PVAc-GOX (acetone), respectively. The impedance measurement results revealed that the linear range of Au/P(ANA-co-CNTA)/PVAc-GOX (DMF) was lower than Au/P(ANA-co-CNTA)/PVAc-GOX (acetone). It could be attributed that smaller fiber diameter resulted in the higher specific surface area. This contributes to increasing the number of available active sites and, thus, increasing the amount of the enzyme loading.

Optimization and characterization of covalent immobilization of glucose oxidase for bioelectronic devices

Enzyme electrodes are widely applied to miniature implantable bioelectronic devices such as biofuel cells and biosensors. The main obstacle associated with miniaturization is the reduced surface area of electrodes for the accommodation of enzymes, leading to poor power output or detection signals. This study aimed to maximize the loading of glucose oxidase (GOx) on the surface of multi-walled carbon nanotubes (MWCNTs), thereby enhancing the generation of electric power or sensing signals. Because the concentrations of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), and glucose oxidase significantly affected the immobilization efficiency, these factors were optimized by the Box–Behnken design. The physically adsorbed enzyme was almost completely removed by washing the GOx-bound MWCNTs with buffer solution containing 5g/L of Tween-20. Enzyme loading was found to be ∼3.3±0.3 mg-GOx/mg-MWCNTs under the optimal conditions (430mM NHS, 52mM EDC and 8.7mg/mL GOx). The formation of carboxyl group on the surface of MWCNTs and the covalent bonding between GOx and MWCNTs, and immobilized GOx were observed by FTIR and AFM, respectively. The biochemical analysis showed that the immobilized GOx possesses high activity for the conversion of glucose into gluconic acid. The cyclic voltammetry data showed that the anodic current density of electrodes loaded with the highest amount of GOx was much higher than those of electrodes loaded with smaller amounts of GOx.

Covalent immobilization of glucose oxidase on mesocellular silica foams: Characterization and stability towards temperature and organic solvents

Glucose oxidase (GOx) immobilization onto mesoporous SBA-15 silica and two mesocellular foams (MCF) characterized by similar surface area and pore volumes but different pore/cell dimensions was examined. The covalent grafting of the enzyme through amide bonds was evidenced by controlling pH conditions, thus preventing GOx leaching. The immobilized protein activity was found to be significantly higher for the mesocellular foam with both cells and windows size larger than the enzyme dimensions. The Michaelis–Menten parameter KM for the immobilized GOx was similar to that of the free enzyme. GOx exhibited higher thermal stability when immobilized on the mesocellular foam compared to the free enzyme. The activity decay of GOx in presence of water soluble organic solvents, i.e., acetonitrile or methanol, was studied. At 50°C, half of the immobilized GOx activity could be retained in 40v/v% MeOH/acetate buffer.

Glucose Chemiluminescence Biosensor Based on Covalent Immobilization of Enzyme in Glutaraldehyde-Functionalized Glass Cell and Direct Coupling of Chitosan-Induced Au/Ag alloy Nanoparticles

We report herein the development of a novel glucose chemiluminescence (CL) biosensor based on covalent immobilization of glucose oxidase (GOD) in glutaraldehyde-functionalized glass cell and direct coupling of chitosan-induced Au/Ag alloy nanoparticles on it, and how it may be useful for determination of glucose due to CL detection of enzymatically generated H2O2. In addition, the nanoalloy offers excellent catalytic activity toward hydrogen peroxide generation in enzymatic reaction between GOD and glucose and increases stability of covalent-linked enzyme. Chitosan molecules act as both the reducing and stabilizing agents for the preparation of nanoparticles (NPs) and also as a coupling agent between GOD and Au/Ag alloy NPs, which made possible the fabrication of a sensitive, accurate and stable biosensor for glucose. Under the optimum conditions, the biosensor can be used for the determination of glucose in the range of 1.2 × 10–6 to 6.25 × 10–3 M with a detection limit of 5.0 × 10–7 M. The CL biosensor exhibited good storage stability, i.e., 90% of its initial response was retained after 2 months storage at pH 7.0. The present CL biosensor has been used to determine glucose in real serum and urine samples and validated against colorimetric spectrophotometry method.

Covalent immobilization of glucose oxidase on amino MOFs via post-synthetic modification

Post Synthetic Modification (PSM) of amino-metal organic frameworks (NH2-MOFs) with the enzyme Glucose Oxidase (GOx) is reported.

A novel glucose sensor based on immobilization of glucose oxidase on the chitosan-coated Fe3O4 nanoparticles and the luminol–H2O2–gold nanoparticle chemiluminescence detection system

A novel and efficient glucose biosensor based on the chemiluminescence (CL) detection of enzymatically generated H2O2 was constructed by the effective immobilization of glucose oxidase (GOX) on the Fe3O4–chitosan (Fe3O4–CS) nanoparticles support. The covalent immobilization of enzyme was performed using glutheraldehyde (GA) as a cross-linking agent. (H2O2 was constructed by one covalent immobilization of glucose oxidase in glutaraldehyde-functionalized glass cell). In addition to catalyzes the luminol CL reaction, gold NPs offer excellent catalytic activity toward hydrogen peroxide generation as a result of enzymatic reaction between glucose oxidase and glucose. Based on response surface methodology (RSM), the optimal immobilization conditions for glucose oxidase were obtained as: enzyme to support ratio, 0.7; Immobilization pH, 6; cross-linking time, 75min; and glutaraldehyde (GA) concentration, 0.2%. The characterization of Fe3O4, Fe3O4–chitosan and Au nanoparticles was analyzed by XRD, FT-IR and SEM. The linear range of this novel method is 1×10−4 to 8.5×10−7molL−1 (R2=0.9956). The detection limit is 4.3×10−7molL−1. The relative standard deviation (RSD) <3.1% was obtained.

Functionalized Polyacrylonitrile Nanofibrous Membranes for Covalent Immobilization of Glucose Oxidase.

Nanofibrous membrane (NFM) with uniform morphology and large surface area was prepared from 10% solution of polyacrylonitrile (PAN) in N,N-dimethylformamide by electrospinning technique. NFM was chemically modified for use as a support for the immobilization of glucose oxidase. Chemical modification of NFM was carried out by two different methods. In the first method, the cyano groups of PAN were modified to amino groups by a two-step process, while in the second method the carboxylic groups were generated first and then further reacted with hexamethylene diamine to create a reactive spacer arm for the immobilization of enzyme. Scanning electron microscopy studies showed that the surface morphology of NFM was not changed by chemical modification and its mechanical strength was improved. The immobilized glucose oxidase (GOx) retained 54 and 60% of its original activity up to 25 cycles with the PAN NFMs modified by the first and the second method, respectively. The GOx-immobilized NFM from the second method showed promising performance with higher enzyme immobilization, activity retention, and favorable kinetic parameters.

Quinone-rich poly(dopamine) magnetic nanoparticles for biosensor applications.

Novel core-shell quinone-rich poly(dopamine)-magnetic nanoparticles (MNPs) were prepared by using an in situ polymerization method. Catechol groups were oxidized to quinone by using a thermal treatment. MNPs were characterized by using X-ray diffraction, X-ray photoelectron spectroscopy, atomic force microscopy, magnetic force microscopy, UV/Vis, Fourier-transform infrared spectroscopy, and electrochemical techniques. The hybrid nanomaterial showed an average core diameter of 17 nm and a polymer-film thickness of 2 nm. The core-shell nanoparticles showed high reactivity and were used as solid supports for the covalent immobilization of glucose oxidase (Gox) through Schiff base formation and Michael addition. The amount of Gox immobilized onto the nanoparticle surface was almost twice that of the nonoxidized film. The resulting biofunctionalized MNPs were used to construct an amperometric biosensor for glucose. The enzyme biosensor has a sensitivity of 8.7 mA M(-1) cm(-2) , a low limit of detection (0.02 mM), and high stability for 45 days. Finally, the biosensor was used to determine glucose in blood samples and was checked against a commercial glucometer.

Co-immobilized glucose oxidase and β-galactosidase on bovine serum albumin coated allyl glycidyl ether (AGE)–ethylene glycol dimethacrylate (EGDM) copolymer as a biosensor for lactose determination in milk

Bovine serum albumin (BSA) was adsorbed on allyl glycidyl ether (AGE)–ethylene glycol dimethacrylate (EGDM) copolymer with 25% crosslink density (AGE-25) at pH 8.0 (16% w/w). The amino, thiol and carboxylic acid functional groups available on protein coated surface were utilized for covalent immobilization of glucose oxidase and β-galactosidase, both independently, and in a step-wise manner on the same matrix, with no more than 10% loss of enzyme activity during immobilization. Glutaraldehyde cross-linking after immobilization provided stable enzyme preparations. The pH-optima of the immobilized enzymes were similar to those for free enzyme but their thermal stability was vastly improved. The co-immobilized enzyme support was used as a biosensor for determination of lactose in milk with excellent reproducibility and reusability.

Chitosan-induced Au/Ag nanoalloy dispersed in IL and application in fabricating an ultrasensitive glucose biosensor based on luminol–H2O2–Cu2+/IL chemiluminescence system

A novel glucose biosensor based on the chemiluminescence (CL) detection of enzymatically generated hydrogen peroxide (H2O2) was constructed by one covalent immobilization of glucose oxidase (GOD) in glutaraldehyde-functionalized glass cell. In following, chitosan-induced Au/Ag nanoparticles dispersed in ion liquid (IL) were synthesised and immobilized on it. Herein, chitosan molecules acted as both the reducing and stabilizing agent for the preparation of NPs and also, as a coupling agent GOD and Au/Ag alloy NPs. In addition to catalyze luminol CL reaction, these NPs offered excellent catalytic activity toward hydrogen peroxide generation in enzymatic reaction between GOD and glucose. The used IL in fabrication of biosensor increased its stability. Also, IL alongside Cu2+ accelerated enzymatic and CL reaction kinetic, and decreased luminol CL reaction optimum pH to 7.5 which would enable sensitive and precision determination of glucose. Under optimum condition, linear response range of glucose was found to be 1.0×10−6–7.5×10−3M, and detection limit was 4.0×10−7M. The CL biosensor exhibited good storage stability, i.e., 90% of its initial response was retained after 2months storage at pH 7.0. The present CL biosensor has been applied satisfactory to analysis of glucose in real serum and urine samples.

Graphene–polyamidoamine dendrimer–Pt nanoparticles hybrid nanomaterial for the preparation of mediatorless enzyme biosensor

A novel inorganic–organic hybrid nanomaterial was prepared by anchoring (3-glycidyloxypropyl)trimethoxysilane at the surface of graphene oxide, further cross-linking with polyamidoamine G-4 dendrimer, and final decoration with platinum nanoparticles. A glassy carbon electrode was coated with this hybrid nanomaterial and then used as support for the covalent immobilization of glucose oxidase. This enzyme electrode was employed to construct a mediatorless glucose amperometric biosensor. The resulting biosensor exhibited good electrocatalytical activity for the oxidation of the H2O2 produced by the enzyme catalyzed reaction, being able to detect glucose when poised at +400mV. The biosensor showed a wide linear response to glucose ranging from 10μM to 8.1mM, high sensitivity of 24.6mA/Mcm2 and low detection limit of 0.8μM. The biosensor was successfully tested for the quantification of glucose in samples of commercial soft drinks.

Covalent immobilization of glucose oxidase on the film prepared by electrochemical polymerization of N-phenylglycine for amperometric glucose sensing

N-Phenylglycine, an aniline-derived amino acid, was polymerized electrochemically. The resulting poly(N-phenylglycine) (PPG) was in the form of a thin film having the conductivity of 2.5×10−3Scm−1. The surface of the PPG film had a fibrous structure, which made the actual area of the surface larger than the apparent one. Glucose oxidase (GOx) was immobilized covalently on the PPG film by the condensation reaction of amino groups of GOx with the carboxyl groups present on the film. The quantity and activity of the immobilized GOx were determined to be 66.4μgcm−2 and 121mUcm−2, respectively. The GOx-immobilized PPG film was applied to amperometric glucose sensing in the presence of p-benzoquinone employed as an electron mediator. It was found that the current response increased with an increase in glucose concentration up to 7.5mM. The sensitivity was 21.0μAmM−1cm−2 with the relative standard deviation of 6.2% (n=5).