Covalent Immobilization Of Horseradish Peroxidase Research Articles

Immobilization of Horseradish Peroxidase on Magnetite-Alginate Beads to Enable Effective Strong Binding and Enzyme Recycling during Anthraquinone Dyes' Degradation.

The aim of this study was to investigate covalent immobilization of horseradish peroxidase (HRP) on magnetic nanoparticles (Mag) encapsulated in calcium alginate beads (MABs) for color degradation, combining easy and fast removal of biocatalyst from the reaction mixture due to its magnetic properties and strong binding due to surface alginate functional groups. MABs obtained by extrusion techniques were analyzed by optical microscopy, FEG-SEM and characterized regarding mechanical properties, magnetization and HRP binding. HRP with initial concentration of 10 mg/gcarrier was successfully covalently bonded on MABs (diameter ~1 mm, magnetite/alginate ratio 1:4), with protein loading of 8.9 mg/gcarrier, immobilization yield 96.9% and activity 32.8 U/g. Immobilized HRP on MABs (HRP-MABs) was then used to catalyze degradation of two anthraquinonic dyes, Acid Blue 225 (AB225) and Acid Violet 109 (AV109), as models for wastewater pollutants. HRP-MABs decolorized 77.3% and 76.1% of AV109 and AB225, respectively after 15 min under optimal conditions (0.097 mM H2O2, 200 mg of HRP-MABs (8.9 mg/gcarrier), 0.08 and 0.1 g/mg beads/dye ratio for AV109 and AB225, respectively). Biocatalyst was used for 7 repeated cycles retaining 75% and 51% of initial activity for AB225 and AV109, respectively, showing potential for use in large scale applications for colored wastewater treatment.

Characterization and application of biochar-immobilized crude horseradish peroxidase for removal of phenol from water

Biochar (BC) has attracted much attention as an environmentally friendly material for application in wastewater treatment. In this study, a suitability of wood-derived BC as a support for covalent immobilization of horseradish peroxidase (HRP) across glutaraldehide as crosslinker, known for the capability to remove phenol from water, was investigated. The efficiency of the immobilized HRP in removal of phenol (2 mM) from water at different reaction conditions (varying dosages of polyethylene glycol (PEG300) 0−750 mg/L; H2O2 1.5–3.5 mM, as well as reaction time 5−120 min) and the general toxicity of bio-treated water (Allium cepa test) were measured. All analyzes were performed for free enzyme as well. The immobilized enzyme showed the highest activity at temperature 30 °C and pH 7.0. The greatest efficiency of immobilized enzyme in phenol removing (90 %) was obtained by applying 2.5 mM H2O2 and 1.5 mg/L of PEG300 at pH 7.0 after 2 h of reaction period. After 4 washings, immobilized HRP retained more than 79 % activity with phenol removal of 64 %. Utilizing immobilized enzyme significantly reduces the toxicity of the tested water (80 %), which further suggested that it might be considered as an environmentally acceptable process for wastewater treatment. Possible degradation products remained in treated water were analyzed in water samples by liquid and gas chromatography with mass spectrometry, including also analysis of volatiles by solid phase microextraction technique; different phenol-base compounds were detected.

Quaternary Ammonium Based Carboxyl Functionalized Ionic Liquid for Covalent Immobilization of Horseradish Peroxidase and Development of Electrochemical Hydrogen Peroxide Biosensor

AbstractA novel electrochemical biosensor was developed using a judiciously designed platform for the rapid and accurate determination of hydrogen peroxide (H2O2). The horseradish peroxidase (HRP) based biosensor was constructed by covalent anchoring of the enzyme to a newly synthesized quaternary ammonium‐based carboxyl functionalized ionic liquid (TBA−COOH−IL) immobilized on a multiwalled carbon nanotube deposited glassy carbon electrode (MWCNT/GCE). A stable amide bond is formed between HRP enzyme and IL by utilizing the terminal −NH2 of HRP and −COOH groups of TBA−COOH−IL, while the pi‐pi stacking holds the TBA−COOH−IL firmly on the MWCNT/GCE and forms HRP/TBA−COOH−IL/MWCNT/GCE. Thus fabricated HRP/TBA−COOH−IL/MWCNT/GCE displayed a well‐resolved redox peak at a formal potential (E°’) of −0.32 V, which corresponds to the concealed FeIII/FeII redox centre of the immobilized HRP enzyme. Further, the developed biosensor was employed for the electrocatalytic determination of H2O2 in static and dynamic conditions, which showed a wide linear range from 0.02 to 4.30 mM with a high sensitivity and low detection limit of 160.6 μA mM−1 cm−2 and 6 μM, respectively. The excellent performance of the fabricated biosensor is attributed to the stable covalent anchoring of freely water‐soluble enzyme on the newly designed, highly conducting and biocompatible platform. Furthermore, the fabricated biosensor exhibited good reproducibility with an extended long‐term stability.

Immobilized Enzyme on Modified Polystyrene Foam Waste: a Biocatalyst for Wastewater Decolorization

In the framework of unremitting efforts to preserve our planet from industrial wastes, this study aims to develop a cheap and efficient biocatalyst for the ecofriendly treatment of dye wastewater. Particularly, packaging waste of expanded polystyrene foam (EPS) was crushed into uniform spheres and easily coated with a layer of the bio-adhesive polydopamine. The successful coating of EPS spheres with a layer of polydopamine was characterized by ATR-FTIR spectroscopy. The polydopamine layer acted as an active platform allowing the covalent immobilization of horseradish peroxidase (HRP) via Schiff base or Michael addition reaction. The immobilization efficiency was about 46%. The immobilized HRP could retain 53% of its activity at high pH (pH 10) however, the reactivity of free HRP was dramatically reduced to 25 %. The immobilized HRP showed remarkable tolerance towards elevated temperature (60 °C) and recorded a higher activity of about 95 % compared to 56 % of free HRP. This in turn makes it a suitable biocatalyst for decolorization of industrial dye wastewater that is typically hot. The immobilized HRP could achieve almost complete decolorization of crystal violet dye within 120 min. Furthermore, the immobilized HRP can be easily separated by skimming and reused for ten cycles with a minimum decolorization efficiency of about 80 % and it can even achieve 60 % of decolorization efficiency after 15 times reuse. Overall, this study constituted the transformation of packaging polystyrene foam waste into an efficient biocatalyst that can be used for the enzymatic treatment of dye wastewater.

Improving stability of biosensor based on covalent immobilization of horseradish peroxidase by γ-aminobutyric acid and application in detection of H2O2

A novel electrochemical biosensor for the determination of hydrogen peroxide (H2O2) has been fabricated through covalently immobilized horseradish peroxidase (HRP) on modified multi walled carbon nanotube (MWCNT) by γ-aminobutyric acid (GABA) on glassy carbon electrode. Using the electrochemical techniques, the electrochemical properties of the biosensor were specified. Cyclic voltammograms of the enzyme film at pH = 7.0 exhibited a pair of quasi-reversible redox peaks according to Fe(III/II) redox process of HRP. The biosensor exhibited good efficiency for finding H2O2 with a broad linear range from 2.0 × 10−7 M to 2.81 × 10−4 M and a detection limit of 0.13 μM. The surface coverage of active HRP, heterogeneous electron transfer rate constant and Michaelis-Menten constant of immobilized HRP were obtained 3.029 × 10−9 mol cm−2, 1.11 s−1, and 0.23 mM respectively. Similarly, the applicability of the suggested biosensor was examined by detecting H2O2 in human plasma samples and the average recovery was obtained as 100.50 ± 0.25. Moreover, the constructed biosensor presented a high stability, reproducibility and repeatability. Finally using docking and the molecular dynamics calculations, it was determined that GABA interacts with lysine residue and it causes HRP to be placed on the electrode with a specific orientation.

Experimental and computational evaluation of area selectively immobilized horseradish peroxidase in a microfluidic device

A microreactor with a square shaped reactor chamber was developed with the aim to correlate enzyme positioning with biocatalytic activity. The enzyme position as an important parameter to improve the contribution of the individual enzymes towards the overall reactor efficacy was therefore evaluated experimentally and by computational fluid dynamics (CFD) simulations. Ultimately, such a correlation would lead to faster development through computational pre-screening and optimized experimental design.In this proof-of-concept study, microreactors were prepared in a 2-step curing process of an off-stoichiometric thiol-ene-epoxy (OSTE+) mixture employing both a thiol-ene (TEC) and a thiol-epoxy curing reaction. Subsequent surface functionalization of the remaining thiol groups on the reactor surface through stenciled photoinitiated TEC enabled the preparation of specific surface patterns in the reactor. Patterns were visualized using an allyl-functional disperse red dye, illustrating the successful preparation of a fully reacted surface, a half covered surface and 2 checkerboard patterns. Similarly, allyl glycidyl ether was exploited to functionalize the microreactor surface with epoxide groups, which were used for covalent immobilization of horseradish peroxidase (HRP) in the same patterns. Biocatalytic activity measurements confirmed a clear dependency of the overall reactor performance depending on the spatial distribution of the immobilized enzymes, where specifically the two checkerboard motifs were identified as being particularly effective compared to enzymes covering homogeneously the entire reactor surface. The performance of the same configurations was additionally determined by 3-dimensional CFD simulations. The computational model predicted the same tendencies for the overall reactor performance as obtained from experimental determination. This good agreement between the obtained experimental and computational results confirmed the high potential of CFD models for predicting and optimizing the biocatalytic performance of such a reactor.

Photoelectrochemical aptasensing of thrombin based on multilayered gold nanoparticle/graphene-TiO2 and enzyme functionalized graphene oxide nanocomposites

A new ternary plasmonic photocatalyst based on multilayered gold nanoparticles decorated graphene (GR)-TiO2 heteronanostructure ((AuNPs)x/GR-TiO2) was fabricated via a simple poly(diallyldimethylammonium chloride) (PDDA) assisted calcination process in combination with the layer-by-layer (LbL) assembly technique. A remarkably enhanced charge separation efficiency of TiO2 was obtained due to the synergistic effect of electron-conducting GR sheets and plasmonic AuNPs. Compared with bare TiO2 and GR-TiO2, the as-prepared (AuNPs)2/GR-TiO2 showed significantly enhanced photocurrent intensity (6-fold of bare TiO2 and 3-fold of GR-TiO2). A sandwich thrombin photoelectrochemical aptasensor was fabricated by using (AuNPs)2/GR-TiO2 immobilized aptamer 1 as the capture probe and graphene oxide, horseradish peroxidase (HRP), and AuNPs triple-labeled aptamer 2 (GO-HRP-AuNPs-apt 2) as the detection probe. On the basis of HRP catalyzed oxidation of 4-chloro-1-naphthol by H2O2 to yield an insulating product on the transducer surface that obstructs the electron transfer of the electron donor (ascorbic acid) to the electrode surface, the detection of thrombin can be carried out by the decrement of photocurrent intensity. The covalent immobilization of HRP on GO can effectively improve the HRP enzyme loading and thus the sensitivity of the sensor. Under the optimum conditions, a linear response was obtained over the range of 0.05–110pM, with a limit of detection (LOD) of 0.02pM (S/N=3). The sensor was successfully used for detection of thrombin in serum samples.

Immobilization of horseradish peroxidase on electrospun poly(vinyl alcohol)–polyacrylamide blend nanofiber membrane and its use in the conversion of phenol

Electrospun nanofibers, with their porous structures, high surface-to-volume ratio, and good mechanical properties, are used as a support material for enzyme immobilization. In this study, the poly(vinyl alcohol) and polyacrylamide bicomponent (PVA–PAAm) nanofibers were fabricated via the electrospinning method. Synthesized PAAm was characterized with size exclusion chromatography (SEC). Nanofibers were characterized by fourier transform infrared (FTIR) spectroscopy, thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and scanning electron microscope (SEM). DSC and TGA analyses showed that the nanofibers were more durable than PVA and PAAm polymers. SEM images demonstrated that all nanofibers possessed uniform and smooth structures (average diameter about 300 nm). FTIR results have shown that PAAm successfully participates in nanofiber structure. The produced nanofibers were used as support material for covalent immobilization of horseradish peroxidase (HRP). The optimum temperature for free HRP was 45 °C, whereas it was 50 °C for the immobilized enzyme. The immobilized HRP showed better storage and thermal stability than free HRP. The kinetic parameters (K m and V max) were found to be 2.42 mM and 0.027 U for the immobilized HRP and 1.86 mM and 0.079 U for the free HRP, respectively. The immobilized enzyme could be used effectively for 25 cycles with 54% retention of the activity. The immobilized HRP was also used for the conversion of phenol. Phenol removal was found to be about 29.68% at 180 min in real wastewater. The novel PVA–PAAm nanofibrous material was successfully used as a support material for covalent immobilization of HRP. Immobilized enzymes such as oxido-reductases onto the PVA–PAAm bicomponent nanofiber could be recommended in the treatment of organic pollutants in industrial effluents.

Amperometric capsaicin biosensor based on covalent immobilization of horseradish peroxidase (HRP) on acrylic microspheres for chilli hotness determination

The evaluation of chilli hotness using amperometric capsaicin biosensor-based enzyme that was immobilized covalently to the surface of modified acrylic microspheres is the first of its kind presented in this work. The immobilization of enzyme covalently to the surface of microspheres via succinimide groups prevented the leaching of the enzyme. The enzymatic reaction between horseradish peroxidase-capsaicin in the presence of hydrogen peroxide, which was mediated by vinyl ferrocene, enabled the current measurement at a low potential (0.22V). Besides, the pungency level of the chilli that was proportional to the capsaicin concentration was measured using this method. This biosensor gave a linear response range towards capsaicin from 0.75–24.94μM (R2=0.992) with a detection limit at 0.39μM. Moreover, the relative standard deviation (RSD) for reproducibility study was 8.2% (n=7). Therefore, this biosensor was successfully applied for evaluation of chilli hotness in chilli sample, as well as in comparison with a standard method that employed the HPLC method.

Preparation of core-shell Fe3O4@poly(dopamine) magnetic nanoparticles for biosensor construction.

Novel core-shell Fe3O4@poly(dopamine) magnetic nanoparticles were prepared through an in situ self-polymerization method. The hybrid nanomaterial showed an average core diameter of 11 ± 3 nm and a polymer thin film thickness of 1.8 ± 0.2 nm. The core-shell nanoparticles were employed as solid supports for the covalent immobilization of horseradish peroxidase (HRP), and the resulting biofunctionalized magnetic nanoparticles were employed to construct an amperometric biosensor for H2O2. The enzyme biosensor showed a high sensitivity of 442.14 mA M-1 cm-2, a low limit of detection of 182 nM, a wide linear range from 6.0 × 10-7 to 8.0 × 10-4 M and high stability for 1 month.

Influence of immobilization protocol on the structure and function of surface bound proteins

A new coupling strategy for biomacromolecules with (3-mercaptopropyl)trimethoxysilane (3MPTMS) and 11-(triethoxysilyl)undecanal (TESU) on gold surfaces is. This immobilization protocol was utilized for the enzyme horseradish peroxidase (HRP). To study the reactions and resulting structures, PM-IRRAS measurements were performed. PM-IRRAS shows there is structure preservation of the HRP when the new coupling strategy is used in contrast to non-specific adsorption on gold. The biological activity of adsorbed and immobilized HRP was measured by the enzyme catalyzed oxidation of 3,5,3′,5′-tetramethylbenzidine. Covalent immobilization of HRP on TESU film compared to physisorption of HRP shows higher enzyme activity on gold surfaces, confirming the structural preservation detected by PM-IRRAS.

Janus Au-mesoporous silica nanoparticles as electrochemical biorecognition-signaling system

Janus Au-mesoporous silica nanoparticles were used as scaffolds to design an integrated electrochemical biorecognition-signaling system. A proof of concept of this strategy, based on the face-selective functionalization of the anisotropic colloid, involves the covalent immobilization of horseradish peroxidase on the mesoporous silica face as enzymatic signaling element, as well as the modification of the Au face with streptavidin and polyethylenglycol chains as biorecognition and solubilizing agents, respectively. The functionalized Janus nanoparticles were successful to recognize biotin on gold surfaces.

Improved Covalent Immobilization of Horseradish Peroxidase on Macroporous Glycidyl Methacrylate-Based Copolymers

A macroporous copolymer of glycidyl methacrylate and ethylene glycol dimethacrylate, poly(GMA-co-EGDMA), with various surface characteristics and mean pore size diameters ranging from 44 to 200nm was synthesized, modified with 1,2-diaminoethane, and tested as a carrier for immobilization of horseradish peroxidase (HRP) by two covalent methods, glutaraldehyde and periodate. The highest specific activity of around 35U g(-1) dry weight of carrier was achieved on poly(GMA-co-EGDMA) copolymers with mean pore diameters of 200 and 120nm by the periodate method. A study of deactivation kinetics at 65°C and in 80% dioxane revealed that periodate immobilization also produced an appreciable stabilization of the biocatalyst, while stabilization factor depended strongly on the surface characteristics of the copolymers. HRP immobilized on copolymer with a mean pore diameter of 120nm by periodate method showing not only the highest specific activity but also good stability was further characterized. It appeared that the immobilization resulted in the stabilization of enzyme over a broader pH range while the Michaelis constant value (K (m)) of the immobilized HRP was 10.8mM, approximately 5.6 times higher than that of the free enzyme. After 6 cycles of repeated use in a batch reactor for pyrogallol oxidation, the immobilized HRP retained 45% of its original activity.

Novel Amperometric Hydrogen Peroxide Biosensor Based on Horseradish Peroxidase Azide Covalently Immobilized on Ethynyl‐Modified Screen‐Printed Carbon Electrode via Click Chemistry

AbstractA novel, simple and versatile protocol for covalent immobilization of horseradish peroxidase (HRP) on screen‐printed carbon electrode (SPCE) based on the combination of diazonium salt electrografting and click chemistry has been successfully developed. The ethynyl‐terminated monolayers are obtained by diazonium salt electrografting, then, in the presence of copper (I) catalyst, the ethynyl modified surfaces reacted efficiently and rapidly with horseradish peroxidase bearing an azide function (azido‐HRP), thus forming a covalent 1,2,3‐triazole linkage by means of click chemistry. All the experimental results suggested that HRP was immobilized onto the electrode surface successfully without denaturation. Furthermore, the immobilized HRP showed a fast electrocatalytic reduction for H2O2. A linear range from 5.0 to 50.0 µM in a phosphate buffer (pH 5.5) with detection limit of 0.50 µM and sensitivity of 0.23 nA/µM were obtained. The heterogeneous electron transfer rate constant Kct was 1.52±0.22 s−1 and the apparent MichaelisMenten constant was calculated to be 0.028 mM. The HRP‐functionalized electrode demonstrated a good reproducibility and long‐term stability.

Mechanisms for Covalent Immobilization of Horseradish Peroxidase on Ion-Beam-Treated Polyethylene

The surface of polyethylene was modified by plasma immersion ion implantation. Structure changes including carbonization and oxidation were observed. High surface energy of the modified polyethylene was attributed to the presence of free radicals on the surface. The surface energy decay with storage time after treatment was explained by a decay of the free radical concentration while the concentration of oxygen-containing groups increased with storage time. Horseradish peroxidase was covalently attached onto the modified surface by the reaction with free radicals. Appropriate blocking agents can block this reaction. All aminoacid residues can take part in the covalent attachment process, providing a universal mechanism of attachment for all proteins. The native conformation of attached protein is retained due to hydrophilic interactions in the interface region. The enzymatic activity of covalently attached protein remained high. The long-term activity of the modified layer to attach protein is explained by stabilisation of unpaired electrons in sp2 carbon structures. A high concentration of free radicals can give multiple covalent bonds to the protein molecule and destroy the native conformation and with it the catalytic activity. The universal mechanism of protein attachment to free radicals could be extended to various methods of radiation damage of polymers.

Bioelectrocatalytic and biosensing properties of horseradish peroxidase covalently immobilized on (3-aminopropyl)trimethoxysilane-modified titanate nanotubes

Titanate nanotubes (TiNT) surface modified with (3-aminopropyl)trimethoxysilane were employed as a support for covalent immobilization of horseradish peroxidase (HRP) by using 1,4-benzoquinone as a coupling agent. Composite film-electrodes consisting of HRP-modified TiNT embedded into the porous carbon powder/Nafion matrix were fabricated and their applicability in direct bioelectrocatalytic reduction of H2O2 and H2O2 biosensing were investigated. An efficient direct electron transfer between the immobilized HRP molecules and the electrode was observed in the presence of H2O2 at potentials lower than 600mV (vs. Hg/Hg2Cl2/3.5M KCl). For the HRP–TiNT-modified electrodes polarized at 0mV, a linear dependence of the bioelectrocatalytic current on the concentration of H2O2 was observed up to the concentration of H2O2 equal to 10μM, with the sensitivity of (1.10±0.01)AM−1cm−2 and the detection limit of 35nM.

Covalent immobilization of horseradish peroxidase via click chemistry and its direct electrochemistry

A simple and versatile approach for covalent immobilization of redox protein on solid surface via self-assembled technique and click chemistry is reported. The alkynyl-terminated monolayers are obtained by self-assembled technique, then, azido-horseradish peroxidase (azido-HRP) was covalent immobilized onto the formed monolayers by click reaction. The modified process is characterized by reflection absorption infrared spectroscopy (RAIR), surface-enhanced Raman scattering spectroscopy (SERS) and electrochemical methods. All the experimental results suggest that HRP is immobilized onto the electrode surface successfully without denaturation. Furthermore, the immobilized HRP shows electrocatalytic reduction for H2O2, and the linear range is from 5.0 to 700μM. The heterogeneous electron transfer rate constant ks is 1.11s−1 and the apparent Michaelis–Menten constant is calculated to be 0.196mM.

Amperometric hydrogen peroxide biosensor based on covalent immobilization of horseradish peroxidase on ferrocene containing polymeric mediator

In this work, a new type of amperometric hydrogen peroxide biosensor was fabricated for the determination of H2O2. Horseradish peroxidase (HRP) was immobilized on a glassy carbon electrode by poly(glycidyl methacrylate-co-vinylferrocene) (poly(GMA-co-VFc)) film. A polymeric electron transfer mediator, containing copolymers of glycidyl methacrylate (GMA) and vinylferrocene (VFc) with different molar ratios, was prepared by free-radical copolymerization. The amperometric response was measured as a function of H2O2 concentration, at a fixed potential of +0.35 V vs. Ag/AgCl in phosphate-buffered saline (pH 7.0). The mediated hydrogen peroxide biosensor showed a fast response of less than 4 s of linear range 2.0–30.0 mM, with a detection of 2.6 μM. The sensitivity of the biosensor for H2O2 was 10.42 nA/mM cm2.

Direct electrochemistry of horseradish peroxidase immobilized on a monolayer modified nanowire array electrode

Vertically aligned nanowire array electrodes (NAEs) were prepared by electrodeposition of gold into an anodic aluminium oxide membrane (AAM), providing an ordered three-dimensional (3D) matrix for immobilization of redox proteins. Third-generation H 2O 2 biosensors were prepared by covalent immobilization of horseradish peroxidase (HRP) on the self-assembled monolayer modified NAEs. Direct electron transfer and electrocatalytic performances of the HRP/NAEs with different nanowire lengths (deposition time of 2, 4 and 5 h) were investigated. Results showed that with longer nanowires, better performances were achieved. The HRP/NAE 5 h (5 h deposition time) exhibited remarkable sensitivity (45.86 μA mM −1 cm −2) towards H 2O 2 with a detection limit of 0.42 μM ( S/ N = 3), linearity up to 15 mM and a response time of 4 s. The ordered 3D gold nanowire array with high conductivity, excellent electron transfer capability and good biocompatibility proved promising for fabricating sensitive, selective, stable and mediator-free enzymatic biosensors.

Development of hydrogen peroxide biosensor based on in situ covalent immobilization of horseradish peroxidase by one-pot polysaccharide-incorporated sol–gel process

A simple and reliable one-pot approach was established for the development of a novel hydrogen peroxide (H 2O 2) biosensor based on in situ covalent immobilization of horseradish peroxidase (HRP) into biocompatible material through polysaccharide-incorporated sol –gel process. Siloxane with epoxide ring and trimethoxy anchor groups was applied as the bifunctional cross-linker and the inorganic resource for organic–inorganic hybridization. The reactivity between amine groups and epoxy groups allowed the covalent incorporation of HRP and the functional biopolymer, chitosan (CS) into the inorganic polysiloxane network. Some experimental variables, such as mass ratio of siloxane to CS, pH of measuring solution and applied potential for detection were optimized. HRP covalently immobilized in the hybrid matrix possessed high electrocatalytic activity to H 2O 2 and provided a fast amperometric response. The linear response of the as-prepared biosensor for the determination of H 2O 2 ranged from 2.0 × 10 −7 to 4.6 × 10 −5 mol l −1 with a detection limit of 8.1 × 10 −8 mol l −1. The apparent Michaelis–Menten constant was determined to be 45.18 μmol l −1. Performance of the biosensor was also evaluated with respect to possible interferences. The fabricated biosensor exhibited high reproducibility and storage stability. The ease of the one-pot covalent immobilization and the biocompatible hybrid matrix serve as a versatile platform for enzyme immobilization and biosensor fabricating.