Immobilization Of Horseradish Peroxidase Research Articles

Enzyme immobilization on polymethacrylate-based monolith fabricated via thermally induced phase separation

Polymer-based monolithic materials with interconnected porous structure have received much attention as high-performance matrices for enzyme immobilization. The present work describes immobilization of horseradish peroxidase (HRP) onto a polymethacrylate-based monolith which was fabricated by thermally induced phase separation (TIPS) method. The poly(glycidyl methacrylate-co-methyl methacrylate) (PGM) monolith was modified with adipic acid dihydrazide (AADH) and ethylenediaminetetraacetic dianhydride (EDTAD) to yield carboxyl group-bearing PGM (PGM-COOH) monolith. This monolith was reacted with N-hydroxysuccinimide (NHS) to activate the carboxyl groups on the monolith and further modified with HRP. The immobilized HRP showed much higher thermal stability than the free one. Furthermore, the immobilized HRP could be reused for at least 6 times with the remaining activity of 52%. The PGM monolith may have a high potential as a solid support for enzyme immobilization.

Immobilized Horseradish Peroxidase (I-HRP) as Biocatalyst for Oxidative Polymerization of 2,6-Dimethylphenol

An enzyme, horseradish peroxidase (HRP), was immobilized on silica nanorods and employed as a catalyst for the oxidative polymerization of 2,6-dimethylphenol. With this catalytic system, the polymer, poly(2,6-dimethyl-1,4-phenylene oxide), was successfully obtained from a water–acetone solvent system. The immobilized HRP exhibited substantially enhanced enzymatic activity toward oxidative polymerization as well as some degree of reusability compared with free HRP.

Two-Dimensional Ordered Silicon Dioxide Cavities Array as Matrix for the Immobilization of Horseradish Peroxidase and Its Direct Electrochemistry

A silicon dioxide (SiO2) cavities array was fabricated on indium-tin oxide (ITO) electrode with polystyrene (PS) particles array as template by using sol-gel technique. The electrochemical properties of SiO2 cavities array indicated that it acted as microelectrode array. Horseradish peroxidase (HRP) was immobilized on the surface of SiO2 cavities by adsorption method. Direct electrochemistry of HRP modified SiO2 cavities array was studied. The results showed that the microenvironment of SiO2 cavities can accelerate electron transfer of HRP efficiently and the electron transfer rate was calculated to be 1.98 s(-1).

An amperometric hydrogen peroxide biosensor based on Co3O4 nanoparticles and multiwalled carbon nanotube modified glassy carbon electrode

Abstract In this work a new type of hydrogen peroxide biosensor was fabricated based on the immobilization of horseradish peroxidase (HRP) by cross-linking on a glassy carbon electrode (GCE) modified with Co 3 O 4 nanoparticles, multiwall carbon nanotubes (MWCNTs) and gelatin. The introduction of MWCNTs and Co 3 O 4 nanoparticles not only enhanced the surface area of the modified electrode for enzyme immobilization but also facilitated the electron transfer rate, resulting in a high sensitivity of the biosensor. The fabrication process of the sensing surface was characterized by scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Amperometric detection of hydrogen peroxide was investigated by holding the modified electrode at −0.30 V (vs. Ag/AgCl). The biosensor showed optimum response within 5 s at pH 7.0. The optimized biosensor showed linear response range of 7.4 × 10 −7 –1.9 × 10 −5 M with a detection limit of 7.4 × 10 −7 . The applicability of the purposed biosensor was tested by detecting hydrogen peroxide in disinfector samples. The average recovery was calculated as 100.78 ± 0.89.

Immobilization of horseradish peroxidase (HRP) on polyimide nanofibers blending with carbon nanotubes

Carbon nanotubes are widely applied in electronics, which offers great potential for enhancing the activity of redox enzyme. In this work, polyimide (PI) was blended with multiwalled carbon nanotubes (MWCNTs) and then electrospun into nanofibers for the immobilization of horseradish peroxidase (HRP), a redox enzyme. SEM and TEM were used to characterize the surface morphology of the PI/MWCNTs nanofibers and to analyze the protruding parts induced by the blending of MWCNT. The results indicate that, compared to the virgin PI nanofibers, the blending of MWCNTs increases the retention activity of immobilized HRP from 2.38% to 12.50%. Circular dichroism and UV–vis molecular absorption spectrometry were used to explore the interactions between HRP and MWCNTs. It seems that MWCNTs increase the enzyme activity by the facilitation of electron transfer among the catalytic intermediates “HRP I”, “HRP II” and MWCNTs.

Simultaneous electropolymerization and electro-click functionalization for highly versatile surface platforms.

Simple preparation methods of chemically versatile and highly functionalizable surfaces remain rare and present a challenging research objective. Here, we demonstrate a simultaneous electropolymerization and electro-click functionalization process (SEEC) for one-pot self-construction of aniline- and naphthalene-based functional polymer films where both polymerization and click functionalization are triggered by applying electrochemical stimuli. Cyclic voltammetry (CV) can be applied for the simultaneous oxidation of 4-azidoaniline and the reduction of Cu(II) ions, resulting in polymerization of the former, and the Cu(I)-catalyzed alkyne/azide cycloaddition ("click" chemistry). Properties of the films obtained can be tuned by varying their morphology, their chemically "clicked" content, or by postconstruction functionalization. To demonstrate this, the CV scan rates, component monomers, and "clicked" molecules were varied. Covalent postconstruction immobilization of horseradish peroxidase was also performed. Consequently, pseudocapacitance and enzyme activity were affected. SEEC provides surface scientists an easy access to a wide range of functionalization possibilities in several fields including sensors, fuel cells, photovoltaics, and biomaterials.

Multi-walled carbon nanotubes in aqueous phytic acid for enhancing biosensor

The poor dispersion of carbon based nanomaterials without strong acid pretreatment in aqueous solution is a fundamental problem, limiting its applications in biology-related fields. A good dispersion of multi-walled carbon nanotubes (MWCNTs) in water was realized by 50 wt.% phytic acid (PA) solution. As an application case, the PA–MWCNTs dispersion in aqueous solution was used for the immobilization of horseradish peroxidase (HRP) and its direct electrochemistry was realized. The constructed biosensor has a sound limit of detection, wide linear range, and high affinity for hydrogen peroxide (H2O2) as well as being free from interference of co-existing electro-active species.

Co-Doped ZnO Nanomaterials As a Novel Platform for Hydrogen Peroxide Biosensing

Co-doped ZnO nanomaterials have been synthesized successfully on indium tin oxide glass by direct electrochemical deposition using as a platform to immobilize horseradish peroxidase enzyme and to enhance the response of hydrogen peroxide. The crystal structures and morphology of Co-doped ZnO were characterized by X-ray diffraction and scanning electron microscopy, whereas surface electronic state and the element ratio were revealed by X-ray photoelectron spectroscopy measurements. Cyclic voltammetric and electrochemical impedance spectroscopy were carried out with the electrodes, and it was found that the Co-doped ZnO nanomaterials had good conductivity and fast electron transfer in electrocatalytic reduction. According to the amperometry response, a broad linear response was in the range of 0.05 to 1.15 mM and the detection limit was 13 mM at a signal-to-noise ratio of 3 (S/N=3) within 10 s at an applied working potential of -0.3V. The sensitivity and value for the electrode were 22.7 mA mM-1 and 2.93 mM, respectively. The proposed biosensor was not affected by other pollutants and maintained nearly 90% of its original activity after a monthly storage. These results indicate that proper doping can improve electron transfer abilities of ZnO and enable this traditional semiconductor material to have better performance in sensing applications.

Direct Electrochemistry of Horseradish Peroxidase Immobilized in a Low Molecular Weight Gel

AbstractThe ternary system of dodecylpyridinium bromide (DDPB)/acetone/H2O with appropriate composition can form a gel spontaneously and the gel is stable in hydrophobic ionic liquid 1‐butyl‐3‐methylimidazolium hexafluorophosphate ([Bmim]PF6). Based on the gelation phenomenon we observed, the low molecular weight gelator (LMWG) was first tried to immobilize horseradish peroxidase (HRP) on glassy carbon electrode (GCE). The scanning electron microscope (SEM) images, the UV‐Vis spectra and the bioactivity measurement indicate that the gel is suitable for the immobilization of HRP. The direct electrochemistry of the HRP‐gel modified GCE (HRP‐gel/GCE) in [Bmim]PF6 shows a pair of well‐defined and quasi‐reversible redox peaks with the heterogeneous electron transfer rate constant (ks) being 14.4 s−1, indicating that the direct electron transfer between HRP and GCE is fast. The HRP‐gel/GCE is stable and reproducible. Also the electrode exhibits good electrocatalytic effect on the reduction of trichloroacetic acid (TCA), showing good promise in bioelectrocatalysis.

Direct Electrochemistry and Electrocatalysis of Horseradish Peroxidase Immobilized in a DNA/Chitosan-Fe3O4 Magnetic Nanoparticle Bio-Complex Film

A DNA/chitosan-Fe3O4 magnetic nanoparticle bio-complex film was constructed for the immobilization of horseradish peroxidase (HRP) on a glassy carbon electrode. HRP was simply mixed with DNA, chitosan and Fe3O4 nanoparticles, and then applied to the electrode surface to form an enzyme-incorporated polyion complex film. Scanning electron microscopy (SEM) was used to study the surface features of DNA/chitosan/Fe3O4/HRP layer. The results of electrochemical impedance spectroscopy (EIS) show that Fe3O4 and enzyme were successfully immobilized on the electrode surface by the DNA/chitosan bio-polyion complex membrane. Direct electron transfer (DET) and bioelectrocatalysis of HRP in the DNA/chitosan/Fe3O4 film were investigated by cyclic voltammetry (CV) and constant potential amperometry. The HRP-immobilized electrode was found to undergo DET and exhibited a fast electron transfer rate constant of 3.7 s−1. The CV results showed that the modified electrode gave rise to well-defined peaks in phosphate buffer, corresponding to the electrochemical redox reaction between HRP(Fe(III)) and HRP(Fe(II)). The obtained electrode also displayed an electrocatalytic reduction behavior towards H2O2. The resulting DNA/chitosan/Fe3O4/HRP/glassy carbon electrode (GCE) shows a high sensitivity (20.8 A·cm−2·M−1) toward H2O2. A linear response to H2O2 measurement was obtained over the range from 2 μM to 100 μM (R2 = 0.99) and an amperometric detection limit of 1 μM (S/N = 3). The apparent Michaelis-Menten constant of HRP immobilized on the electrode was 0.28 mM. Furthermore, the electrode exhibits both good operational stability and storage stability.

Immobilization of chemically modified horse radish peroxidase within activated alginate beads

Immobilization of horse radish peroxidase (HRP) within alginate beads was improved by chemical modification of the enzyme and polysaccharide chains. HRP and alginate were oxidized by periodate and subsequently modified with ethylenediamine. Highest specific activity of 0.43 U/ml of gel and 81 % of bound enzyme activity was obtained using aminated HRP and alginate oxidized by periodate. Immobilized enzyme retained 75 % of original activity after 2 days of incubation in 80 % (v/v) dioxane and had increased activity at basic pH values compared to native enzyme. During repeated use in batch reactor for pyrogallol oxidation immobilized peroxidase retained 75 % of original activity.

Simple approach for the immobilization of horseradish peroxidase on poly-l-histidine modified reduced graphene oxide for amperometric determination of dopamine and H2O2

In this work, immobilization of horseradish peroxidase (HRP) on poly-l-histidine (P-l-His) modified reduced graphene oxide (RGO) was demonstrated.

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.

A third-generation hydrogen peroxide biosensor based on horseradish peroxidase immobilized by sol–gel thin film on a multi-wall carbon nanotube modified electrode

A third-generation H2O2biosensor was developed by using tetraethoxy-silicone sol–gel film to immobilize horseradish peroxidase (HRP) on a multi-walled carbon nanotubes (MWNTs) modified glass carbon electrode.

Direct electrochemistry of horseradish peroxidase based on hierarchical porous calcium phosphate microspheres

Biomorphic calcium phosphate (CaP) microspheres with hierarchical porous structure were synthesized using natural cole pollen grains as templates and were further employed for the immobilization of horseradish peroxidase (HRP). Scanning electron microscopy and Fourier transform infrared spectroscopy revealed (a) the porous structure of the CaP microspheres, (b) the effective immobilization, and (c) the retention of the conformation of HRP on CaP. The immobilized HRP was placed on a glassy carbon electrode where it underwent a direct, fully reversible, and surface-controlled redox reaction with an electron transfer rate constant of 1.96 s−1. It also exhibits high sensitivity to the reduction of H2O2. The response to H2O2 is linear in the 5.00 nM to 1.27 μM concentration range, and the sensitivity is 30357 μA⋅mM−1⋅cm−2. The detection limit (at an SNR of 3) is as low as 1.30 nM. The apparent Michaelis–Menten constant (KMapp) of the immobilized enzyme is 0.92 μM. This new CaP with hierarchical porous structure therefore represents a material that can significantly promote the direct electron transfer between HRP and an electrode, and is quite attractive with respect to the construction of biosensors.

Immobilization of horseradish peroxidase in phospholipid-templated titania and its applications in phenolic compounds and dye removal

In this study, horseradish peroxidase (HRP) was encapsulated in phospholipid-templated titania particles through the biomimetic titanification process and used for the treatment of wastewater polluted with phenolic compounds and dye. The encapsulated HRP exhibited improved thermal stability, a wide range of pH stability and high tolerance against inactivating agents. It was observed an increase in Km value for the encapsulated HRP (8.21mM) when compared with its free counterpart. For practical applications in the removal of phenolic compounds and dye by the encapsulated HRP, the removal efficiency for phenol, 2-chlorophenol, Direct Black-38 were 92.99%, 87.97%, and 79.72%, respectively, in the first treatment cycle. Additionally, the encapsulated HRP showed better removal efficiency than free HRP and a moderately good capability of reutilization.

A simple and sensitive immunoassay for the determination of human chorionic gonadotropin by graphene-based chemiluminescence resonance energy transfer

In this study, we report a strategy of chemiluminescence resonance energy transfer (CRET) using graphene as an efficient long-range energy acceptor. Magnetic nanoparticles were also used in CRET for simple magnetic separation and immobilization of horseradish peroxidase (HRP)-labeled anti-HCG antibody. In the design of CRET system, the sandwich-type immunocomplex was formed between human chorionic gonadotropin (HCG, antigen) and two different antibodies bridged the magnetic nanoparticles and graphene (acceptors), which led to the occurrence of CRET from chemiluminescence light source to graphene. After optimizing the experimental conditions, the quenching of chemiluminescence signal depended linearly on the concentration of HCG in the range of 0.1mIUmL−1–10mIUmL−1 and the detection limit was 0.06mIUmL−1. The proposed method was successfully applied for the determination of HCG levels in saliva and serum samples, and the results were in good agreement with the plate ELISA with colorimetric detection. It could also be developed for detection of other antigen–antibody immune complexes by using the corresponding antigens and respective antibodies.

Preparation of boronate-functionalized molecularly imprinted monolithic column with polydopamine coating for glycoprotein recognition and enrichment

A novel imprinting strategy using reversible covalent complexation of glycoprotein was described for creating glycoprotein-specific recognition cavities on boronate-functionalized monolithic column. Based on it, a molecularly imprinted monolithic column was prepared by self-polymerization of dopamine (DA) on the surface of 4-vinylphenylboronic acid (VPBA)-based polymeric skeletons after reversible immobilization of horseradish peroxidase (HRP). Due to the combination of boronate affinity and surface imprinting of DA, the stable and accessible recognition sites in the as-prepared imprinted monolith could be obtained after the removal of the template, which facilitated the rebinding of the template and provided good reproducibility and lifetime of use. The recognition behaviors of proteins on the bare VPBA-based, HRP-imprinted and nonimprinted monolithic columns were evaluated in detail and the results showed that the HRP-imprinted monolith exhibited higher recognition ability toward the template than another two monolithic columns. Not only nonglycoproteins but also glycoproteins can be well separated with the HRP-imprinted monolith. In addition, the feasibility of the HRP-imprinted monolith, adopted as an in-tube solid phase microextraction (in-tube SPME), was further assessed by selective extraction and enrichment of HRP from human serum. The good results demonstrated its potential in glycoproteome analysis.

Improved activity of immobilized horseradish peroxidase on gold nanoparticles in the presence of bovine serum albumin

The using of macromolecular additives is known to be a simple and effective way to improve the activity of immobilized enzymes on solid support, yet the mechanism has not been well understood. Taking horseradish peroxidase (HRP) as an example, only 30 % of its catalytic activity was kept after being immobilized on the surface of 25-nm Au nanoparticles, mainly attributed to the conformational change of the heme-containing active site. The catalytic activity of HRP was significantly improved to 80 % when a certain amount of bovine serum albumin (BSA) was added at the initial stage of the immobilization. Systematic spectral investigation indicated that the addition of BSA inhibited the tertiary structure change around the active site, which was a prerequisite for improved activity of the immobilized HRP. Steady-state kinetic analyses revealed that the introduction of BSA could effectively improve the turnover rate of substrate to product in spite of slight reduced affinity to substrates, which also contributed to the improved catalytic activity.

Titanium dioxide nanorod-based amperometric sensor for highly sensitive enzymatic detection of hydrogen peroxide

Titanium dioxide nanorods (TNR) were grown on a titanium electrode by a hydrothermal route and further employed as a supporting matrix for the immobilization of nafion-coated horseradish peroxidase (HRP). The strong electrostatic interaction between HRP and TNR favors the adsorption of HRP and facilitates direct electron transfer on the electrode. The electrocatalytic activity towards hydrogen peroxide (H2O2) was investigated via cyclic voltammetry and amperometry. The biosensor exhibits fast response, a high sensitivity (416.9 μA·mM−1), a wide linear response range (2.5 nM to 0.46 mM), a detection limit as low as 12 nM, and a small apparent Michaelis-Menten constant (33.6 μM). The results indicate that this method is a promising technique for enzyme immobilization and for the fabrication of electrochemical biosensors.