Direct Electrochemistry Of Horseradish Peroxidase Research Articles

Preparation and Application of Electrochemical Horseradish Peroxidase Sensor Based on a Black Phosphorene and Single-Walled Carbon Nanotubes Nanocomposite.

To design a new electrochemical horseradish peroxidase (HRP) biosensor with excellent analytical performance, black phosphorene (BP) nanosheets and single-walled carbon nanotubes (SWCNTs) nanocomposites were used as the modifier, with a carbon ionic liquid electrode (CILE) as the substrate electrode. The SWCNTs-BP nanocomposite was synthesized by a simple in situ mixing procedure and modified on the CILE surface by the direct casting method. Then HRP was immobilized on the modified electrode with Nafion film. The electrocatalysis of this electrochemical HRP biosensor to various targets was further explored. Experimental results indicated that the direct electrochemistry of HRP was realized with a pair of symmetric and quasi-reversible redox peaks appeared, which was due to the presence of SWCNTs-BP on the surface of CILE, exhibiting synergistic effects with high electrical conductivity and good biocompatibility. Excellent electrocatalytic activity to trichloroacetic acid (TCA), sodium nitrite (NaNO2), and hydrogen peroxide (H2O2) were realized, with a wide linear range and a low detection limit. Different real samples, such as a medical facial peel solution, the soak water of pickled vegetables, and a 3% H2O2 disinfectant, were further analyzed, with satisfactory results, further proving the potential practical applications for the electrochemical biosensor.

Facile strategy for immobilizing horseradish peroxidase on a novel acetate functionalized ionic liquid/MWCNT matrix for electrochemical biosensing

Facile yet simple platforms for the immobilization of biomolecules have always been a substantial requirement for the fabrication of proficient biosensors. In this study, we report a naphthyl substituted acetate functionalized ionic liquid (NpAc-IL) for the covalent anchoring of horseradish peroxidase (HRP), using which the direct electrochemistry of HRP was successfully accomplished and a H2O2 biosensor was developed. The naphthyl substitution on the NpAc-IL was utilized for the π-π stacking with the MWCNT modified GCE and the terminal -OCH3 group of NpAc-IL was used for the covalent attachment with the free -NH2 group of HRP via amide bond formation. High conducting nature of the newly designed ionic liquid (NpAc-IL), facilitated an improved communication with the deeply buried redox centre of the HRP, while the covalent bonding provided enhanced stability to the fabricated biosensor by stably holding the water soluble HRP enzyme on the electrode surface. Furthermore, incorporation of MWCNT on the sensor setup synergistically enhanced the sensitivity of the developed biosensor. Under optimized conditions, the fabricated biosensor showed an enhanced electrocatalytic reduction of H2O2 in the range of 0.01 to 2.07 mM with a limit of detection and sensitivity of 2.7 μM and 55.98 μA mM−1 cm−2 respectively. Further, the proposed biosensor was utilized for the sensing of H2O2 spiked in real samples. Moreover, the newly fabricated biosensor demonstrated excellent stability with improved sensitivity and selectivity towards H2O2 reduction. The superior analytical characteristics are attributed to the facile fabrication strategy using this newly developed acetate functionalized ionic liquid platform.

Thiolated DAB Dendrimer-Gold Nanoparticles Self-Assembled Monolayer as Covalent Support for Direct Electrochemistry of HRP and Sensing Applications

A thiolated DAB dendrimer has been employed to bond gold nanoparticles of several sizes in order to obtain an electrocatalytic framework for the covalent immobilization and direct electrochemistry of horseradish peroxidase (HRP). This biosensor must represent the basis for developing a lot of oxidase-peroxidase bienzymatic biosensors and heavy metals biosensors based on the HRP inhibition. The kinetic study of the modified electrodes showed that the 5 nm and 16 nm gold nanoparticles are the most efficient to contact with the HRP active centre and the optimized biosensor allow to measure hydrogen peroxide at -0.3 V applied potential in linear ranges of 1-5000 and 1-140 or 140-5000 respectively with high sensitivities of 418.6 and 266.3 u A mM-1 cm-2 respectively too with low detection limits of 5 and 9 nM and fast response. The obtained apparent Michaelis-Menten constants, were 0.16 and 0.84 mM respectively. Both are significantly lower than the intrinsic K´M revealing the very high enzymatic efficiency of the developed devices.

Thiolated DAB Dendrimers-Gold Nanoparticles as Self-Assembled Layers for the Direct Electrochemistry of HRP

In this work, three generations of a thiolated DAB dendrimer have been used as bonding-layer between electrodeposited and colloidal gold nanoparticles of several sizes to develop electrocatalytic self-assembled layers for the covalent immobilization and direct electrochemistry of horseradish peroxidase (HRP). This study covers the kinetic, electrochemical and analytical characterization of all the prepared biosensors with the aim of developing the best electrocatalytic basis for future oxidase-peroxidase bienzymatic biosensors or biosensors based on the HRP inhibition. The best results were obtained with the dendrimer of third generation and 16 nm colloidal gold nanoparticles, which showed a homogeneous electron transfer rate constant of 422 s−1. This self-assembled monolayer was the most efficient to contact with the HRP active centre and allowed to determine hydrogen peroxide at an applied potential −0.3 V in three linear ranges of 0.4-200; 200–700 and 700–4000 μM with high sensitivities of 590; 441 and 303 μA mM−1 cm−2 respectively, low detection limit of 20 nM and fast response. The obtained apparent overall rate and Michaelis-Menten constants for this optimal biosensor were 0.047 A M−1 and 1.16 mM respectively. This last one, is significantly lower than the intrinsic K'M revealing the very high enzymatic efficiency of the developed devices.

One-Step Preparation of Direct Electrochemistry HRP Biosensor via Electrodeposition

A horseradish peroxidase (HRP) biosensor was constructed by one step electrodeposition method and the direct electrochemistry of HRP in the biosensor was studied. In the biosensor HRP was immobilized on surface of the glassy carbon electrode (GCE) during the process of the gold nanoparticles (AuNPs) deposition with the help of surfactant cetyltrimethylammonium bromide (CTAB), through which HRP-CTAB-AuNPs/GCE was successfully constructed. HRP-CTAB-AuNPs/GCE can directly catalyze the reduction of peroxides without the electron mediator. SEM and electron dispersive spectroscopy (EDS) were used to characterize the modified electrode. The preparation process of the modified electrode was studied by electrochemical impedance spectroscopy. The electrochemical properties of the modified electrode to hydrogen peroxide, tert-butyl hydroperoxide and cumyl hydroperoxide were studied by electrochemical cyclic voltammetry and current-time curve. Results showed that the modified electrode exhibited good catalytic performances for the detection of hydrogen peroxide, tert-butyl hydroperoxide and cumyl hydroperoxide. The sensitivities for H2O2, tert-butyl hydroperoxide and cumyl hydroperoxide are 0.0187 A·L/mol, 0.000340A·L/mol and 0.0052 A·L/mol, respectively. The detection limits are 2.3 × 10−7 mol/L, 8.6 × 10−6 mol/L and 3.6 × 10−6 mol/L, respectively.

A novel nitrite biosensor based on the direct electrochemistry of horseradish peroxidase immobilized on porous Co3O4 nanosheets and reduced graphene oxide composite modified electrode

Porous Co3O4 hexagonal nanosheets (PCHNSs) with a well-defined morphology have been synthesized successfully via a hydrothermal process and followed by calcinations. Co3O4 nanosheets immobilizing horseradish peroxidase (HRP), together with reduced graphene oxide, have been used to modify glassy carbon electrode to fabricate a mediator-free biosensor. The morphology and structure of PCHNSs are studied by XRD, FESEM, and HRTEM techniques. Spectroscopic and electrochemical results reveal that Co3O4/rGO composite film is an excellent immobilization matrix with biocompatibility for enzymes, affording good enzymes’ bioactivity and stability. Meanwhile, due to the unique structure of the modified electrode, the direct electron transfer of HRP is facilitated and the prepared nitrite biosensors display good performance—a wide linear range of 1–5400μM, an extremely low limit of detection (LOD) as 0.21μM and a limit of quantification as (LOQ) as 0.58μM. This work suggests that Co3O4/rGO composite film is a potential matrix for enzymes’ immobilization and biosensors’ preparation.

A novel biosensor based on the direct electrochemistry of horseradish peroxidase immobilized in the three-dimensional flower-like Bi2WO6 microspheres

Three-dimensional flower-like Bi2WO6 microspheres (3D-Bi2WO6 MSs) have been synthesized through a simple hydrothermal method. The morphology and structure of 3D-Bi2WO6 MSs were characterized by scanning electron microscopy (SEM) and X-ray diffraction (XRD). The 3D-Bi2WO6 MSs subsequently were used to immobilize horseradish peroxidase (HRP) and fabricate a mediator-free biosensor for the detection of H2O2. Spectroscopic and electrochemical results reveal that 3D-Bi2WO6 MSs constitute an excellent immobilization matrix with biocompatibility for enzymes. Meanwhile, due to unique morphology of the flower-like microspheres, the direct electron transfer of HRP is facilitated and the prepared biosensors display good performances for the detection of H2O2 with a wide linear range, including two linear sections: 0.5–100μM (R2=0.9983) and 100–250μM (R2=0.9981), as well as an extremely low method detection limit of 0.18μM.

Direct Electrochemistry of Horseradish Peroxidase on NiO Nanoflower Modified Electrode and Its Electrocatalytic Activity

Direct Electrochemistry of Horseradish Peroxidase on NiO Nanoflower Modified Electrode and Its Electrocatalytic Activity

Polyferrocenyl Polycyclosiloxane/Gold Nanoparticles: An Efficient Electrocatalytic Platform for Immobilization and Direct Electrochemistry of HRP

Sensitivity and enzyme stability are the main targets in the biosensors improvement. This work achieves both by means of the combination of two efficient electrocatalysts: ferrocene and gold nanoparticles (AuNPs). The AuNPs were electrodeposited onto a polyferrocenyl polycyclosiloxane polymer (FPP) in order to obtain an electrocatalytic framework for the covalent immobilization and direct electrochemistry of horseradish peroxidase (HRP). The developed surface is like that obtained with graphene modified electrodes, with very high specific area. The kinetic study of electrodes modified with and without HRP showed that the FPP/AuNPs system greatly improves other more complex systems based on the direct HRP electro-reduction by AuNPs, allowing the use of lower measuring potentials, with improved sensitivity, wider linear ranges and lower detection limits. The obtained apparent Michaelis-Menten constant, KM,app, was 0.32 mM, significantly lower than the intrinsic KM, revealing the very high enzymatic efficiency of the developed device. The HRP/AuNPs/FPP/Pt electrode was used as biosensor for hydrogen peroxide with two wide linear ranges of 0–200 μM and 200–6800 μM, and a high sensitivity of 685.71 μA mM−1 cm−2, a low detection limit of 50 nM and long-term stability. The new device has been successfully used too as for the determination of organic hydroperoxides.

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).

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.

Amperometric carbohydrate antigen 19-9 immunosensor based on three dimensional ordered macroporous magnetic Au film coupling direct electrochemistry of horseradish peroxidase

A sandwich-type electrochemical immunosensor for the detection of carbohydrate antigen 19-9 (CA 19-9) antigen based on the immobilization of primary antibody (Ab1) on three dimensional ordered macroporous magnetic (3DOMM) electrode, and the direct electrochemistry of horseradish peroxidase (HRP) that was used as both the label of secondary antibody (Ab2) and the blocking reagent. The 3DOMM electrode was fabricated by introducing core–shell Au–SiO2@Fe3O4 nanospheres onto the surface of three dimensional ordered macroporous (3DOM) Au electrode via the application of an external magnet. Au nanoparticles functionalized SBA-15 (Au@SBA-15) was conjugated to the HRP labeled secondary antibody (HRP-Ab2) through the Au–SH or Au–NH3+ interaction, and HRP was also used as the block reagent. The formation of antigen–antibody complex made the combination of Au@SBA-15 and 3DOMM exhibit remarkable synergistic effects for accelerating direct electron transfer (DET) between HRP and the electrode. Under the optimal conditions, the DET current signal increased proportionally to CA 19-9 concentration in the range of 0.05 to 15.65UmL−1 with a detection limit of 0.01UmL−1. Moreover, the immunosensor showed high selectivity, good stability, satisfactory reproducibility and regeneration. Importantly, the developed method was used to assay clinical serum specimens, achieving a good relation with those obtained from the commercialized electrochemiluminescent method.

Comparison investigation of several hydrogen peroxide biosensor based on the direct electrochemistry of horseradish peroxidase immobilised on self-assembled monolayers

Horseradish peroxidase was immobilised onto self-assembled monolayers (SAMs) by electrostatic adsorption, acylimide and Sciff base covalent-linkage, respectively. The effects of different immobilised methods on the direct electrochemistry of HRP and electrocatalysis towards H2O2 have been evaluated. The experimental results reveal that the direct electrochemistry of HRP can be achieved by the method of acylimide linkage. The electron transfer coefficient a and rate constant ks have been calculated to be 0.70 and 33.88 s-1 for HRP-GSH/Au, 0.52 and 11.35 s-1 for HRP-Cys/Au, respectively. The response is linear to H2O2 concentration from 1 µM to 1.4 mM and lower detection limit of 0.46 µM for HRP-GSH/Au, 1.5µM to 1.2 mM and lower detection limit of 0.76 µM for HRP-Cys/Au. The biosensor showed longer stability and favourable reproducibility and anti-interfering ability.

Electrosynthesis of ERGO-NP Nanocomposite Films for Bioelectrocatalysis of Horseradish Peroxidase towards H2O2

A simple and sensitive amperometric sensor was developed based on electrochemical co-reduction of graphene oxide/nanoparticle (ERGO-NP) composite films by chronoamperometry method on indium tin oxide (ITO) electrodes. By analyzing the properties of the composite films with experimental results and theoretical investigations, a comprehensive report on the physical and electrochemical interfacial properties of graphene mixed gold nanoparticles composite electrode is presented. The developed electrodes were applied for the detection of hydrogen peroxide (H2O2) based on the direct electrochemistry of horseradish peroxidase (HRP). Standard carbodiimide chemistry enabled the covalent tethering of HRP on to the modified film thus maintaining its native structures, consequently facilitating the direct electron transfer between the protein and the underlying surface. The electrochemical result revealed that the ERGO-NP/ITO electrodes exhibited much higher conductivity (ca. 5 times) when compared with unmodified electrode. The developed sensor using amperometric measurements revealed high sensitivity (1808.9 μA.mM−1 cm−2), with detection limit (0.6 μM) achieved under optimized conditions toward H2O2. Further, the sensor showed good specificity, stability and reproducibility toward H2O2 detection, therefore results proved that well-dispersed, high surface area and highly conductive modified surface could be a promising material for protein adsorption and fabrication of electrochemical biosensors.

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.

Preparation of Magnetic Ordered Mesoporous Carbon Composite and Its Application in Direct Electrochemistry of Horseradish Peroxidase

AbstractA novel magnetic ordered mesoporous carbon composite was prepared. Electrochemical measurements showed that the ordered mesoporous carbon composite provided an excellent matrix for the co‐adsorption of horseradish peroxidase (HRP). HRP could be separated and collected by the application of a magnetic field and its direct electron‐transfer could be achieved in the solution, not on the electrode thereby preventing the degradation of the enzyme. The cyclic voltammetric experimental results of HRP indicated that HRP displayed a pair of stable peaks with a formal potential of −0.306 V in PBS. The resulting biosensor exhibited fast amperometric response to hydrogen peroxide.

Self-Assembled Au Electrode for Direct Electrochemistry of Horseradish Peroxidase and Detection of Hydrogen Peroxide

The H2O2 biosensor was prepared by continuous processes: 2,3-dimercaptosuccinic acid (DMSA) self-assembly monolayers gold nanoparticles (AuNPs) film by electrodepositioncovalent immobilization of horseradish peroxidase (HRP). In pH 7.2 phosphoric buffer solution (PBS), the HRP-AuNPs-DMSA-Au electrode exhibited a pair of well-defined cyclic voltammetric peaks in H2O2 solution, and the oxidation peak is about +0.4V versus saturated calomel electrode (SCE). The resulting substrates were characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The biosensor exhibited remarkable sensitivity towards H2O2 and a wide dynamic range of 2-800 μM. The electrode retained 90% of its initial activity after 30 days of storage at 4°C.

Fabrication of hydrogen peroxide biosensor based on Ni doped SnO2 nanoparticles

Ni doped SnO2 nanoparticles (0–5wt%) have been prepared by a simple microwave irradiation (2.45GHz) method. Powder X-ray diffraction (XRD) and transmission electron microscopy (TEM) studies confirmed the formation of rutile structure with space group (P42/mnm) and nanocrystalline nature of the products with spherical morphology. Direct electrochemistry of horseradish peroxidase (HRP)/nano-SnO2 composite has been studied. The immobilized enzyme retained its bioactivity, exhibited a surface confined, reversible one-proton and one-electron transfer reaction, and had good stability, activity and a fast heterogeneous electron transfer rate. A significant enzyme loading (3.374×10−10molcm−2) has been obtained on nano-Ni doped SnO2 as compared to the bare glassy carbon (GC) and nano-SnO2 modified surfaces. This HRP/nano-Ni–SnO2 film has been used for sensitive detection of H2O2 by differential pulse voltammetry (DPV), which exhibited a wider linearity range from 1.0×10−7 to 3.0×10−4M (R=0.9897) with a detection limit of 43nM. The apparent Michaelis–Menten constant (KMapp) of HRP on the nano-Ni–SnO2 was estimated as 0.221mM. This excellent performance of the fabricated biosensor is attributed to large surface-to-volume ratio and Ni doping into SnO2 which facilitate the direct electron transfer between the redox enzyme and the surface of electrode.

A novel approach to construct a horseradish peroxidase|hydrophilic ionic liquids|Au nanoparticles dotted titanate nanotubes biosensor for amperometric sensing of hydrogen peroxide

The direct electrochemistry of horseradish peroxidase (HRP) on a novel sensing platform modified glassy carbon electrode (GCE) has been achieved. This sensing platform consists of Nafion, hydrophilic room-temperature ionic liquid (RTIL) and Au nanoparticles dotted titanate nanotubes (GNPs-TNTs). The composite of RTIL and GNPs-TNTs was immobilized on the electrode surface through the gelation of a small amount of HRP aqueous solution. The composite was characterized by transmission electron microscopy (TEM), powder X-ray diffraction (XRD) and infrared spectroscopy (IR). UV–Vis and IR spectroscopy demonstrated that HRP in the composite could retain its native secondary structure and biochemical activity. The HRP-immobilized electrode was investigated by cyclic voltammetry and chronoamperometry. The results from both techniques showed that the direct electron transfer between the nanocomposite modified electrodes and heme in HRP could be realized. The biosensor responded to H2O2 in the linear range from 5×10−6 to 1×10−3molL−1 with a detection limit of 2.1×10−6molL−1 (based on the S/N=3).

Direct electrochemistry of horseradish peroxidase immobilized on electrografted 4-ethynylphenyl film via click chemistry

In this paper, a simple two-step approach for redox protein immobilization was introduced. Firstly, alkynyl-terminated film was formed on electrode surface by electrochemical reduction of 4-ethylnylphenyl (4-EP) diazonium compound. Then, horseradish peroxidase (HRP) modified with azido group was covalently immobilized onto the electrografted film via click reaction. Reflection absorption infrared (RAIR) spectroscopy and electrochemical methods were used to characterize the modification process. The results indicate that HRP retains its native structure and shows fast direct electron transfer. Moreover, the immobilized HRP shows excellent electrocatalytic reduction activity toward H 2O 2 with a linear range of 5.0 × 10 −6 to 9.3 × 10 −4 mol L −1.