Direct Electron Transfer Of Horseradish Peroxidase Research Articles

Electrochemical Biosensor Based on Horseradish Peroxidase and Black Phosphorene Quantum Dot Modified Electrode.

Black phosphorene quantum dots (BPQDs) were prepared by ultrasonic-assisted liquid-phase exfoliation and centrifugation with morphologies proved by TEM results. Furthermore, an electrochemical enzyme sensor was prepared by co-modification of BPQDs with horseradish peroxidase (HRP) on the surface of a carbon ionic liquid electrode (CILE) for the first time. The direct electrochemical behavior of HRP was studied with a pair of well-shaped voltammetric peaks that appeared, indicating that the existence of BPQDs was beneficial to accelerate the electron transfer rate between HRP and the electrode surface. This was due to the excellent properties of BPQDs, such as small particle size, high interfacial reaction activity, fast conductivity, and good biocompatibility. The presence of BPQDs on the electrode surface provided a fast channel for direct electron transfer of HRP. Therefore, the constructed electrochemical HRP biosensor was firstly used to investigate the electrocatalytic behavior of trichloroacetic acid (TCA) and potassium bromate (KBrO3), and the wide linear detection ranges of TCA and KBrO3 were 4.0-600.0 mmol/L and 2.0-57.0 mmol/L, respectively. The modified electrode was applied to the actual samples detection with satisfactory results.

Surface modification of cerasomes with AuNPs@poly(ionic liquid)s for an enhanced stereo biomimetic membrane electrochemical platform

A novel liposomal nanocomposite, Au@PIL-cerasome, with biocompatibility and conductivity was fabricated via the self-assembly of cerasomes and gold nanoparticles (AuNPs) stabilized by poly(ionic liquid)s (PILs). The surface charge, morphology and chemical composition of the nanocomposites were characterized by the zeta potential, UV–vis, TEM, SEM and EDS. The nanocomposites exhibited structural stability directly on the surface of solid electrodes, without fusion. Electrochemical impedance experiments demonstrated that the nanocomposites had an enhanced conductivity compared with unmodified cerasomes. Horseradish peroxidase (HRP), as a reporter, was immobilized on the nanocomposites without denaturation or inactivation. The direct electron transfer of HRP was achieved, and the HRP/Au@PIL-cerasome/GCE exhibited an amplified current and improved electrocatalytic activity. Activity towards H2O2 displayed a linear range over 10–70 μM and a detection limit of 3.3 μM. Activity towards NO2− displayed linear ranges over 1–5 mM and 5–1280 mM, and the limit of detection was 0.11 mM. In addition, the electrode was stable and reproducible, with 6% RSD. Such multi-component liposomal nanocomposites with an enhanced electrical performance pave a better way for building novel and straightforward 3D stereo biomimetic electrochemical platforms and even molecular communication systems to investigate information transduction between cells.

Advantages of an Electrochemical Method Compared to the Spectrophotometric Kinetic Study of Peroxidase Inhibition by Boroxine Derivative.

In this study, boroxine derivative (K2[B3O3F4OH]) was tested as an inhibitor of horseradish peroxidase (HRP) by spectrophotometric and electrochemical methods. The activity of horseradish peroxidase was first studied under steady-state kinetic conditions by a spectrophotometric method which required the use of guaiacol as a second substrate to measure guaiacol peroxidation. The results of this method have shown that, by changing the concentration of guaiacol as the literature suggests, a different type of inhibition is observed than when changing the concentration of hydrogen peroxide as the substrate. This suggests that guaiacol interferes with the reaction in some way. The electrochemical method involves direct electron transfer of HRP immobilized in Nafion nanocomposite films on a glassy carbon (GC) electrode, creating a sensor with an electro-catalytic response to the reduction of hydrogen peroxide. The electrochemical method simplifies kinetic assays by removing the requirement of reducing substrates.

Direct electron transfer of horseradish peroxidase at Co3O4–graphene nanocomposite modified electrode and electrocatalysis

In this paper, the mixture of Co3O4–graphene nanocomposite and horseradish peroxidase (HRP) was spread on the surface of carbon ionic liquid electrode (CILE). Then, Nafion film was used for the immobilization. The results of spectroscopy proved that HRP kept up its native structure in the complex material. Direct electrochemistry of HRP resulted in a couple of quasi-reversible redox waves on cyclic voltammograms, reflecting the realization of direct electron transfer of HRP with electrode. The improvement in electrochemical responses was due to the usage of highly conductive Co3O4–graphene nanocomposite with biocompatible interface. Electrochemical parameters such as the electron transfer coefficient (α) was estimated as 0.47, and the apparent heterogeneous electron transfer rate constant (ks) was calculated as 2.90 s−1. The HRP modified electrode exhibited good electrochemical catalytic ability toward the reduction of trichloroacetic acid and NaNO2. As a consequence, an updated third-generation electrochemical HRP biosensor with Co3O4–GR/CILE was constructed successfully.

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.

An intimately bonded titanate nanotube–polyaniline–gold nanoparticle ternary composite as a scaffold for electrochemical enzyme biosensors

In this work, titanate nanotubes (TNTs), polyaniline (PANI) and gold nanoparticles (GNPs) were assembled to form a ternary composite, which was then applied on an electrode as a scaffold of an electrochemical enzyme biosensor. The scaffold was constructed by oxidatively polymerising aniline to produce an emeraldine salt of PANI on TNTs, followed by gold nanoparticle deposition. A novel aspect of this scaffold lies in the use of the emeraldine salt of PANI as a molecular wire between TNTs and GNPs. Using horseradish peroxidase (HRP) as a model enzyme, voltammetric results demonstrated that direct electron transfer of HRP was achieved at both TNT-PANI and TNT-PANI-GNP-modified electrodes. More significantly, the catalytic reduction current of H2O2 by HRP was ∼75% enhanced at the TNT-PANI-GNP-modified electrode, compared to that at the TNT-PANI-modified electrode. The heterogeneous electron transfer rate constant of HRP was found to be ∼3 times larger at the TNT-PANI-GNP-modified electrode than that at the TNT-PANI-modified electrode. Based on chronoamperometric detection of H2O2, a linear range from 1 to 1200 μM, a sensitivity of 22.7 μA mM−1 and a detection limit of 0.13 μM were obtained at the TNT-PANI-GNP-modified electrode. The performance of the biosensor can be ascribed to the superior synergistic properties of the ternary composite.

Preparation of graphene nanoplatelet–titanate nanotube composite and its advantages over the two single components as biosensor immobilization materials

A novel nanocomposite consisting of graphene nanoplatelets (GNPs) and titanate nanotubes (TNTs) have been synthesized successfully utilizing the hydrothermal method. The GNP–TNT composite was characterized by transmission electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy and electrochemical impedance spectroscopy. The voltammetric characterization of GNP–TNT composite, pure GNPs and pure TNTs modified horseradish peroxidase (HRP) biosensors were conducted to select the most suitable electrode immobilization material for enzyme biosensors. The GNPs was firstly eliminated owing to its extremely high background charging current, distinct electrochemical interference from its surface functional groups and low signal to noise ratio. Next, the direct electron transfer of HRP on electrode and the catalytic current of HRP towards H2O2 was increased around 45% and 72% respectively on GNP–TNT composite modified electrodes compared with those on pure TNTs modified electrodes. GNP–TNT composite modified HRP biosensor also exhibited superiority over pure TNTs modified HRP biosensor in the analytical performance. The precision and stability study provided additional evidence for the feasibility of using GNP–TNT composite as electrode modification material.

In situ synthesized gold nanoparticles for direct electrochemistry of horseradish peroxidase

In order to construct a three-dimensional electrode, in situ electrochemical deposition of gold nanoparticles onto a gold electrode coated with a self-assembled monolayer of 3-mercaptopropionic acid (Au NP/MPA/Au) was conducted. Horseradish peroxidase (HRP) was then immobilized into this three-dimensional electrode, leading to the realization of direct electron transfer of HRP. Scanning electron microscopy and electrochemical impedance spectroscopy were applied to characterize the electrode before and after HRP immobilization. The apparent Michaelis-Menten constant for immobilized HRP on a Au NP/MPA/Au electrode is 0.78mM, indicating high enzymatic activity of HRP. The HRP modified electrode was further utilized as a sensing platform for the electrocatalytic reduction and detection of hydrogen peroxide. For the detection of hydrogen peroxide on this electrode, the sensitivity is 311.72μAmM−1cm−2, the detection limit is 0.16μM, and the dynamic range is from 0.48μM to 1.2mM. Therefore this electrode is a promising device for sensitive and reproducible detection of H2O2.

Immobilization of Horseradish Peroxidase on TiO<sub>2</sub> Nanotube Arrays via Seeded-Growth Synthesis

Horseradish peroxidase (HRP) was successfully immobilized on vertically oriented TiO2nanotube arrays (NTAs), which was prepared by seeded-growth mechanism. The nanotubular structure of TiO2was characterized by scanning electron microscope (SEM). After encapsulated HRP on TiO2nanotube arrays, the direct electron transfer of HRP was observed.

A amperometric biosensor for hydrogen peroxide by adsorption of horseradish peroxidase onto single-walled carbon nanotubes

Development of a highly sensitive nanostructured electrochemical biosensor based on the integrated assembly of horseradish peroxidase (HRP) and single-walled carbon nanotubes (SWNTs) is described. In this study, we describe the use of a sodium cholate suspension-dialysis method to adsorb the horseradish peroxidase (HRP) onto single-walled carbon nanotubes (SWNTs). We demonstrate that HRP–SWNTs conjugates can be assembled into amperometric biosensors which l-cysteine were assembled on a gold electrode through the covalent bond of S–Au and was used as a substrate for the immobilization of enzymes. Direct electron transfer of HRP is realized at SWNTs, and both anodic and cathodic currents of the redox reaction at the l-cysteine–HRP–SWNTs-modified gold film upon electrocatalysis are amplified. Meanwhile, experimental results reveal that HRP is stably immobilized onto the SWNTs and maintains inherent enzymatic activity toward H 2O 2. The modified electrode shows high sensitivity toward H 2O 2. A linear response to hydrogen peroxide measurement is obtained over the range from 1.0 × 10 −12 to 1.0 × 10 −11 M and an amperometric detection limit of 2.1 × 10 −13 M due to its bioelectrocatalytic reduction based on direct electron transfer between gold electrode and the active site of the HRP. The biosensor displays excellent operational, storage stability and highly sensitive. The excellent performance validates the integrated assembly as an attractive sensing element for the development of a new hydrogen peroxide amperometric biosensor.

A novel electrochemical sensor surface for the detection of hydrogen peroxide using cyclic bisureas/gold nanoparticle composite

A novel electrochemical sensor surface with enhanced sensitivity for the detection of hydrogen peroxide has been developed based on the layer-by-layer assembly of mercapto propionic acid (MPA), cystine-based polymethylene-bridged cyclic bisureas (CBU)/gold nanoparticle (AuNP) and horseradish peroxidase (HRP) on gold electrode. Possibility of a large number of hydrogen bonds, allowed by the chemical and sterical structure of the CBU ensures the proper immobilization of the enzyme in favorable orientation and retention of enzymatic activity. Efficient electron tunneling property of AuNP together with its electrocatalytic activity leads to higher sensitivity in the detection of H2O2. In cyclic voltammetry measurements a cathodic current due to direct electron transfer of HRP is observed which, indicates excellent electrocatalytic activity of the sensor surface. The biosensor surface modified with gold nanoparticle and CBU showed a lower detection limit of 50nM for hydrogen peroxide. Chronoamperometry is performed at −0.3V and Michaelis–Menten constant KMapp value is estimated to be 4.5μM. The newly developed sensor surface showed very high stability, reproducibility and high sensitivity.

Nanosheet-based titania microspheres with hollow core-shell structure encapsulating horseradish peroxidase for a mediator-free biosensor

Nanosheet-based titania (TiO2) microspheres with a hollow core-shell structure have been synthesized and employed to immobilize horseradish peroxidase (HRP) in order to fabricate a mediator-free biosensor. The morphology and structure of the TiO2 microspheres were characterized by X-ray diffraction, scanning electron microscopy and transmission electronic microscopy. A possible growth mechanism has been proposed. Spectroscopic and electrochemical measurements revealed that the TiO2 microspheres are an immobilization support with biocompatibility for enzymes, affording good enzyme stability and bioactivity. Due to the nanosheet-based hollow core-shell structure of the TiO2 microspheres, the direct electron transfer of HRP is facilitated and the resulting biosensor displayed good performance for the detection of H2O2, with both a low detection limit of 0.05 μM and a wide linear range of 0.4–140 μM, as well as a fast response and excellent long-term stability. The nanosheet-based TiO2 microspheres with hollow core-shell structure, can be used for the efficient entrapment of other redox-active proteins and have wide potential applications in biosensors, biocatalysis, biomedical devices and bioelectronics.

Effect of the structure of imidazolium cations in [BF4]−-type ionic liquids on direct electrochemistry and electrocatalysis of horseradish peroxidase in Nafion films

The direct electrochemistry and bioelectrocatalysis of horseradish peroxidase (HRP) in Nafion films at glassy carbon electrode (GCE) was investigated in three [BF(4)](-)-type room-temperature ionic liquids (ILs) to understand the structural effect of imidazolium cations. The three ILs are 1-ethyl-3-methylimidazolium tetrafluoroborate ([Emim][BF(4)]), 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim][BF(4)]) and 1-hexyl-3-methylimidazolium tetrafluoroborate ([Hmim][BF(4)]). A small amount of water in the three ILs is indispensable for maintaining the electrochemical activity of HRP in Nafion films, and the optimum water contents decrease with the increase of alkyl chain length on imidazole ring. Analysis shows that the optimum water contents are primarily determined by the hydrophilicity of ILs used. In contrast to aqueous medium, ILs media facilitate the direct electron transfer of HRP, and the electrochemical parameters obtained in different ILs are obviously related to the nature of ILs. The direct electron transfer between HRP and GCE is a surface-confined quasi-reversible single electron transfer process. The apparent heterogeneous electron transfer rate constant decreases gradually with the increase of alkyl chain length on imidazole ring, but the changing extent is relatively small. The electrocatalytic reduction current of H(2)O(2) at the present electrode decreases obviously with the increase of alkyl chain length, and the mass transfer of H(2)O(2) via diffusion in ILs should be responsible for the change. In addition, the modified electrode has good stability and reproducibility; the ability to tolerate high levels of F(-) has been greatly enhanced due to the use of Nafion film. When an appropriate mediator is included in the sensing layer, a sensitive nonaqueous biosensor could be fabricated.

Three-dimensional network polyamidoamine dendrimer-Au nanocomposite for the construction of a mediator-free horseradish peroxidase biosensor

A three-dimensional network PAMAM-Au nanocomposite (3D-PAMAM-Au NC) was prepared by using the first generation polyamidoamine dendrimer (G1 PAMAM) as the dispersant agent. The resultant 3D-PAMAM-Au NC was successfully used as an immobilization matrix for the construction of a reagentless mediator-free horseradish peroxidase (HRP)-based H(2)O(2) biosensor on a multi-walled carbon nanotubes (MWCNTs) modified glassy carbon electrode. With the advantages of the three-dimensional network, the organic-inorganic hybrid materials dramatically facilitate the direct electron transfer of HRP, and good bioelectrocatalytic activity towards H(2)O(2) was demonstrated. Under optimum conditions, the current response of the enzyme modified electrode at -0.30 V was detected. The current response is linearly correlated to H(2)O(2) concentration within the range of 18.00 μM to 20.80 mM with a correlation coefficient of 0.9992 and a sensitivity of 377.78 μA mM(-1) cm(-2). The detection limit was down to 6.72 μM (S/N = 3). Furthermore, the biosensor exhibits some other excellent characteristics, such as high selectivity, short response time, and long-term stability. The 3D-PAMAM-Au NC has proved to be a promising biosensing platform for the construction of mediator-free biosensors, and may find wide potential applications in biosensors, biocatalysis, bioelectronics and biofuel cells.

A highly soluble poly(3,4-ethylenedioxythiophene)-poly(styrene sulfonic acid)/Au nanocomposite for horseradish peroxidase immobilization and biosensing

A highly soluble poly(3,4-ethylenedioxythiophene)-poly(styrene sulfonic acid)/Au (PEDOT-PSS/Au) nanocomposite was prepared via one-step chemical synthesis and the matrix was characterized by UV–vis spectroscopy (UV–vis), Fourier transform infrared spectroscopy (FT-IR), scanning electron microscope (SEM) and transmission electron microscope (TEM). Due to the excellent aqueous compatibility and biocompatibility, the PEDOT/PSS-Au nanocomposite can be used as biomaterial for enzymes immobilization. In this system, redox enzyme, horseradish peroxidase (HRP) was integrated with PEDOT/PSS-Au nanocomposite and the direct electron transfer of HRP was observed. Moreover, we find that the HRP/PEDOT-PSS/Au modified electrode shows excellent electrocatalytic ability for H 2O 2 and the formal Michaelis–Menten constant ( K m app ) was 0.78 mmol/L. The response currents have good linear relation with the concentrations of H 2O 2 with a linear range from 2.0 × 10 −7 to 3.8 × 10 −4 mol/L.

A novel hydrogen peroxide biosensor based on Au–graphene–HRP–chitosan biocomposites

Graphene was prepared successfully by introducing –SO 3 − to separate the individual sheets. TEM, EDS and Raman spectroscopy were utilized to characterize the morphology and composition of graphene oxide and graphene. To construct the H 2O 2 biosensor, graphene and horseradish peroxidase (HRP) were co-immobilized into biocompatible polymer chitosan (CS), then a glassy carbon electrode (GCE) was modified by the biocomposite, followed by electrodeposition of Au nanoparticles on the surface to fabricate Au/graphene/HRP/CS/GCE. Cyclic voltammetry demonstrated that the direct electron transfer of HRP was realized, and the biosensor had an excellent performance in terms of electrocatalytic reduction towards H 2O 2. The biosensor showed high sensitivity and fast response upon the addition of H 2O 2, under the conditions of pH 6.5, potential −0.3 V. The time to reach the stable-state current was less than 3 s, and the linear range to H 2O 2 was from 5 × 10 −6 M to 5.13 × 10 −3 M with a detection limit of 1.7 × 10 −6 M (S/N = 3). Moreover, the biosensor exhibited good reproducibility and long-term stability.

One-pot construction of mediatorless bi-enzymatic glucose biosensor based on organic–inorganic hybrid

A new methodology for the fabrication of bienzymatic amperometric glucose biosensor based on the use of an organic–inorganic hybrid is presented. The fabrication involves a self-assembly directed one-pot electrochemical process. Bi-enzymes, horseradish peroxidase (HRP) and glucose oxidase (GOx) are immobilized into the porous and electroactive silica–polyaniline hybrid composite through electrochemical polymerization of N[3-(trimethoxysilyl)propyl]aniline in the presence of enzymes. The modified electrode is designated as PTMSPA/HRP-GOx. The direct electron transfer of HRP is achieved at the modified electrode. Also, the electrode exhibits excellent bio-electro-catalytic activity for the reduction of hydrogen peroxide. The response current at PTMSPA/HRP-GOx modified electrode revealed a good linear relationship with concentration of glucose range between 1 and 20mM with a response time of 7s. Thus, the modified electrode shows the combined advantages of polyaniline and silica networks through synergistic influence from the individual components. The PTMSPA assembly has shown the potential for a third generation amperometric biosensor.

Evaporative deposition of lipophilic quantum dots for an enzyme modified electrode

Lipophilic quantum dots were placed on the surface of a horseradish peroxidase (HRP) modified glassy carbon (GC) electrode by evaporative deposition. Direct electron transfer of immobilized HRP was found to occur. The deposition of quantum dots (QDs) was characterized by various ways, and the results indicated that the direct electron transfer of HRP was promoted preferably when QDs were deposited for 7 h at 35 °C. Under these conditions, the average coverages of QDs and HRP on the electrode surface is 56 nmol cm−2 and 65 pmol cm−2, respectively, and the resulting heterogeneous electron transfer rate constant k of HRP is 6.0 s−1. This work provides a new clue for research on the direct electrochemistry of proteins, and on biosensors based on it.

Amperometric hydrogen peroxide biosensor based on the immobilization of horseradish peroxidase on core–shell organosilica@chitosan nanospheres and multiwall carbon nanotubes composite

The application of the composites of multiwall carbon nanotubes (MWNTs) and core–shell organosilica@chitosan crosslinked nanospheres as an immobilization matrix for the construction of an amperometric hydrogen peroxide (H 2O 2) biosensor was described. MWNTs and positively charged organosilica@chitosan nanospheres were dispersed in acetic acid solution (0.6 wt%) to achieve organosilica@chitosan/MWNTs composites, which were cast onto a glass carbon electrode (GCE) surface directly. And then, horseradish peroxidase (HRP), as a model enzyme, was immobilized onto it through electrostatic interaction between oppositely charged organosilica@chitosan nanospheres and HRP. The direct electron transfer of HRP was achieved at HRP/organosilica@chitosan/MWNTs/GCE, which exhibited excellent electrocatalytic activity for the reduction of H 2O 2. The catalysis currents increased linearly to H 2O 2 concentration in a wide range of 7.0 × 10 −7 to 2.8 × 10 −3 M, with a sensitivity of 49.8 μA mM −1 cm −2 and with a detection limit of 2.5 × 10 −7 M at 3 σ. A Michaelies–Menten constant K M a p p value was estimated to be 0.32 mM, indicating a high-catalytic activity of HRP. Moreover, the proposed biosensor displayed a rapid response to H 2O 2 and possessed good stability and reproducibility. When used to detect H 2O 2 concentration in disinfector samples and sterilized milks, respectively, it showed satisfactory results.