Horseradish Peroxidase Molecules Research Articles

Direct Electrochemistry and Electrocatalytic Behavior of Horseradish Peroxidase on Attapulgite Clay Modified Electrode

A novel third-generation hydrogen peroxide (H(2)O(2)) biosensor was developed by immobilizing horseradish peroxidase (HRP) on a biocompatible attapulgite (ATP) modified glassy carbon (GC) electrode. The ATP could provide a biocompatible microenvironment for enzyme molecules, greatly amplify the coverage of HRP molecules on the electrode surface, and most importantly facilitate the direct electron transfer between HRP and the electrode. The biosensor construction process was followed by atomic force microscopy (AFM). Cyclic voltammetry was employed to characterize the properties of the biosensor. A linear calibration plot of the enzyme electrode was obtained over the range of 5 µM to 0.3 mM for H(2)O(2) with a detection limit of 5 µM. Furthermore, the biosensor showed high sensitivity, good reproducibility, and fine long-term stability.

Amperometric biosensor for hydrogen peroxide based on horseradish peroxidase onto gold nanowires and TiO2 nanoparticles

An electrochemical biosensor for determination of hydrogen peroxide (H(2)O(2)) was fabricated, based on the electrostatic immobilization of horseradish peroxidase (HRP) with one-dimensional gold nanowires (Au NWs) and TiO(2) nanoparticles (nano-TiO(2)) on a gold electrode. The nano-TiO(2) can give a biocompatible microenvironment and compact film, and the Au NWs can provide fast electron transferring rate and greatly add the amount of HRP molecules immobilized on the electrode surface. Au NWs were characterized by ultraviolet-visible spectra and transmission electron microscope. The electrode modification process was probed by cyclic voltammetry and electrochemical impedance spectroscopy. Chronoamperometry was used to study the electrochemical performance of the resulting biosensor. Under optimal conditions, the linear range for the determination of H(2)O(2) was from 2.3 × 10(-6) to 2.4 × 10(-3) M with a detection limit of 7.0 × 10(-7) M (S/N = 3). Moreover, the proposed biosensor showed superior stability and high sensitivity.

Plasmon-Enhanced Colorimetric ELISA with Single Molecule Sensitivity

Robust but ultrasensitive biosensors with a capability of detecting low abundance biomarkers could revolutionize clinical diagnostics and enable early detection of cancer, neurological diseases, and infections. We utilized a combination of localized surface plasmon resonance (LSPR) refractive index sensing and the well-known enzyme-linked immunosorbent assay to develop a simple colorimetric biosensing methodology with single molecule sensitivity. The technique is based on spectral imaging of a large number of isolated gold nanoparticles. Each particle binds a variable number of horseradish peroxidase (HRP) enzyme molecules that catalyze a localized precipitation reaction at the particle surface. The enzymatic reaction dramatically amplifies the shift of the LSPR scattering maximum, λ(max), and makes it possible to detect the presence of only one or a few HRP molecules per particle.

Molecular and cellular mechanism of the effect of La(III) on horseradish peroxidase

Horseradish is an important economic crop. It contains horseradish peroxidase (HRP) and lots of nutrients, and has specific pungency. Lanthanum is one of the heavy metals in the environment. It can transfer through the food chain to humans. In this paper, the molecular and cellular mechanism of the toxic effects of La(III) on HRP in vivo was investigated with an optimized combination of biophysical, biochemical, and cytobiological methods. It was found that La(III) could interact with O and/or N atoms in the backbone/side chains of the HRP molecule in the cell membrane of horseradish treated with 80 microM La(III), leading to the formation of a new complex of La and HRP (La-HRP). The formation of the La-HRP complex causes the redistribution of the electron densities of atoms in the HRP molecule, especially the decrease in the electron density of the active center, Fe(III), in the heme group of the La-HRP molecule compared with the native HRP molecule in vivo. Therefore, the electron transfer and the activity of HRP in horseradish treated with 80 microM La(III) are obviously decreased compared with those of the native HRP in vivo. This is a possible molecular and cellular mechanism for the toxic effect of La(III) on HRP in vivo. It is suggested that the accumulation of La in the environment, especially the formation of the La-HRP complex in vivo, is harmful to organisms.

The quasi-one-dimensional assembly of horseradish peroxidase molecules in presence of the alternating magnetic field

Here we reported the assembly of horseradish peroxidase molecules mediated by the alternating magnetic field. The field can make quasi-one-dimensional assembly conformations. We proved the phenomenon was relative to the magnetic property of HRP molecules. Importantly, the electromagnetic field had little influence on the biological activity of HRP molecules.

Mixed monolayers of ferrocenylalkanethiol and encapsulated horseradish peroxidase for sensitive and durable electrochemical detection of hydrogen peroxide.

This paper describes the construction of a mixed monolayer of ferrocenylalkanethiol and encapsulated horseradish peroxidase (HRP) at a gold electrode for amperometric detection of H(2)O(2) at trace levels. By tuning the alkanethiol chain lengths that tether the HRP enzyme and the ferrocenylalkanethiol (FcC(11)SH) mediator, facile electron transfer between FcC(11)SH and HRP can be achieved. Unlike most HRP-based electrochemical sensors, which rely on HRP-facilitated H(2)O(2) reduction (to H(2)O), the electrocatalytic current is resulted from an HRP-catalyzed oxidation reaction of H(2)O(2) (to O(2)). Upon optimizing other experimental conditions (surface coverage ratio, pH, and flow rate), the electrocatalytic reaction proceeding at the electrode was used to attain a low amperometric detection level (0.64 nM) and a dynamic range spanning over 3 orders of magnitude. Not only does the thin hydrophilic porous HRP capsule allow facile electron transfer, it also enables H(2)O(2) to permeate. More significantly, the enzymatic activity of the encapsulated HRP is retained for a considerably longer period (>3 weeks) than naked HRP molecules attached to an electrode or those wired to a redox polymer thin film. By comparing to electrodes modified with denatured HRP that are subsequently encapsulated or embedded in a poly-L-lysine matrix, it is concluded that the encapsulation has significantly preserved the native structure of HRP and therefore its enzymatic activity. The electrode covered with FcC(11)SH and encapsulated HRP is shown to be capable of rapidly and reproducibly detecting H(2)O(2) present in complex sample media.

Inhibition mechanism of lanthanum ion on the activity of horseradish peroxidase in vitro

In order to understand the inhibition mechanism of lanthanum ion (La 3+) on the activity of horseradish peroxidase (HRP), the effects of La 3+ on the activity, electron transfer and conformation of HRP in vitro were investigated by using cyclic voltammetry (CV), atomic force microscopy (AFM), circular dichroism (CD), high performance liquid chromatography (HPLC), matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF/MS) and inductively coupled plasma mass spectrometry (ICP-MS). It was found that La 3+ can combine with the amide groups of the polypeptide chain in HRP molecule, forming the complex of La 3+ and HRP (La-HRP). The formation of the La-HRP complex causes the destruction of the native structure of HRP molecule, leading to the decrease in the non-planarity of the porphyrin ring in the heme group of HRP molecule, and then in the exposure extent of active center, Fe(III) of the porphyrin ring of HRP molecule. Thus, the direct electrochemical and catalytic activities of HRP are decreased. It is a possible inhibition mechanism of La 3+ on the activity of peroxidase.

A quantitative study of the enzymatic activity of horseradish peroxidase at a planar poly(acrylic acid) brush

The enzymatic activity of horseradish peroxidase (HRP) has been quantified at a planar poly(acrylic acid) (PAA) brush. A PAA brush is known to exhibit an unusual protein binding affinity, since proteins adsorb at low ionic strength only. At elevated ionic strengths of a few 100 mM proteins desorb, and the PAA brush becomes largely protein resistant. In this study, HRP was used to catalyze the oxidation of Amplex Red to resorufin by hydrogen peroxide. The fluorescence of resorufin was recorded using a microplate reader. As compared to a bare silica surface, the enzymatic activity of HRP is higher by more than one order of magnitude at a PAA brush. This increase results from a higher degree of adsorption and a reasonable preservation of the HRP activity. Upon adsorption at a PAA brush from a 20 mU/mL (0.1 μg/mL) solution, the molecular enzymatic activity of HRP is reduced to about 11%. However, when a HRP molecule is desorbed from a PAA brush by increasing the ionic strength, a gain of the molecular enzymatic activity by only 52% can be observed. Overall, this study illustrates the potential applicability of a PAA brush as a biocompatible material coating.

Interaction between lanthanum ion and horseradish peroxidase in vitro

The interaction between lanthanum ion (La(3+)) and horseradish peroxidase (HRP) in vitro was investigated using a combination of biophysical and biochemical methods. When the molar ratio of La(3+) and HRP is low, it was found that the interaction between La(3+) and HRP mainly depends on the electrostatic attraction, van der waals force and hydrogen bond etc. Thus, the interaction is weak and the La-HRP complex cannot be formed in vitro. As expected, the interaction can change the conformation of HRP molecule, leading to the increase in the non-planarity of the porphyrin ring in the heme group of HRP molecule, and then in the exposure degree of the active center, Fe(III) of the porphyrin ring of HRP molecule. Therefore, the catalytic activity of HRP for the H(2)O(2) reduction is improved. When the molar ratio of La(3+) and HRP is high, La(3+) can strongly coordinate with O and/or N in the amide group of the polypeptide chain of HRP molecule, forming the La-HRP complex. The formation of the La-HRP complex causes the change in the conformation of HRP molecule, leading to the decrease in the non-planarity of the porphyrin ring in the heme group of HRP molecule, and then in the exposure degree of the active center, Fe(III) of the porphyrin ring of HRP molecule. Thus, the catalytic activity of HRP for the H(2)O(2) reduction is decreased comparing with that of HRP in the absence of La(3+). The results can provide some references for understanding the interaction mechanism between trace elements ions and peroxidase in living organisms.

Colorimetric multiplexed immunoassay for sequential detection of tumor markers

In this paper, a very simple and easily-operated colorimetric multiplexed immunoassay method for sequential detection of tumor markers has been presented. Magnetic microparticles which are conjugated with biotinylated antibodies are firstly added into the test solution. After fast magnetic collection, these complexes are separated from non-specific proteins. Through different enzymatic reactions of 3,3′,5,5′-tetramethylbenzidine (TMB) and o-phenylenediamine (OPD) catalyzed by horseradish peroxidase molecules which are loaded on the surfaces of gold nanoparticles, two antigens carcinoembryonic antigen and α-fetoprotein can be detected even with naked eyes. The detection limit obtained from the spectrophotometric measurements is as low as 0.02ng/mL. This proposed method also has high specificity and reproducibility, as well as excellent efficiency of 94min for the detection of serum samples. So, this new multiplexed immunoassay method might be a promising approach for the diagnosis of cancer and some other diseases in clinical applications.

Simple Approach for Efficient Encapsulation of Enzyme in Silica Matrix with Retained Bioactivity

We developed an alcohol-free sol-gel approach to encapsulate biomolecules such as horseradish peroxidase (HRP) in an electrochemically induced three-dimensional porous silica matrix by a one-step process. In this sol-gel process, the electrochemically generated hydroxyl ions at the electrode surface by applying cathodic current promote the hydrolysis of ammonium fluorosilicate to produce silica, and simultaneously the generated hydrogen bubbles play an important role in forming porous silica matrix. If HRP is mixed with ammonium fluorosilicate solution, it can be encapsulated in the forming silica matrix. Since there is no ethanol involved in the entire procedure, bioactivities of the encapsulated HRP can be effectively retained. As revealed by scanning electron microscopy (SEM) characterization, the resultant silica matrix has interconnected and network-like porous structures. Macroporous holes induced by hydrogen bubbles scattering on the relatively flat areas of porous structure can be observed. Such structure free from cracks provides effective mass transport and long-term stability. Scanning electrochemical microscope (SECM) characterization shows that the immobilized HRP molecules uniformly distribute in the silica matrix. The present HRP electrochemical biosensor exhibits a quick response (within 5 s) to H(2)O(2) in the concentration range from 0.02 to 0.20 mM (correlation coefficient of 0.9934) with a detection limit of 3 microM. The apparent Michaelis-Menten constant is 0.88 mM. The present alcohol-free sol-gel approach is effective for biomolecule encapsulation and is promising for the construction of biosensors, bioelectronics, and biofuel cells.

Mechanistic Aspects of Horseradish Peroxidase Elucidated through Single-Molecule Studies

Many individual horseradish peroxidase (HRP) molecules were isolated and observed simultaneously by fluorescence microscopy in an array of 50 000 femtoliter chambers chemically etched into the surface of a glass optical fiber bundle. The substrate turnovers of hundreds of individual HRP molecules were readily analyzed, and the large number of molecules observed provided excellent statistics. In contrast to other enzymes used for single-molecule studies, the rates of product formation in the femtoliter array were, on average, 10 times lower than in bulk solution. We attribute this phenomenon to the particular redox-reaction mechanism of HRP that involves two separate steps of product formation. HRP first oxidizes fluorogenic substrate molecules like Amplex Red to radical intermediates. Two radical molecules subsequently undergo an enzyme-independent dismutation reaction, the rate of which is decreased when confined to a femtoliter chamber resulting in less product. This two-step reaction mechanism of the widely used Amplex Red, as well as other fluorogenic substrates, is often overlooked. The mechanism not only affects single-molecule studies with HRP but also bulk reactions at low substrate turnover rates.

Horseradish peroxidase microcapsules based on layer-by-layer polyelectrolyte deposition

Polyelectrolyte multilayer microcapsules have been prepared for encapsulating horseradish peroxidase (HRP). The CaCO 3 microparticles containing HRP molecules were prepared and subsequently enwrapped with layer-by-layer (LbL) deposited multilayer films. The LbL films were constructed via an alternate electrostatic adsorption of poly(allylamine) and poly(styrene sulfonate). The CaCO 3 templates were then dissolved in an ethylenediaminetetraacetic acid solution, leaving HRP in the microcapsules. Optical microscope and fluorescence microscope observations indicated that the microcapsules are homogeneous spheres with an average diameter about 7 μm. The encapsulated HRP showed catalytic activity in the oxidation of pyrogallol, suggesting the microcapsules provide a suitable environment for enzymatic reaction. A leakage of HRP from the microcapsules was negligibly small. Therefore, the HRP-containing microcapsules is promising for detection and treatment of phenolic compounds.

Colloidal Crystals from Surface-Tension-Assisted Self-Assembly: A Novel Matrix for Single-Molecule Experiments

In this work, we develop a new method of creating colloidal crystals with cavities for the entrapment and long-term observation of single biomolecules. Colloidal crystals are first fabricated using surface-tension-assisted self-assembly. Surface tension helps to reduce the interparticle distance between dispensed colloids. Subsequently, the colloids are used as a matrix in which single fluorescently tagged molecules can be tracked using fluorescence microscopy. This method has a high efficiency of self-assembly for small volumes (4 microL) of colloidal suspensions (polystyrene colloids with diameters of 1000, 500, 200, and 100 nm) at low concentration (1% w/w). The spatial hindrance effect on the diffusion of molecules and their entrapment is discussed on the basis of fluorescence correlation spectroscopy results from the diffusion of molecules with different hydrodynamic radii in the cavities of colloidal crystals formed from micrometer- to nanometer-sized polystyrene spheres. Single horseradish peroxidase molecules turning over fluorescent products are tracked over a few seconds. This shows that colloidal crystals can be used to test the function of single molecules of enzymes and protein under controlled spatial confinement.

Inhibition Mechanism of TbIII on Horseradish Peroxidase Activity

The inhibition mechanism of Tb(III) on horseradish peroxidase (HRP) in vitro was discussed. The results from MALDI-TOF/MS and X-ray photoelectron spectroscopy (XPS) showed that Tb(III) mainly interacts with the O-containing groups of the amides in the polypeptide chains of the HRP molecules and forms the complex of Tb(III)-HRP, and, in the complex, the molar ratio Tb(III)/HRP is 2 : 1. The results from CD and atomic force microscopy (AFM) indicated that the coordination effect between Tb(III) and HRP can lead to the conformation change in the HRP molecule, in which the contents of alpha-helix and beta-sheet conformation in the peptide of the HRP molecules is decreased, and the content of the random coil conformation is increased. Meanwhile, the coordination effect also leads to the decrease in the content of inter- and intrapeptide-chain H-bonds in the HRP molecules, resulting in the HRP molecular looseness and/or aggregation. Thus, the conformation change in the HRP molecules can significantly decrease the electrochemical reaction of HRP and its electrocatalytic activity for the reduction of H2O2.

Magnetically Enhanced Dielectrophoretic Assembly of Horseradish Peroxidase Molecules: Chaining and Molecular Monolayers

Synergy in assembly: The combination of a magnetostatic field with dielectrophoresis can assemble biological molecules—something not possible when these techniques are used in isolation. The AFM image shows horseradish peroxidase (HRP) monolayers. Electromagnetic treatment does not affect the molecular structure, as indicated by the uninhibited catalytic activity of HRP after treatment.

An ultrasensitive chemiluminescence immunosensor for PSA based on the enzyme encapsulated liposome

A highly sensitive chemiluminescence immunosensor for the detection of prostate-specific antigen (PSA) was developed based on a novel amplification procedure with the application of enzyme encapsulated liposome. Horseradish peroxidase (HRP) encapsulated and antibody-modified liposome acts as the carrier of a large number of markers and specific recognition label for the amplified detection of PSA. In the detection of PSA, the analyte was first bound to the specific capture antibody immobilized on the microwell plates, and then sandwiched by the antibody-modified liposomes encapsulating HRP. The encapsulated markers, HRP molecules were released by the lysis of the specifically bound liposomes in the microwell with Triton X-100 solution. Then, the analyte PSA could be determined via the chemiluminescence signal of HRP-catalyzed luminol/peroxide/enhancer system. The “sandwich-type” immunoassay provides the amplification route for the PSA detection in ultratrace levels. The CL emission intensity exhibits dynamic correlation to PSA concentration in the range from 0.74 pg/ml to 0.74 μg/ml with readily achievable detection limit of 0.7 pg/ml.

Light‐Induced Triggering of Peroxidase Activity Using Quantum Dots

Light‐Induced Triggering of Peroxidase Activity Using Quantum Dots

Evaluation of Enzymatic Activity on Nanoscale Polystyrene-block-Polymethylmethacrylate Diblock Copolymer Domains

Understanding structural and functional changes of polymeric surface-bound proteins is extremely important as polymers play an increasingly significant role as arrays and substrates in proteomics applications. We carried out, for the first time, quantitative activity measurements of horseradish peroxidase (HRP) enzymes immobilized selectively on the polystyrene domains of microphase-separated polystyrene-block-polymethylmethacrylate ultrathin films. The specific enzymatic activity of HRP adsorbed on the diblock copolymer surface was evaluated and compared to that of HRP in free solution. We demonstrate that the polymeric surface-bound HRP molecules maintain approximately 85% of their activity in free solution. The unique advantages of diblock copolymer templates, involving nanoscale self-assembly and largely retained protein functionality, make the spontaneously constructed enzyme nanoarrays highly suitable as proteomics substrates. Our novel assembly method of providing functional enzymes on diblock copolymer thin films can be greatly beneficial for high-throughput and high-density protein assays.

Immobilized Horseradish Peroxidase as a Reusable Catalyst for Emulsion Polymerization

The study on the adsorption of horseradish peroxidase (HRP) onto silicon wafers was carried out by means of in situ ellipsometry, atomic force microscopy (AFM) and contact angle measurements. A smooth HRP layer adsorbed onto Si wafers. The enzymatic activity of free or adsorbed HRP was determined by the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and by the emulsion polymerization of ethylene glycol dimethacrylate (EGDMA). Upon adsorbing, HRP molecules might have undergone some conformational changes, which caused a small reduction of enzymatic activity in comparison to that observed for HRP solution. However, it was possible to reuse the same HRP-covered Si wafer as catalyst in the polymerization of EGDMA three times.