Imaging of Activity of Horseradish Peroxidase at β-Cyclodextrin Polymer by Scanning Electrochemical Microscopy

The activity of horseradish peroxidase at b-cyclodextrin polymer was imaged by scanning electrochemical microscopy using 3, 3', 5, 5'-tetramethylbenzide and H2O2 as the substrates.

Effects of different valences of cerium ion on conformation of Horseradish Peroxidase

Our previous studies demonstrated that Ce^4＋could induce reactive oxygen species （ROS） burst as a signal to promote pacilitaxel biosynthesis in suspension cultured Taxus cuspidate cells. To further understand the mechanism of cerium ions inducing ROS burst, circular dichroism （CD）, synchronous fluorescence, and electron paramagnetic resonance （EPR） were used to detect them inducing conformational change of horseradish peroxidase （HRP）. Horseradish peroxidase activity was reduced by 78% by 0.1 mmol/L Ce^4＋, whereas it was only reduced by 28% by 0.1 mmol/L Ce^3＋. Circular dichroism spectra showed that the percentage of transition from helical content and other structure to β strands and flturns was 23.1 when induced by Ce^4＋, whereas it was only 13.2 when induced by Ce^3＋. In synchronous fluorescence spectra, Ce^4＋ led to red shift and intensity-elevation of tryptophan fluorescence emission maximum, whereas in the case of Ce^3＋, the results were a contrast to the above. Furthermore, g factor （gx and gy） in electron paramagnetic resonance （EPR） induced by Ce^4＋ and Ce^3＋ was significantly different. These results indicated that the different valence of cerium ion induced various conformations of HRP, and Ce^4＋ was more effective than Ce^3＋. This suggested that Ce^4＋ affected the burst of ROS through changing the conformation of oxidoreductase.

Development of an Amperometric Hydrogen Peroxide Biosensor based on the Immobilization of Horseradish Peroxidase onto Nickel Ferrite Nanoparticle-Chitosan Composite

Nickel ferrite(NiFe_2O_4) nanoparticles have been dispersed in chitosan solution in order to fab ricate nanocomposite films.Horseradish peroxidase(HRP) has been immobilized onto this chitosan NiFe_2O_4 nanocomposite film via physical adsorption.The size of the NiFe_2O_4 nanoparticles has been estimated us ing X ray diffraction pattern and scanning electron microscopy(SEM) to be 40±9 nm.The chitosan NiFe_2O_4 nanocomposite film and HRP/chitosan NiFe_2O_4 bioelectrode have been characterized using SEM technique.The HRP/chitosan NiFe_2O_4 nanocomposite bioelectrode has a response time of 4 s,linearity as 0.3 to 12 m M of H2O2,sensitivity as 22 n A/m M.The effects of p H and the temperature of the immobilized HRP electrode have also been studied.

Hydrogen Peroxide Sensor Based on Horseradish Peroxidase Combined with CaCO_3 Microspheres and Gold Nanoparticles

Direct electrochemistry and electrocatalysis of horseradish peroxidase（HRP） were achieved by entrapping the enzyme between CaCO3 microspheres and gold nanoparticles through forming sandwich configuration （CaCO3-HRP-AuNPs）. Polyanion, poly（styrene sulfonate）（PSS）, was hybrid with CaCO3 microspheres to increase the surface negative charges for binding with HRP through electrostatic interaction. After the bioconjugate CaCO3 PSS-HRP was entrapped in chitosan based sol-gel（CS-GPTMS） film, HRP was encapsulated by in situ formation of an outer layer of AuNPs through electrochemical reduction of HAuCl4. The composite film containing AuNPs, CaCO3-PSS-HRP bioconjugates and CS-GPTMS can provide favorable microenvironment for HRP to perform direct electron transfer at glassy carbon electrode（GCE）. HRP retained its bioelectrocatalytic activity and lead to sensitive and fast amperometric response for the determination of H2O2. H2O2 could be detected in a very wide linear range from 5.0×10-6 mol/L to 7.1×10-2 mol/L. The sandwich configuration of CaCO3-biomolecules-AuNPs could serve as a versatile platform for enzyme immobilization and biosensing.

INHIBITION AND RADIATION SENSITIZING EFFECT OF INDOLEACETIC ACID COMBINED WITH HORSERADISH PEROXIDASE ON HELA CELLS

Objective To observe the inhibition and radiation-sensitizing effect of Indoleacetic acid (IAA) combined with horseradish peroxidase(HRP)on Hela cells. Methods Hela cells were cultured in vitro and classified into control group, drug group incubated with different doses of IAA(30, 60, 90μmol·L -1) plus 1.2μg·mL -1 HRP, radiation group (6MV-X, 4Gy ) and group of radiation plus IAA plus HRP(same dose as above). All the above were treated for 24-96 hours.The growth inhibition, radiation-sensitizing effect were observed with methyl thiazolyl tetrazolium (MTT) photocolorimetric assay and trypan blue dye assay. The-effect on cell proliferation cycle was determined by flow cytometry. Results The antiproliferation activities showed a significant time-effect and dose-effect relationship to some extent after Hela cells were treated with IAA combined with HRP. The group of radiation plus 60μmol·L -1 IAA plus 1.2μg·mL -1 HRP and radiation plus 90μmol·L -1 IAA plus 1.2μg·mL -1 HRP showed an obvious radiation sensitizing effect. After treatment with 90μmol·L -1 IAA plus 1.2 μg·mL -1 HRP for 72 hours, the determination of cell cycle showed that the percentages of the cells on stages G 2-M and S were all higher than those of the control group. For the group of radiation plus IAA combined with HRP, the percentages of the cells on stages G 2-M were higher than those of the radiation group. Conclusion The above findings suggest that IAA combined with HRP has an inhibitive and killing effect on Hela cells. The effect was stronger during the cell cycles of G 2-M and S. It also has a radiation sensitizing effect. Its mechanism might be that Hela cells were blocked on stages G 2-M, and it presents a collaborative killing effect during miototic time.

A Chemiluminescence Optical Fiber Glucose Biosensor Based on Co-immobilizing Glucose Oxidase and Horseradish Peroxidase in a Sol-gel Film

An optical fiber bienzyme sensor based on the luminol chemiluminescent reaction was developed and demonstrated to be sensitive to glucose. Glucose oxidase(GOD) and horseradish peroxidase(HRP) were co-immobilized by microencapsulation in a sol-gel film derived from tetraethyl orthosilicate(TEOS). The calibration plots for glucose were established by the optical fiber glucose sensor fabricated by attaching the bienzyme silica gel onto the glass window of the fiber bundle. The linear range was 0 2-2 mmol/L and the detection limit was approximately 0 12 mmol/L. The relative standard deviation was 5.3% ( n =6). The proposed biosensor was applied to glucose assay in ofloxacin injection successfully.

Direct electrochemistry and electrocatalysis of horseradish peroxidase immobilized on L-glutathione self-assembled monolayers

A novel hydrogen peroxide biosensor has been fabricated based on covalently linked horseradish peroxidase （HRP） onto L- glutathione self-assembled monolayers （SAMs）. The SAMs-based electrode was characterized by electrochemical methods, and direct electrochemistry of HRP can be achieved with formal potential of-0.242 V （vs. saturated Ag/AgCl） in pH 7 phosphate buffer solution （PBS）, the redox peak current is linear to scan rate and rate constant can be calculated to be 0.042 s^-1. The HRP-SAMs- based biosensors show its better electrocatalysis to hydrogen peroxide in the concentration range of 1 × 10^-6 mol/L to 1.2 × 10^-3 mol/L with a detection limit of 4 × 10^-7 mol/L. The apparent Michealis-Menten constant is 3.12 mmol/L. The biosensor can effectively eliminate the interferences of dopamine, ascorbic acid, uric acid, catechol and p-acetaminophen.

Study of the mechanism on the apoptosis induced in Human leukemia cell line K562 by the combination of indole-3-acetic acid and horseradish peroxidase

Objective To investigate the mechanisms of apoptosis induced in Human leukemia cell line K562 by the combination of indole-3-acetic acid and horseradish peroxidase.Methods Human leukemia cell line K562 were exposed to indole-3-acetic acid(IAA) at 20,40,60,80 or 100mol/L and horseradish peroxidase(HRP) at 1.2g/mL for varying times.MTT assay was applied to detect the cell proliferation.Flow cytometry was performed to detect the arrest of cell cycle.Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL) assay was used to measure apoptosis.2,7-dichlorofluorescin diacetate(DCFH-DA) uptake was measured to determine free radical by confocal microscope.Content of malondiadehyde(MDA) and activity of superoxide dismutase(SOD) were measured by biochemical methods.Results IAA/HRP initiated growth inhibition of K562 cells in a dose-and time-dependent manner.Flow cytometry revealed that cell cycle arrested at G_1/G_0 after 24 hours treatment.After 72 hours treatment,apoptotic rate of 100 mol/L IAA group increased to 43.9%,which was 5 times that of control(P<0.01).Content of MDA and activity of SOD increased respectively in treatments compared with control.Meanwhile,IAA/HRP stimulated the formation of free radical,which was increased by IAA concentration-dependently.Conclusion The combination of IAA and HRP can inhibit the growth of Human leukemia cell line K562 in vitro by inducing apoptosis which is associated with the increase of free radical.The combination of IAA and HRP might be a promising chemopreventive and chemotherapeutic agent against human leukemia.

The Difference of Sensitivity between BXPC-3 and K562 Cells by Treatments with Combination of Indole-3-acetic Acid and Horseradish Peroxidase

The difference of sensitivity to indole- 3-acetic acid （ IAA ） combined with horseradish peroxidase （HRP） in K562 and BXPC- 3 cells was investigated. The cell proliferation was determined by MTF assay. The cell cycle and apoptosis of K562 and BXPC-3 cells were examined by a fluorescence flow cytometer （FCM） and terminal deoxynacleotidyl transferase-mediated dUTP nick-end labeling （TUNEL） respectively. The experimental results show that IAA and HRP could inhibit BXPC- 3 cell proliferation greatly compared with K562 cell during the first 48 h . The cell cycle was arrested predominantly at G2/ M phase in K562 and BXPC- 3 cells. The cell apoptosis of K562 and BXPC- 3 was induced by IAA/ HRP. There was a significant difference between the two cell lines since BXPC-3 cells were more sensitive than K562 cells by treatments with combination of IAA and HRP.

Third Generation Horseradish Peroxidase Biosensor Based on Self-assembling Carbon Nanotubes to Gold Electrode Surface

A third-generation horseradish peroxidase (HRP) biosensor has been developed by adsorbing HRP on multi-wall carbon nanotube (MWNTs) monolayer modified gold electrode surface. The assembly process was investigated by electrochemical and spectroscopic techniques. Results showed that the immobilized HRP exhibited direct electrochemical behavior toward the reduction of H2O2. The resulting biosensor shows a fast amperometric response (<2 s) to H2O2. The linear response range was from 5.0×10-7～1.0×10-5 mol/L with a detection limit of 1.0×10-7mol/L. Moreover, the biosensor has a good reproducibility, and long-term stability.

New fluorimetric assay of horseradish peroxidase using sesamol as substrate and its application to EIA

Horseradish peroxidase (HRP) is generally used as a label enzyme in enzyme immunoassay (EIA).The procedure used for HRP detection in EIA is critical for sensitivity and precision.This paper describes a novel fluorimetric assay for horseradish peroxidase (HRP) using sesamol as substrate.The principle of the assay is as follow:sesamol (3,4-methylenedioxy phenol) is reacted enzymatically in the presence of hydrogen peroxide to produce dimeric sesamol.The dimer is fluorescent and can be detected sensitively at ex.347 nm,em.427 nm.The measurable range of HRP was 1.0×10-18 to 1.0×10-15 mol/assay,with a detection limit of 1.0×10-18 mol/assay.The coefficient of variation (CV,n=8) was examined at each point on the standard curve,with a mean CV percentage of 3.8%.This assay system was applied to thyroid stimulating hormone (TSH) EIA using HRP as the label enzyme.

Nitration of Phenol Catalyzed by Horseradish Peroxidase

Horseradish peroxidase, an acidic peroxidase from the horseradish, is one of the most important enzymes as analytical reagent. The enzymatic nitration of phenol by oxidation of nitrite was studied using horseradish peroxidase in the presence of H2O2. The results showed that nitration occur at 2- and 4- positions of phenol. There were also minor products of hydroquinone and catechol. The influence of various reaction parameters, including pH, organic solvent, and concentration of H2O2, on nitration products were discussed. The best nitration pH was 7.0, and H2O2 should be added to the reaction mixture slowly.

Electrochemical Detection of Horseradish Peroxidase at Zeptomole Level

An electrochemical method for determination of horseradish peroxidase (HRP) was developed using a capillary catalytic system. HRP can be measured in several minutes with a detection limit of 4.8×10-12 mol/L or 47 zmol (S/N=3).

Direct Electrochemical Reaction of Horseradish Peroxidase Immobilized on the Surface of Active Carbon Powders

It is reported for the first time that horseradish peroxidase (HRP) immobilized on the active carbon can undergo a direct quasi-reversible electrochemical reaction. In addition, the immobilized HRP showed the stable bioelectrocatalytic activity for the reduction of H2O2.

Activation of Carcinogenic Non-aminoazo Compounds in Horseradish Peroxidase/H_2O_2 System

Eriochrome black T and Nitrosulfophenol S were advocated as the chemical models of carcinogenic non-aminoazo compounds. The main products of their oxidative cleavage in horseradish peroxidase/H2O2 system was identified as the benezenediazonium ion, the ultimate carcinogens, which could bind to DNA. The reaction conditions were investigated preliminarily. Some inhibitors and inducers of the reaction were discovered.

Engineering trienzyme cascade-triggered fluorescent immunosensor platform by sequentially integrating alkaline phosphatase, tyrosinase and horseradish peroxidase

Mulit-enzyme cascades are a major type of chemical transformations and play a crucial role in biological signal transduction and metabolism. Herein, a trienzyme cascade-triggered fluorescent immunosensor platform was constructed by sequentially integrating alkaline phosphatase(ALP), tyrosinase(TYR)and horseradish peroxidase(HRP). The proposed platform was based on HRP-induced a rapid in situ fluorogenic reaction between dopamine(DA) and 1,5-dihydroxynaphthalene(DHA) to produce a strong yellow azamonardine fluorescent compound(AFC). The obtained AFC was clearly characterized by highresolution mass spectrum,1H NMR,^(13)C NMR and theoretical calculations. The integration of the twoenzyme system(TYR and HRP) or three-enzyme system(ALP, TYR and HRP) led to a maximum of 400.0-fold and 250.0-fold fluorescence enhancements, respectively. Using cardiac troponin I(c Tn I) as the model antigen, a trienzyme cascade-triggered fluorescent immunosensor platform was developed for quantitative detecting c Tn I in a wide linear range from 2 ng/m L to 150 ng/m L with a detection limit of 0.67ng/m L. In addition, the proposed platform was successfully applied in detection of c Tn I in serum of clinical patients. Overall, the developed fluorescent immunosensor performs powerful implications for researching enzyme cascade systems in the field of biomedicine.

Nitration Reaction Catalyzed by Horseradish Peroxidase in the Presence of H2O2 and NaNO2

The enzymatic nitration of phenol and m-cresol catalyzed by horseradish peroxidase was studied in the presence of H2O2 and NaNO2. The results showed that the nitration products of phenol were 2-nitro and 4-nitrophenols. There was also a small amount of by-products of hydroquinone and catechol. The influences of various reaction parameters, including pH, organic solvent type, and concentrations of NaNO2 and H2O2, on the nitration products were investigated. The yields of 4-nitrophenol and 2-nitrophenol were 14% and 12%, respectively. The nitration products of m-cresol were 4-nitro-m-cresol and 6-nitro-m-cresol, and the yields of 4-nitro-m-cresol and 6-nitro-m-cresol were 19% and 30%, respectively.

Methylene green-mediated sensor highly sensitive to hydrogen peroxide based on entrapment of horseradish peroxidase in composite membrane of regenerated silk fibroin and poly(vinyl alcohol)

A novel composite membrane of poly(vinyl alcohol) and regenerated silk fibroin was employed to immobilize horseradish peroxidase (HRP) and IR was used to give a useful insight into the structure of the composite membrane before and after ethanol treatment. A methylene green-mediated sensor sensitive to hydrogen peroxide was fabricated, based on the composite membrane as immobilization matrix for HRP. Cyclic voltammetry and amperometric measurement were for the first time utilized to demonstrate the suitability of methylene green as an electron transfer mediator between immobilized HRP and a glassy carbon electrode in bioelectrocatalytic reduction of hydrogen peroxide. Performance and characteristics of the sensor were evaluated in respect to response time, detection limit, applied potential and concentration of the mediator. The sensor possesses a variety of characteristics including good sensitivity, rapid response time and low detection of limit of 0.1 mu mol/L.

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

The 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 hexafluorophos-phate([Bmim]PF_(6)).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 elec-tron 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]PF_(6)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.

Effect of Tetrabutylammonium Bromide on Enzymatic Polymerization of Phenol Catalyzed by Horseradish Peroxidase

In this article tetrabutylammonium bromide （TBAB） was first added in buffer to compose a convenient and environmentally friendly system, and enzymatic polymerization of phenol catalyzed by horseradish peroxidase （HRP） could proceed efficiently in this system. When TBAB was added, the most conversion of phenol could reach 99.1%. The phenol polymer was considered to consist of a mixture of phenylene （Ph） and oxyphenylene （Ox） units by IR analysis, and the ratio of phenylene to oxyphenylene units （Ph/Ox） was measured by titration. Moreover, the effects of the dosage of horseradish peroxidase （HRP） and pH value on the conversion of phenol were investigated. The reaction performed very effectively in this novel system when the addition of HRP was only 0.2 mg. In all cases, the weight-average molecular weight calculated by GPC-SLS was in a range from 12000 Da to 30000 Da. The phenol polymer prepared in the present research possessed good thermal stability shown by TG analysis.