Quantitative analysis of catalysis and inhibition at horseradish peroxidase monolayers immobilized on an electrode surface

Out of several tries, biotinylation of the electrode surface by means of a sacrificial biotinylated immunoglobulin, followed by the anchoring of an avidin-enzyme conjugate appears as the best procedure for depositing a horseradish peroxidase (HRP) monolayer onto an electrode surface, allowing a high-yield immobilization of the enzyme within a stable and highly catalytic coating. Cyclic voltammetry is an efficient means for analyzing the catalytic reduction of H(2)O(2) at such HRP monolayer electrodes in the presence of [Os(III)(bpy)(2)pyCl](2+) (with bpy = bipyridine and py = pyridine) as a one-electron reversible cosubstrate. The odd shapes of current-potential responses, unusual bell-shaped variation of the peak or plateau current with the substrate concentration, hysteresis and trace crossing phenomena, and dependence or lack of dependence with the scan rate, can all be explained and quantitatively analyzed in the framework of the same catalysis/inhibition mechanism as previously demonstrated for homogeneous systems, taking substrate and cosubstrate mass transport of into account. According to H(2)O(2) concentration, limiting-behavior analyses based on the dominant factors or complete numerical simulation were used in the treatment of experimental data. The kinetic characteristics derived from these quantitative treatments implemented by the determination of the amount of enzyme deposited by the newly developed droplet depletion method allowed a comparison with homogeneous characteristics to be drawn. It shows that HRP remains nearly fully active once anchored on the electrode surface through the avidin-biotin linkage. On the basis of this full mechanistic and kinetic characterization, the analytical performances in H(2)O(2) detection and amperometric immunosensor applications are finally discussed.     

Electrocatalytic activity of horseradish peroxidase/chitosan/carbon microsphere microbiocomposites to hydrogen peroxide

Colloidal carbon microspheres (CMS) are dispersed in chitosan (CHIT) solution to form an organic-inorganic hybrid with excellent micro-environment for the immobilization of biomolecules. A novel amperometric biosensor for the determination of hydrogen peroxide (H(2)O(2)) has been constructed by entrapping horseradish peroxidase (HRP) in as-synthesized CMS/CHIT hybrid. The modification of glassy carbon electrode is made by a simple solution-evaporation method. The electrochemical properties of the biosensor are characterized in electrochemical methods. The proposed biosensor shows high sensitive determination and fast response to H(2)O(2) at -0.15 V. The constructed HRP/CHIT/CMS/GC electrode also exhibits a fine linear correlation with H(2)O(2) concentration. The calculated value of the apparent Michaelis-Menten constant, 2.33 mM, suggests that the HRP in CMS/CHIT hybrid keeps its native bioactivity and has high affinity for H(2)O(2).     

An amperometric horseradish peroxidase inhibition biosensor for the determination of phenylhydrazine

An amperometric horseradish peroxidase (HRP) inhibition biosensor has been substantially constructed by the help of N,N-dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS). The preparation steps and the biosensor response to phenylhydrazine were monitored by electrochemical impedance spectroscopy (EIS), cyclic voltammetry, and chronoamperometry. The proposed biosensor could be applied to determine phenylhydrazine in a 0.10 M phosphate buffer solution containing 1.2 mM hydroquinone and 0.50 mM H(2)O(2) by phenylhydrazine, inhibiting the catalytic activity of the HRP enzyme in the reduction of H(2)O(2). The system was optimized to realize a reliable determination of phenylhydrazine in the range of 2.5 x 10(-7) to 1.1 x 10(-6) M with a detection limit of 8.2 x 10(-8) M and a correlation coefficient of 0.999. The modified electrode displayed good reproducibility, sensitivity and stability for the determination of phenylhydrazine.     

基于聚硫堇和层层组装碳纳米管/HRP的过氧化氢传感器研究

将电聚合和层层组装方法有效结合构筑了聚硫堇（PTH）电子介体修饰的碳纳米管（CNTs）/辣根过氧化物酶（HRP）多层膜无试剂H2O2传感器.利用电化学阻抗谱对CNTs/HRP多层膜的组装过程进行监测,用循环伏安法和计时电流法研究了该HRP电极的电化学行为.探讨了酶组装层数、工作电位、pH值和碳纳米管对电极响应的影响.该...     

Elementary steps of enzymatic oxidation of nifedipine catalyzed by horseradish peroxidase

The elementary steps of the enzymatic oxidation of nifedipine (NF) catalyzed by horseradish peroxidase (HRP) have been described based on analysis of kinetic magnetic field effects (MFEs). It has been shown that the first step of the catalytic cycle is single electron transfer resulting in formation of NF*(+) radical cation and ferroperoxidase (Per(2+)). As a result, comparison with an earlier studied oxidation reaction of NADH catalyzed by HRP evidenced that the enzymatic oxidations of two substrates-native, NADH, and its synthetic analogue, NF-catalyzed by HRP in the absence of H(2)O(2) follow identical mechanisms.     

Electrochemical surface plasmon resonance detection of enzymatic reaction in bilayer lipid membranes

In this paper, electrochemical surface plasmon resonance (SPR) method was first used to detect enzymatic reaction in bilayer lipid membrane (BLM) based on immobilizing horseradish peroxidase (HRP) in the BLMs supported by the redox polyaniline (PAn) film. By SPR kinetic curve in situ monitoring the redox transformation of PAn film resulted from the reaction between HRP and PAn, the enzymatic reaction of HRP with H(2)O(2) was successfully analyzed by electrochemical SPR spectroscopy. The results show that this BLM supported on PAn film cannot only preserve the bioactivity of HRP immobilized in the membrane, but also provide a channel for the transfer of electrons between HRP and PAn on electrode surface. These characteristics enabled the development of SPR biosensor for sensitively detecting H(2)O(2). H(2)O(2) has been detected by electrochemical SPR spectroscopy in the concentration range of 5 x 10(-5)M to 2 x 10(-3)M. After each of detections, the SPR sensor surface was completely regenerated by electrochemically reducing the oxidized PAn to its reduced state. This method provides a novel route for enhancing the detection of small ligand of enzymatic reaction in BLM by electrochemical SPR spectroscopy.     

A novel application of horseradish peroxidase: Oxidation of alcohol ethoxylate to alkylether carboxylic acid

A novel application of horseradish peroxidase （HRP） in the oxidation of alcohol ethoxylate to alkylether carboxylic acid in the present of H2O2 was reported in this paper. We propose the mechanism for the catalytic oxidation reaction is that the hydrogen transfers from the substrate to the ferryl oxygen to form the α-hydroxy carbon radical intermediate. The reaction offers a new approach for further research structure and catalytic mechanism of HRP and production of alkylether carboxylic acid.     

Spectroscopic studies of interactions involving horseradish peroxidase and Tb3+

The spectroscopic properties of interactions involving horseradish peroxidase (HRP) and Tb(3+) in the simulated physiological solution was investigated with some electrochemical and spectroscopic methods, such as cyclic voltammetry (CV), circular dichroism (CD), X-ray photoelectron spectroscopy (XPS) and synchronous fluorescence (SF). It was found that Tb(3+) can coordinate with oxygen atoms in carbonyl groups in the peptide chain of HRP, form the complex of Tb(3+) and HRP (Tb-HRP), and then lead to the conformation change of HRP. The increase in the random coil content of HRP can disturb the microstructure of the heme active center of HRP, in which the planarity of the porphyrin cycle in the heme group is increased and then the exposure extent of the electrochemical active center is decreased. Thus Tb(3+) can inhibit the electrochemical reaction of HRP and its electrocatalytic activity for the reduction of H(2)O(2) at the Au/Cys/GC electrode. The changes in the microstructure of HRP obstructed the electron transfer of Fe(III) in the porphyrin cycle of the heme group, thus HRP catalytic activity is inhibited. The inhibition effect of Tb(3+) on HRP catalytic activity is increased with the increasing of Tb(3+) concentration. This study would provide some references for better understanding the rare earth elements and heavy metals on peroxidase toxicity in living organisms.     

Electrochemical studies on reconstituted horseradish peroxidase modified carbon paste electrodes

Horseradish peroxidase (HRP) is a heme protein that acts specifically on H(2)O(2) as the electron acceptor. Hemin (Ferriprotoporhyrin-IX) is the prosthetic group of the enzyme. A direct molecular wire to the redox center of the enzyme is expected to enhance the electrochemical response of the enzyme. Native HRP was immobilized onto the surface of glassy carbon (GC) matrix using a 16-atom spacer arm. We have also immobilized the redox center of the enzyme (hemin) through one of the propionate groups onto the surface of glassy carbon matrix using an 11-atom spacer arm with amino terminus. Apoperoxidase was isolated according to the Teale's method and was allowed to reconstitute with the hemin-bound matrix for enzyme reconstitution. The HRP paste and reconstituted-HRP (rec-HRP) paste electrodes were used to study the electrochemical response to substrate H(2)O(2) using electrochemical techniques like cyclic voltammetry (CV) and flow injection (FI) studies. Flow injection studies using HRP paste electrode showed a linearity from 25 to 200 microM H(2)O(2). The rec-HRP paste showed approximately 100 times increase in the electron transfer rates compared to native HRP paste, and substrate linearity from 25 to 100 microM was observed.     

Zirconia nanoparticles enhanced grafted collagen tri-helix scaffold for unmediated biosensing of hydrogen peroxide

A novel, biocompatible, thermally steady, and nontoxic zirconia enhanced grafted collagen tri-helix scaffold was prepared on a graphite electrode. This scaffold provided a microenvironment for loading biomolecules and helped to retain their natural structure. UV-vis spectroscopy and scanning electron microscopy were used to characterize the scaffold and the structure of immobilized biomolecules. Using horseradish peroxidase (HRP) as an example, this scaffold accelerated its electron transfer and led to its direct electrochemical behavior with a good thermal stability up to 80 degrees C. The surface electron-transfer rate constant of the immobilized HRP was (5.55 +/- 0.43) s(-)(1) in 0.1 M pH 7.0 PBS at 18 degrees C. The immobilized HRP showed an electrocatalytic activity to the reduction of hydrogen peroxide (H(2)O(2)) without aid of an electron mediator. The linear response range of the biosensor for H(2)O(2) was from 1.0 to 73.0 microM with a correlation coefficient of 0.999 (n = 14), a limit of detection down to 0.25 microM and an apparent Michaelis-Menten constant of (0.28 +/- 0.02) mM. The biosensor exhibited high sensitivity, acceptable stability, and reproducibility. The ZrO(2) grafted collagen provided an excellent matrix for protein immobilization and biosensor preparation.     

Prosthetic heme modification during halide ion oxidation. Demonstration of chloride oxidation by horseradish peroxidase

Myeloperoxidase (MPO), eosinophil peroxidase (EPO), and chloroperoxidase can oxidize iodide, bromide, and chloride, but most peroxidases, including the prototypical horseradish peroxidase (HRP), reportedly only oxidize iodide and, in some cases, bromide. We report here that incubation of HRP with Br(-) and H(2)O(2) at acidic pH results in both bromination of monochlorodimedone and modification of the heme group. Mass spectrometry indicates that the heme 2- and 4-vinyl groups are modified by either replacement of a vinyl hydrogen by a bromide or addition of HOBr to give a bromohydrin. These reactions do not occur if protein-free heme and Br(-) are co-incubated with H(2)O(2) or if the HRP reaction is carried out at pH 7. Surprisingly, similar prosthetic heme modifications occur in incubations of HRP with H(2)O(2) and Cl(-). A mechanism is proposed involving oxidation of Br(-) or Cl(-) to give HOBr or HOCl, respectively, followed by addition to a vinyl group. In the reaction with Cl(-), a meso-chloro heme adduct is also formed. This first demonstration of Cl(-) oxidation by HRP, and the finding that prosthetic heme modification occurs when Br(-) or Cl(-) is oxidized in the absence of a cosubstrate, show that only modest tuning is required to achieve the unique chloride oxidation activity of MPO and EPO. The results raise the question of how the prosthetic hemes of MPO and EPO, whose function is to produce oxidized halide species, escape modification.     

Spectroscopic studies of 1-aminocyclopropane-1-carboxylic acid oxidase: molecular mechanism and CO(2) activation in the biosynthesis of ethylene

1-Aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACCO) catalyzes the last step in the biosynthesis of the gaseous plant hormone ethylene, which is involved in development, including germination, fruit ripening, and senescence. ACCO is a mononuclear non-heme ferrous enzyme that couples the oxidation of the cosubstrate ascorbate to the oxidation of substrate ACC by dioxygen. In addition to substrate and cosubstrate, ACCO requires the activator CO(2) for continuous turnover. NIR circular dichroism and magnetic circular dichroism spectroscopies have been used to probe the geometric and electronic structure of the ferrous active site in ACCO to obtain molecular-level insight into its catalytic mechanism. Resting ACCO/Fe(II) is coordinatively saturated (six-coordinate). In the presence of CO(2), one ferrous ligand is displaced to yield a five-coordinate site only when both the substrate ACC and cosubstrate ascorbate are bound to the enzyme. The open coordination position allows rapid O(2) activation for the oxidation of both substrates. In the absence of CO(2), ACC binding alone converts the site to five-coordinate, which would react with O(2) in the absence of ascorbate and quickly deactivate the enzyme. These studies show that ACCO employs a general strategy similar to other non-heme iron enzymes in terms of opening iron coordination sites at the appropriate time in the reaction cycle and define the role of CO(2) as stabilizing the six-coordinate ACCO/Fe(II)/ACC complex, thus preventing the uncoupled reaction that inactivates the enzyme.     

聚合物纳米组装体修饰丝网印刷电极构建过氧化氢传感器

通过在聚合物结构中同时引入生物亲和单体和电活性单体,使得聚合物组装体在修饰丝网印刷电极时兼具提高比表面积、保持酶活和促进电子转移功能,从而发展了一种简单、高效构建电化学传感器的方法。 以苯乙烯(St)、丙烯酸(AA)、N-乙烯基咔唑(VCz)和甲基丙烯酸二甲氨基乙酯(DMAEMA)为单体自由基聚合合成双亲性无规共聚物Poly(St-co-AA-co-VCz-co-DMAEMA)(PSACD),将聚合物在选择性混合溶剂DMF/H₂O中自组装得到聚合物纳米粒子PSACD NPs。 利用纳米粒度分析仪、扫描电子显微镜(SEM)对其进行表征。 依次将PSACD NPs水分散液、辣根过氧化物酶(HRP)溶液和全氟磺酸-聚四氟乙烯共聚物(Nafion,NF)溶液滴涂在丝网印刷碳电极(SPCE)上,制备得到过氧化氢生物传感器。 通过计时电流法对传感器性能进行研究,表明该传感器对H₂O₂在0.02~7.48 mmol/L有良好的线性响应,且响应时间短(<2 s),具有良好的选择性和稳定性。     

Characterization of G-quadruplex/hemin peroxidase: substrate specificity and inactivation kinetics

Recently, G-quadruplex/hemin (G4/hemin) complexes have been found to exhibit peroxidase activity, and this feature has been extensively exploited for colorimetric detection of various targets. To further understand and characterize this important DNAzyme, its substrate specificity, inactivation mechanism, and kinetics have been examined by comparison with horseradish peroxidase (HRP). G4/hemin DNAzyme exhibits broader substrate specificity and much higher inactivation rate than HRP because of the exposure of the catalytic hemin center. The inactivation of G4/hemin DNAzyme is mainly attributed to the degradation of hemin by H(2)O(2) rather than the destruction of G4. Both the inactivation rate and catalytic oxidation rate of G4/hemin DNAzyme depend on the concentration of H(2)O(2), which suggests that active intermediates formed by G4/hemin and H(2)O(2) are the branch point of catalysis and inactivation. Reducing substrates greatly inhibit the inactivation of G4/hemin DNAzyme by rapidly reacting with the active intermediates. A possible catalytic and inactivation process of G4/hemin has been proposed. These results imply a potential cause for the hemin-mediated cellular injury and provide insightful information for the future application of G4/hemin DNAzyme.     

基于石墨烯-壳聚糖-辣根过氧化物酶的H2O2生物传感器的研制

制备了石墨烯-壳聚糖(GR-CS)纳米复合材料,并将之与辣根过氧化物酶(HRP)混合,构建了基于石墨烯-壳聚糖-辣根过氧化物酶的生物传感器(GR-CS-HRP/GC)。探针及循环伏安研究表明,该界面具有优异的电子传导能力、较大的比表面积和良好的生物相容性,对H2O2的还原显示出较好的电催化活性,在工作电位为-0.2 V,0.05 mol/L的磷酸盐缓冲盐溶液(PBS,pH 6.8)中,该酶传感器对过氧化氢响应灵敏度高,检测范围宽,测定H2O2的线性范围为5.0×10-7～2×10-3mol/L(相关系数为0.998)。检出限为2.0×10-7mol/L(S/N=3)。并且表现出良好的稳定性和高选择性。该电极用于实际样品中H2O2的测定,结果令人满意。     

基于纳米金自组装膜固定辣根过氧化物酶的生物传感研究

将辣根过氧化物酶(HRP)通过纳米技术和自组装技术固定于电极表面,制得了酶修饰电极.纳米金与HRP形成了静电复合物并高效地保持了HRP的生物活性,以对苯二酚作为电子媒介体,差示脉冲伏安法(DPV)研究生物酶电极测定H2O2的线性范围为5.0×10-6～1.0×10-3 mol/L,检测限为2.5×10-6 mol/L,线性方程为△I=0.34765+4.05553CH2O2(mM).酶电极的表观米氏常数(K(app))为0.0675 mmol/L.实验同时证明该生物酶电极具有良好的稳定性和使用寿命.     

Amperometric biosensor for hydrogen peroxide based on direct electrocatalysis by hemoglobin immobilized on gold nanoparticles/1,6-diaminohexane modified glassy carbon electrode.

A facile strategy of an amperometric biosensor for hydrogen peroxide based on the direct electrocatalysis of hemoglobin (Hb) immobilized on gold nanoparticles (GNPs)/1,6-diaminohexane (DAH) modified glassy carbon electrode (GCE) has been described. A uniform monolayer film of DAH was initially covalently bound on a GCE surface by virtue of the electrooxidation of one amino group of DAH, and another amino group was modified with GNPs and Hb, successively. The fabrication process was characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and scanning electron microscopy (SEM). The proposed biosensor exhibited an effective and fast catalytic response to the reduction of H2O2 with good reproducibility and stability. A linear relationship existed between the catalytic current and the H2O2 concentration in the range of 1.5x10(-6) to 2.1x10(-3) M with a correlation coefficient of 0.998 (n=24). The detection limit (S/N=3) was 8.8x10(-7) M.     

基于仿生聚多巴胺膜和纳米金的酶固定化平台的构建

首次以仿生聚多巴胺膜为功能基底膜并结合使用纳米金, 构建了一种高导电性、稳健的酶生物分子固定化平台. 以固定辣根过氧化物酶(HRP)为例, 发展了一种新的电化学酶传感器用于H2O2的测定. 结果表明, 酶传感器借助聚多巴胺膜对基底电极的高结合力及其高生物亲和性与电活性, 并协同纳米金的“电子通道”作用, 不仅可以实现酶分子在电极表面的大量而高活性的固定化, 而且能促进电子在酶活性中心和电极表面间的快速传递. 与采用其它常见聚合物材料(例如壳聚糖)的酶传感器比较, 以聚多巴胺/纳米金固定化平台发展的酶传感器具有更优良的检测H2O2的性能. 其对H2O2的检测线性范围为4.0×10－7～4.5×10－4 mol•L－1, 检测限为3.7×10－7 mol•L－1, 灵敏度为100.2 μA•L•mmol－1. 此外, 该酶传感器还具有优良的检测重现性和存贮稳定性, 以及较好的抗干扰能力.     

Direct electrochemistry and electrocatalysis of horseradish peroxidase in alpha-zirconium phosphate nanosheet film

Alpha-zirconium phosphate nanosheets (ZrPNS) derived via the delamination of layered alpha-zirconium phosphate (alpha-ZrP) have been proven to be efficient support matrixes for the immobilization of horseradish peroxidase (HRP). X-ray powder diffraction (XRD) results revealed that ZrPNS in HRP-ZrPNS film remained unorderly structured for the effect of HRP. Fourier transform infrared (FTIR) spectra results revealed that HRP remained the secondary structure in HRP-ZrPNS film. The direct electrochemistry of HRP was realized in HRP-ZrPNS film on a glassy carbon electrode (GCE), showing a pair of well-defined, nearly reversible cyclic voltammetry (CV) peaks for the HRP heme Fe(III)/Fe(II) redox couple. The average surface concentration (Gamma(*)) of electroactive HRP in HRP-ZrPNS film was estimated to be 1.35x10(-10) mol cm(-2), which indicated a high loading of enzyme molecules in HRP-ZrPNS film. Based on these, a third generation reagentless biosensor was constructed for the determination of hydrogen peroxide (H(2)O(2)). The response time of the biosensor was less than 3 s, and the linear response range of the biosensor for H(2)O(2) was from 1.3x10(-6) to 1.6x10(-2) M with a correlation coefficient of 0.9997.