μM For H2O2 Research Articles

Visual fluorescence detection of H2O2 and glucose based on "molecular beacon"-hosted Hoechst dyes.

In this work, a label-free molecular beacon (MB)-like biosensor is designed for the determination of H2O2 and glucose based on the fluorescence regulation of Hoechst dyes hosted by the designed AT-rich single-stranded DNA (ssDNA), in which Hg(2+) and cysteine (Cys) act as activators. The designed AT-rich ssDNA (ATprobe) can be directed to form a hairpin with an Hg(2+)-induced T-Hg(2+)-T complex, which provides a medium for enhancing the fluorescence of Hoechst dyes significantly. On the other hand, Cys can effectively grab Hg(2+) from the T-Hg(2+)-T complex by thiol-Hg(2+) interactions, destructing the hairpin and then switching the Hoechst dyes to the fluorescence "off" state. Combined with these properties, we have demonstrated its application for label-free fluorescence "turn on" detection of H2O2. The sensing mechanism is based on the specific reaction between H2O2 and Cys catalyzed by I(-), the resulting disulfide reverses the Cys-mediated fluorescence decrease of the MB-hosted Hoechst dyes. The approach achieves a low detection limit of 0.1 μM for H2O2. Moreover, this method is further applied to the noninvasive detection of glucose in artificial saliva and urine samples, combining with glucose oxidase (GOx) for the oxidation of glucose and formation of H2O2. Compared to traditional methods, the proposed design is cost-effective, simple to prepare and manipulate without fluorescence labeling or chemical modification.

AgNP-DNA@GQDs hybrid: new approach for sensitive detection of H2O2 and glucose via simultaneous AgNP etching and DNA cleavage.

A growing body of evidence suggests that hydrogen peroxide (H2O2) plays an active role in the regulation of various physiological processes. Development of sensitive probes for H2O2 is an urgent work. In this study, we proposed a DNA-mediated silver nanoparticle and graphene quantum dot hybrid nanocomposite (AgNP-DNA@GQDs) for sensitive fluorescent detection of H2O2. The sensing mechanism is based on the etching effect of H2O2 to AgNPs and the cleavage of DNA by as-generated hydroxyl radicals (•OH). The formation of AgNP-DNA@GQDs nanocomposite can result in fluorescence quenching of GQDs by AgNPs through the resonance energy transfer. Upon H2O2 addition, the energy transfer between AgNPs and GQDs mediated by DNA was weakened and obvious fluorescence recovery of GQDs could be observed. It is worth noting that the reaction product •OH between H2O2 and AgNPs could cleave the DNA-bridge and result in the disassembly of AgNP-DNA@GQDs to achieve further signal enhancement. With optimal conditions, the approach achieves a low detection limit of 0.10 μM for H2O2. Moreover, this nanocomposite is further extended to the glucose sensing in human urine combining with glucose oxidase (GOx) for the oxidation of glucose and formation of H2O2. The glucose concentrations in human urine are detected with satisfactory recoveries of 94.6-98.8% which holds potential for ultrasensitive quantitative analysis of glucose and supplies valuable information for diabetes mellitus research and clinical diagnosis.

A Novel Electrochemical Biosensor Based on a Modified Gold Electrode for Hydrogen Peroxide Determination in Different Beverage Samples

In the present work, an electrochemical biosensor is constructed based on a Nafion nano composite polymer, toluidine blue (TB) and catalase enzyme-modified gold electrode (AuE). The TB molecules were strongly adsorbed on the Nafion/AuE. The electrochemical properties of this biosensor were examined. Cyclic voltammetry was used at various scan rates to investigate the redox properties of the Nafion and TB-modified AuE (Nafion/TB/AuE). The electron transfer coefficient, α, and the electron transfer rate constant, k s, were found to be 0.48 and 12.1 ± 0.3 s−1 in pH 7.0, respectively. The Nafion, TB, and catalase-modified AuE (Nafion/TB/catalase/AuE) exhibited excellent electrocatalytic response to the reduction of hydrogen peroxide (H2O2). Using cyclic voltammetry, kinetic parameters such as electron transfer coefficient, α, and heterogeneous rate constant, k’, were determined for the reduction of H2O2 at this biosensor surface. Differential pulse voltammetry exhibited two linear dynamic ranges and a detection limit of 0.25 μM for H2O2.

Fe3O4 peroxidase mimetics as a general strategy for the fluorescent detection of H2O2-involved systems

Enzyme mimetics have recently attracted considerable interest because of their high stability and low cost. We developed a general H2O2-involved fluorescence system using Fe3O4 magnetic microspheres as peroxidase mimetics and benzoic acid (BA) as indicator. Glucose and p-nitrophenol were used as models to determine the characteristics and effectiveness of the system. Glucose oxidase hydrolyzes glucose, in the presence of oxygen, to H2O2 followed by the activation of Fe3O4 MMs, resulting in the catalyzed oxidation of benzoic acid. Glucose can be determined by the quantitative fluorescence production. p-Nitrophenol is determined as model compounds which competes with benzoic acid for H2O2 resulting in the decreased catalytic oxidation of benzoic acid with the Fe3O4 MMs. The detection limit of the Fe3O4/H2O2/BA system is 0.008μM for H2O2, 0.025μM for glucose and 0.05μM for p-nitrophenol. Furthermore, the system had high sensitivity, good selectivity and was capable of sensing glucose in human serum and p-nitrophenol in water samples. The proposed system has great potential in the chemical/biological sensing of a variety of analytes associated with reactions that produce or consume H2O2.

A nitrite and hydrogen peroxide sensor based on Hb adsorbed on Au nanorods and graphene oxide coated by polydopamine

A novel hemoglobin (Hb) electrode modified with Au nanorods (AuNRs) and graphene oxide sheets (GOs) coated by polydopamine (Pdop) (GOs@Pdop) was fabricated and used for highly selective and sensitive determination of nitrite (NO2−) and hydrogen peroxide (H2O2). The electrochemical characteristics of the sensor were studied by cyclic voltammetry and chronoamperometry. The GOs@Pdop composite was characterized by Fourier transform infrared (FTIR), ultraviolet-vis absorption spectroscopy (UV-vis) and transmission electron microscopy (TEM). The system was optimized to realize a reliable determination of NO2− at 0.7 V with a detection limit of 2.5 × 10−8 M and H2O2 at –0.2 V with a detection limit of 2.0 × 10−6 M (S/N = 3). The apparent Michaelis–Menten constants (KappM) were calculated to be 0.13 mM for NO2− and 0.70 mM for H2O2. Moreover, the sensor obtained from this study exhibited high sensitivity, good reproducibility, and long-term stability.

Horseradish peroxidase and chitosan: Activation, immobilization and comparative results

Recently, horseradish peroxidase (HRP) was immobilized on activated wool and we envisioned that the use of chitosan would be interesting instead of wool owing to its simple chemical structure, abundant nature and biodegradability. In this work, HRP was immobilized on chitosan crosslinked with cyanuric chloride. FT-IR spectroscopy and scanning electron microscopy were used to characterize immobilized HRP. The number of ten reuses of immobilized HRP has been detected. The pH was shifted from 5.5 for soluble HRP to 5.0 for immobilized enzyme. The soluble HRP had an optimum temperature of 30°C, which was shifted to 35°C for immobilized enzyme. The soluble HRP and immobilized HRP were thermal stable up to 35 and 45°C, respectively. The apparent kinetic constant values (Km) of soluble HRP and chitosan–HRP were 35mM and 40mM for guaiacol and 2.73mM and 5.7mM for H2O2, respectively. Immobilization of HRP partially protected them from metal ions compared to soluble enzyme. The chitosan–HRP was remarkably more stable against urea, Triton X-100 and organic solvents. Chitosan–HRP exhibited large number of reuses and more resistance to harmful compounds compared with wool–HRP. On the basis of results obtained in the present study, chitosan–HRP could be employed in bioremediation application.

Immobilization of horseradish peroxidase on activated wool

This work is aimed to immobilize partially purified horseradish peroxidase (HRP) on wool activated by multifunctional reactive center, namely cyanuric chloride. The effect of cyanuric chloride concentration, pH and enzyme concentration on immobilization of HRP was studied. FT-IR and SEM analyses were detected for wool, activated wool and immobilized wool-HRP. The wool-HRP, prepared at 2% (w/v) cyanuric chloride and pH 5.0, retained 50% of initial activity after seven reuses. The wool-HRP showed broad optimum pH at 7.0 and 8.0, which was higher than that of the soluble HRP (pH 6.0). The soluble HRP had an optimum temperature of 30°C, which was shifted to 40°C for immobilized enzyme. The soluble and wool-HRP were stable up to 30 and 40°C after incubation for 1h, respectively. The apparent kinetic constant values (Kms) of wool-HRP were 10mM for guiacol and 2.5mM for H2O2, which were higher than that of soluble HRP. The wool-HRP was remarkably more stable against proteolysis mediated by trypsin. The wool-HRP exhibited more resistance to heavy metal induced inhibition. The wool-HRP was more stable to the denaturation induced by urea, Triton X-100, isopropanol, butanol and dioxan. The wool-HRP was found to be the most stable under storage. In conclusion, the wool-HRP could be more suitable for several industrial and environmental purposes.

Detoxification of Pesticides Aqueous Solution Using Horseradish Peroxidase

There are pesticide residues in agriculture wastewater and that compounds must be removed before discharge of wastewater in native waters. Thus the aim of this study was to remove toxic pesticide in waste water by the addition of horseradish peroxidase enzyme. The process of pesticide (methyl-parathion (O,O-Diethyl- O-4-nitro-phenylthiophosphate), atrazine (1-chloro-3-ethylamino-5-isopropylamino-2,4,6-triazine) and triazophos (O,O-diethyl O-1-phenyl-1H-1,2,4- triazol-3-yl phosphorothioate) removal from synthetic wastewater using horseradish peroxidase and hydrogen peroxide has been analyzed. The technical feasibility of the process was studied using 0.001-3.0 mM synthetic pesticides solutions. Experiments were carried out at different time, HRP and H2O2 dose and pH to determine the optimum removing conditions. The removal of the three pesticides increases with an increase in HRP and hydrogen peroxide dose. The optimum HRP dose is 2.0 U L(-1) and 10 mM for H2O2. The contact needed to reach equilibrium was found to be 360 min. Maximum removal was achieved up to 74% at pH 8. Also, Chemical Oxygen Demand (COD) of the effluent reduced at the end of 6 h from 2111-221 mg L(-1) (at pH 8). Tests based upon horseradish peroxidase, at optimized parameters, show the reduction of toxicity to non-toxic levels.

Synthesis of Small-Sized Freestanding Co3O4 Nanosheets with Improved Activity for H2O2 Sensing and Oxygen Evolution

Small-sized freestanding Co3O4 nanosheets have been successfully synthesized on conductive substrate via a novel and simple electrochemical-based strategy. The as-synthesized sheets have a small size of 30 ± 10 nm in edge length and about 10 ± 5 nm in thickness with most sheets lying perpendicular to the substrate. The combined properties of products such as small size, two-dimension shape and freestanding pose could lead to integrative advantages of high surface-to-volume ratio, the maximized exposed surfaces without particles aggregation, strong affinity and good conductivity. As for electrochemical tests, the as-synthesized Co3O4 nanosheets show an improved sensitivity of ∼0.56 A M−1 cm−2 with lower detection limit of 1 μM at concentration range of 0–1.8 mM for H2O2 sensing, and also exhibit good performance for oxygen evolution with low evolution over-potential, high catalytic efficiency and durable stability.

Nanostructured biosensors built with layer-by-layer electrostatic assembly of hemoglobin and Fe3O4@Pt nanoparticles

This paper reports layer-by-layer (LBL) films fabricated with hemoglobin and core–shell nanoparticles with Fe3O4 as the core covered by Pt (Fe3O4@Pt) and their applications in biosensing. The characterization of {Hb/Fe3O4@Pt}n LBL films at different layers revealed that the formation of films is step-by-step and uniform. Meanwhile, at glassy carbon electrodes modified with {Hb/Fe3O4@Pt}n film at different layers there was a pair of well-defined and nearly reversible peaks in cyclic voltammetry (CV). CV results indicated that the electroactivity of the structure with four bilayers was the best. The {Hb/Fe3O4@Pt}4 film modified electrode could be used to detect H2O2 and nitrite with the linear range from 0.125μM to 0.16mM for H2O2 and 1.5μM to 0.12mM for nitrite as well as the detection limits of 0.03μM for H2O2 and 0.29μM for nitrite (S/N=3). The biosensors also exhibited good reproducibility, high selectivity, and long-term stability. Our investigation showed that the strategy taking advantages of Fe3O4@Pt and LBL assembly is ideal for direct electrochemistry of redox proteins as well as the sensitive and stable mediator-free biosensors.

Direct Electrochemistry and Application in Electrocatalysis of Hemoglobin in a Polyacrylic Resin‐Gold Colloid Nanocomposite Film

AbstractA novel nanocomposite integrating the good biocompatibility of polyacrylic resin nanoparticles (PAR) and the good conductivity of colloidal gold nanoparticles was proposed to construct the matrix for the immobilization of hemoglobin (Hb) on the surface of a glassy carbon electrode (GCE). UV‐vis spectra demonstrated that Hb preserved its native structure after being entrapped into the composite film. The direct electrochemistry of hemoglobin (Hb) in this nanocomposite films showed a pair of well‐defined and quasi‐reversible cyclic voltammetric peaks with a formal potential of −0.307 mV and a constant electron transfer rate of 2.51±0.2 s−1. The resultant amperometric biosensor showed fast responses to the analytes with excellent detection limits of 0.2 µM for H2O2 and 0.89 µM for TCA (S/N=3), and high sensitivity of 1108.6 for H2O2 and 77.14 mA cm−2 M−1 for TCA, respectively. The linear current response was found in the range from 0.59 to 7.3 µM (R2=0.9996) for H2O2 and from 5 to 85 µM (R2=0.9996) for TCA, while the superior apparent Michaelis–Menten constant was 0.012 mM for H2O2 and 0.536 mM for TCA, respectively. Therefore, the PAR‐Au‐Hb nanocomposite as a novel matrix opens up a possibility for further study on the direct electrochemistry of other proteins.

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.

Determination of glucose by flow injection analysis with amperometric detection at Fe(III)-(tris(3,5-dimethyl-1-pyrazolyl)borate)2 modified electrodes

A modified screen printed electrode with (Fe(tris(3,5-di- methyl-1-pyrazolyl)borate)2) + (FeCl4) - , installed as part of a flow in- jection system, was used as an amperometric detector to determine hydrogen peroxide and glucose after on-line reaction with glucose oxi- dase. The influence of experimental variables on sensitivity, such as the flow rate, injection volume and reaction coil length, was evaluated using a two-level factorial experimental design. Under optimal condi- tions, detection limits of 15 µM and 0.5 mM for H2O2 and glucose, respectively, were obtained. A sampling rate of 60 determinations h -1 was achieved; the relative standard deviation of analytical mea- surements was <5.0%. The method was validated by comparing the

Determination of optimum process parameters for peroxidase‐catalysed treatment of bisphenol A and application to the removal of bisphenol derivatives

Systematic investigations were carried out to determine the optimum process parameters such as the hydrogen peroxide (H2O2) concentration, concentration and molar mass of poly(ethylene glycol) (PEG) as an additive, pH value, temperature and enzyme dose for treatment of bisphenol A (BPA) with horseradish peroxidase (HRP). The HRP‐catalysed treatment of BPA was effectively enhanced by adding PEG, and BPA was completely converted into phenoxy radicals by HRP dose of 0.10 U/cm3. The optimum conditions for HRP‐catalysed treatment of BPA at 0.3 mM was determined to be 0.3 mM for H2O2 and 0.10 mg/cm3 for PEG with a molar mass of 1.0 × 104 in a pH 6.0 buffer at 30 °C. Different kinds of bisphenol derivatives were completely or effectively treated by HRP under the optimum conditions determined for treatment of BPA, although the HRP dose was further increased as necessary for some of them. The aggregation of water‐insoluble oligomers generated by the enzymatic radicalization and radical coupling reaction was enhanced by decreasing the pH values to 4.0 with HCl after the enzymatic treatment, and BPA and bisphenol derivatives were removed from aqueous solutions by filtering out the oligomer precipitates.

Synergistic antifungal activity of sodium hypochlorite, hydrogen peroxide, and cupric sulfate against Penicillium digitatum.

Oxidizing compounds such as sodium hypochlorite (NaCIO) and hydrogen peroxide (H2O2) are widely used in food sanitization because of their antimicrobial effects. We applied these compounds and metals to analyze their antifungal activity against Penicillium digitatum, the causal agent of citrus green mold. The MICs were 300 ppm for NaClO and 300 mM for H2O2 when these compounds were individually applied for 2 min to conidia suspensions. To minimize the concentration of these compounds, we developed and standardized a sequential treatment for conidia that resulted in loss of viability on growth plates and loss of infectivity on lemons. The in vitro treatment consists of preincubation with 10 ppm of NaClO followed by incubation with 100 mM H2O2 and 6 mM CuSO4 (cupric sulfate). The combination of NaClO and H2O2 in the presence of CuSO4 produces a synergistic effect (fractional inhibitory concentration index of 0.36). The sequential treatment applied in situ on lemon peel 24 h after the fruit was inoculated with conidia produced a significant delay in the fungal infection. The in vitro treatment was effective on both imazalil-sensitive and imazalil-resistant strains of P. digitatum and Geotrichum candidum, the causal agent of citrus sour rot. However, this treatment inhibited 90% of mycelial growth for Penicillium italicum (citrus blue mold). These results indicate that sequential treatment may be useful for postharvest control of citrus fruit diseases.

Hydrogen peroxide degradation by immobilized cells of alkaliphilic Bacillus halodurans

Whole cells of Bacillus halodurans LBK 261 were used as a source of catalase for degradation of hydrogen peroxide. The organism, B. halodurans grown at 55°C and pH 10, yielded a maximum catalase activity of 275 U g−1 (wet wt.) cells. The catalase in the whole cells was active over a broad range of pH with a maximum at pH 8–9. The enzyme was optimally active at 55°C, but had low stability above 40°C. The whole cell biocatalyst exhibited a Km of 6.6 mM for H2O2 and Vmax of 707 mM H2O2 min−1 g−1 wet wt. cells, and showed saturation kinetics at 50 mM H2O2. The cells were entrapped in calcium alginate and used for H2O2 degradation at pH 9 in batch and continuous mode. In the batch process, the immobilized preparation containing 1.5 g (wet wt.) cells could be recycled at least four times for complete degradation of the peroxide in 50 mL solution at 25°C. An excess of immobilized biocatalyst could be used in a continuous stirred tank reactor for an average of 9 days at temperatures upto 55°C, and in a packed bed reactor (PBR) for 5 days before the beads started to deform.

Radical induced damage ofMicrococcus luteusbacteria monitored using FT‒IR spectroscopy

Oxidative damage induced by ascorbic acid (AA) and hydrogen peroxide (H2O2) was monitored by Fourier transform infrared spectroscopy (FT‒IR); it appeared as a rapid and convenient means to follow the biochemical changes generated in the culture media of the yellow-pigmentedMicrococcus luteus. Beyond a threshold of 20 mM for AA and of 40 mM for H2O2(final concentration), antioxidant systems were overwhelmed and significant changes were observed in the bacterial spectra, particularly in the 1430–900 cm−1region; this spectral window provided large information about carboxylate groups, phosphate-carrying compounds and polysaccharides implicated in the radical process. The spectroscopic results indicated that for the same final concentration, the toxicity of H2O2was less important than that of AA towardM. luteuscells, although H2O2had a more damaging effect on proteins. Thus, FT-IR spectroscopy was an appropriate physico-chemical tool suitable in biochemical and clinical research for early characterization of any type of radical aggression, and for rapid detection of the damage intensity.

5-Aminosalicylic acid prevents oxidant mediated damage of glyceraldehyde-3-phosphate dehydrogenase in colon epithelial cells

BackgroundReactive oxygen and nitrogen derived species produced by activated neutrophils have been implicated in the damage of mucosal proteins including the inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the active inflammatory...

The effect of polychlorophenols on the content of protein fractions as well as on enzyme activities and substrate concentrations of bovine lenses in vitro

The lactoperoxidase-catalyzed transformations of penta-, 2,3,4,6,-tetra-, 2,4,6,-tri, 2,4,-di- and 4-monochlorophenol were followed spectrophotometrically. Apparent stoichiometries of chlorophenol: H2O2 ranged from 1:1 for the tri- and tetrachlorophenol at pH 7 to 5:2 for pentachlorophenol at pH 4. The initial velocity (ν0) was only slightly influenced by changes in [H2O2] ⩾ 5 μM. ν0 responded to [chlorophenol] according to the empirical expression ν0 = [lactoperoxidase]·(k1[chlorphenol] + k2[chlorophenol]2). The constant k1 was found to be 5.8 · 105, 1.8 · 106, 1.9 · 106 M−1 · s−1 for the protonated forms of penta-, tetra- and trichlorophenol, respectively, at pH 7. With the di- and monochlorophenol the solution soon became opaque, and the reaction ceased. The results show that more than one reaction occurs. Some comparisons were also made with horseradish peroxidase A and C. Cetyltrimethylammonium bromide prevented opaqueness, but was shown to be a substrate for lactoperoxidase. Assuming an average concentration of 0.1 μM for H2O2 and pentachlorophenol in man, the metabolic rate becomes 30 ng/h per g peroxidase-containing tissue, possibly with deposition of the products.

The influence of dexamethasone disodium phosphate on the regeneration of corneal endothelium in vitro

The lactoperoxidase-catalyzed transformations of penta-, 2,3,4,6,-tetra-, 2,4,6,-tri, 2,4,-di- and 4-monochlorophenol were followed spectrophotometrically. Apparent stoichiometries of chlorophenol: H2O2 ranged from 1:1 for the tri- and tetrachlorophenol at pH 7 to 5:2 for pentachlorophenol at pH 4. The initial velocity (ν0) was only slightly influenced by changes in [H2O2] ⩾ 5 μM. ν0 responded to [chlorophenol] according to the empirical expression ν0 = [lactoperoxidase]·(k1[chlorphenol] + k2[chlorophenol]2). The constant k1 was found to be 5.8 · 105, 1.8 · 106, 1.9 · 106 M−1 · s−1 for the protonated forms of penta-, tetra- and trichlorophenol, respectively, at pH 7. With the di- and monochlorophenol the solution soon became opaque, and the reaction ceased. The results show that more than one reaction occurs. Some comparisons were also made with horseradish peroxidase A and C. Cetyltrimethylammonium bromide prevented opaqueness, but was shown to be a substrate for lactoperoxidase. Assuming an average concentration of 0.1 μM for H2O2 and pentachlorophenol in man, the metabolic rate becomes 30 ng/h per g peroxidase-containing tissue, possibly with deposition of the products.