Defect and Additional Active Sites on the Basal Plane of Manganese-Doped Molybdenum Diselenide for Effective Enzyme Immobilization: In Vitro and in Vivo Real-Time Analyses of Hydrogen Peroxide Sensing.

The defect engineering makes the new concepts and designs to further enhance the electrocatalytic activity of layered structures. In this work, we demonstrated the synthesis of Mn-doped MoSe2 and reported the resultant defective sites. Subsequently, the MnMoSe2 was developed as a new type of electrocatalyst for electrochemical biosensors. The formation of defect/distortion and effective immobilization of myoglobin (Mb) were evidently confirmed by using the transmission electron microscopy and UV-vis spectroscopy analyses, respectively. The result of electrochemical impedance spectroscopy analysis reveals that the Mn doping not only helps  to enzyme immobilization but also enhances the electronic conductivity of layered material.  Owing to the multiple signal amplification strategies, the proposed Mb-immobilized MnMoSe2 (Mb@MnMoSe2) exhibited an ultralow detection limit (0.004 µM) and a higher sensitivity (222.78 µA µM-1 cm-2) of H2O2. In real-sample analysis, the Mb@MnMoSe2 showed a feasible recovery range of H2O2 detection in human serum (95.6-102.1%), urine (101.2-102.3%), and rain water (100.7-102.1%) samples. On the other hand, an in vivo study using HaCaT (7.1 × 105/mL) and RAW 264.7 (1 × 106/mL) living cells showed the feasible current responses of 0.096 and 0.085 µA, respectively. Finally, the Mn doping gives a new opportunity to fabricate a promising electrocatalyst for H2O2 biosensing.

A new 3D printed radial flow-cell for chemiluminescence detection: Application in ion chromatographic determination of hydrogen peroxide in urine and coffee extracts.

A new polymer flow-cell for chemiluminescence detection (CLD) has been designed and developed by diverging multiple linear channels from a common centre port in a radial arrangement. The fabrication of radial flow-cell by 3D PolyJet printing and fused deposition modeling (FDM) has been evaluated, and compared with a similarly prepared spiral flow-cell design commonly used in chemiluminescence detectors. The radial flow-cell required only 10 h of post-PolyJet print processing time as compared to ca. 360 h long post-PolyJet print processing time required for the spiral flow-cell. Using flow injection analysis, the PolyJet 3D printed radial flow-cell provided an increase in both the signal magnitude and duration, with an average increase in the peak height of 63% and 58%, peak area of 89% and 90%, and peak base width of 41% and 42%, as compared to a coiled-tubing spiral flow-cell and the PolyJet 3D printed spiral flow-cell, respectively. Computational fluid dynamic (CFD) simulations were applied to understand the origin of the higher CLD signal obtained with the radial flow-cell design, indicating higher spatial coverage near the inlet and lower linear velocities in the radial flow-cell. The developed PolyJet 3D printed radial flow-cell was applied in a new ion chromatography chemiluminescence based assay for the detection of H2O2 in urine and coffee extracts.

Use of crude extract of lentil plant (Lens culinaris Medikus) in peroxidase-based analyses: fast kinetic determination of hydrogen peroxide and sarcosine in urine.

Peroxidase-catalysed reactions are used in a wide variety of analytical applications, most of them based on the final quantification of hydrogen peroxide. Clinical tests for glucose, cholesterol, creatine, creatinine or uric acid in blood or urine and enzyme-linked immunosorbent assays for pesticides, hepatitis or acquired immune deficiency syndrome are good examples of such applications. The most widely used and commercially available peroxidase for biotechnological processes and analytical applications is horseradish peroxidase followed, although in much lower proportion, by soybean peroxidase. The high commercial interest in peroxidases has led to the search for new sources of these enzymes. This work describes the analytical use of lentil plant peroxidase (LPP), which is a new peroxidase extracted from lentil plants (Lens culinaris Medikus); an abundant post-harvest agricultural waste in the area of Castilla y León (Spain). A procedure for the quantification of hydrogen peroxide in urine is first proposed using crude extract of lentil plant instead of the purified enzyme. This procedure is then applied to the determination of sarcosine; a natural amino acid that has attracted considerable interest in clinical diagnostics since urinary sarcosine was proposed and later questioned as a biomarker for prostate cancer. Under the action of sarcosine oxidase, sarcosine is oxidized by molecular oxygen to give glycine, formaldehyde and hydrogen peroxide that is quantified according to the previously proposed procedure. The limit of detection for both hydrogen peroxide and sarcosine is around 5 × 10(-7) M. In the determination of sarcosine, the high selectivity of the overall enzymatic reaction, the simple sample treatment and instrumentation, the high-sample throughput and the use of LPP in the plant extract instead of the purified enzyme provide a rapid and inexpensive procedure with characteristics very suitable for routine analysis in a clinical laboratory.

Functionalization of carbon buckypaper for the sensitive determination of hydrogen peroxide in human urine.

Here we report on a new approach for the electrochemical detection of hydrogen peroxide (H(2)O(2)) based on the co-immobilization of horseradish peroxidase and methylene blue on the functionalized carbon buckypaper supported by a titanium substrate. Cyclic voltammetry was used to study and optimize the performance of the resulting electrochemical biosensor. The proposed biosensor exhibited high analytical performance towards the quantification of H(2)O(2) at the physiological pH 7.4. Under optimized conditions, the biosensor shows a wide linear response range from 0.1 × 10(-6) to 5 × 10(-4)M concentrations of H(2)O(2). The detection limit was determined to be 7.5 × 10(-8)M (based on S/N=3). Reproducibility and stability of the fabricated biosensor were examined with satisfactory results. The biological relevance of the developed electrochemical biosensor has been further studied by the determination of H(2)O(2) in human urine samples of normal volunteers prior to and following the ingestion of coffee. Increased levels of urinary H(2)O(2) concentration suggest that oxidative stress is induced by coffee drinking in humans. There is considerable interest in oxidative stress as relates to human physiology. The sensitive determination of H(2)O(2) in human urine may serve as a valuable biomarker to effectively elucidate specific levels of oxidative stress in vivo.

Effect of oestrogen on reactive oxygen species production in the aortas of ovariectomized Dahl salt-sensitive rats.

OBJECTIVE: In the present study, we examined whether ovariectomy increases reactive oxygen species (ROS) and the expression of nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase and modulates the scavenger enzymes for ROS in the aortas of Dahl salt-sensitive (DSS) rats fed a high salt diet. METHODS: DSS female rats were ovariectomized and fed a high salt diet (8% NaCl), or a high salt diet plus oestrogen supplement for 4 weeks. Urinary levels of hydrogen peroxide (H2O2) were measured by using 2',7'-dichlorofluorescein. The expression of an NADPH oxidase subunit p22phox, extracellular superoxide dismutase (ecSOD), glutathione peroxidase (GPx)1, GPx4 and monocyte chemoattractant protein 1 (MCP-1) messenger RNA was assessed by reverse transcription-polymerase chain reaction. The expression of MCP-1, and macrophage infiltration were examined by immunohistochemical analysis. RESULTS: Ovariectomy increased superoxide production and the expression of NADPH oxidase subunit p22phox mRNA and protein in the aortas of DSS rats fed a high salt diet. In contrast, ovariectomy reduced the expression of ecSOD mRNA and protein and the expression of GPx1 and GPx4 mRNA in the aorta. Ovariectomy increased MCP-1 mRNA and protein expression and ED1-positive cells in the aorta. CONCLUSIONS: Ovariectomy leads to an amplification of oxidative stress in DSS rats fed a high salt diet synergistically by an increase in the ROS-generating system and a decrease in the ROS-eliminating system, as shown in the increase in superoxide production and the urinary excretion of H2O2. Oestrogen supplementation counteracted these alterations, showing how oestrogen is antioxidative.

Effects of thiol antioxidant on reduced nicotinamide adenine dinucleotide phosphate oxidase in hypertensive Dahl salt-sensitive rats.

Recent studies implicate of reactive oxygen species (ROS) in hypertension; however, whether reactive oxygen species promote hypertensive derangements is not fully clear. We thus investigated the effects of an antioxidant, N-acetyl-L-cysteine, on hypertensive Dahl salt-sensitive rats. High-salt intake for 4 weeks markedly elevated systolic arterial pressure, urinary excretion of protein, 8-isoprostane, and H(2)O(2), and the enzyme activity of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase along with the elevated expression of its subunits gp91phox and p47phox at the levels of mRNA and protein. Supplement with N-acetyl-L-cysteine reduced the increase in systolic arterial pressure and counteracted the elevation of urinary excretion of protein, 8-isoprostane, and H(2)O(2), and the increases in NADPH oxidase activity/expression in high-salt-loaded Dahl salt-sensitive rats. N-acetyl-L-cysteine supplement ameliorated plasma and urinary levels of thromboxane B(2) (an end metabolite of thromboxane A(2)), associated with improvement of both the abnormal contraction and the impaired nitric oxide-dependent relaxation in renal arteries. These results revealed that oxidative stress mediates hypertensive changes in Dahl salt-sensitive rats, because thiol antioxidant N-acetyl-L-cysteine attenuated the augmentation of local ROS production by diminishing the elevation of NADPH oxidase expression and ameliorated renal/vascular hypertensive changes.

Establishing biomarkers of oxidative stress: the measurement of hydrogen peroxide in human urine.

Hydrogen peroxide (H(2)O(2)) can be detected in freshly-voided human urine from healthy subjects and has been proposed as a "biomarker" of oxidative stress. This paper summarizes our studies to examine the extent to which urinary H(2)O(2) measurement fulfils the criteria for the "ideal biomarker". Levels of H(2)O(2), standardised for creatinine, varied widely between subjects. In most subjects, levels also varied considerably when measurements were made at different times and on different days. A reproducible increase in urinary H(2)O(2) was detected in all subjects examined after drinking coffee, a beverage rich in H(2)O(2). By contrast, green tea decreased urinary H(2)O(2) levels. We conclude that the H(2)O(2) in coffee is not excreted into urine. Instead, hydroxyhydroquinone from coffee is absorbed, excreted and oxidises in urine to produce H(2)O(2). No other confounders of urinary H(2)O(2) have been identified to date. Work is underway to compare H(2)O(2) levels with variations in other biomarkers of oxidative damage, to test the possibility that there are daily or other periodic variations in oxidative damage rates.

Increased urinary hydrogen peroxide levels caused by coffee drinking.

Experiments with volunteers in Singapore have demonstrated that coffee drinking increases urinary hydrogen peroxide levels (Long, Halliwell, Free Rad. Res., 32, 463-467 (2000)). We re-examined the effect of coffee drinking of healthy Japanese subjects on urinary hydrogen peroxide levels. A cup of brewed or canned coffee commercially available in Japan generated 120-420 micro mol hydrogen peroxide in incubation in a neutral medium at 37 degrees C for 6 h. The increased levels were higher than those obtained from a cup of green tea extract or a glass of red wine. After the subject drank a cup of coffee, apparent hydrogen peroxide levels (micro mol/g creatinine) in urine collected 1-2 h after coffee drinking increased 3-10-fold compared to the levels before coffee drinking. The increased urinary hydrogen peroxide levels are likely derived mainly from 1,2,4-benzenetriol excreted in urine, because the major component that generates hydrogen peroxide is found to be 1,2,4-benzenetriol, and storing urine collected after coffee drinking increased hydrogen peroxide levels in a time-dependent fashion. Total hydrogen peroxide equivalent levels excreted in 3 h-urine after coffee drinking were estimated to be 0.5-10% that of coffee consumed. A residual amount of hydrogen peroxide may be retained or consumed in human bodies.

Multivitamin solutions for enteral supplementation: a source of peroxides.

OBJECTIVE: We investigated whether solutions of enteral vitamin supplementation are involved in the generation of peroxides and whether that contamination is biologically significant. METHODS: Peroxide contents of oral multivitamin preparations were measured over 3 wk after the initial opening of the containers. In selected premature infants (younger than 35 wk gestation), urinary peroxides were measured after initiating oral multivitamin supplementation. RESULTS: Peroxides in multivitamin solutions for enteral use are predominantly organic peroxides because they resist catalase. After the initial opening of the containers, there was a two-fold increase in total peroxides levels (P < 0.05) even in the preparation without riboflavin, a catalyst for the generation of peroxides. Initiation of oral vitamin supplementation was associated with increased (P < 0.05) urine peroxide levels. The high organic peroxide load did not correlate with its urinary excretion, mostly in the form of H(2)O(2). The excretion of H(2)O(2) corresponded to its oral intake from the multivitamin solution. CONCLUSIONS: Compared with parenteral multivitamin solutions, the enteral preparations contained higher organic peroxide levels starting with the initial opening of the bottles. The increased urinary excretion of H(2)O(2) after enteral multivitamin supplementation suggested a systemic diffusion of peroxides or of components of the multivitamin preparation responsible for the generation of peroxides. This oxidant load was not quenched by the immature antioxidant defenses of premature infants.

Coffee drinking increases levels of urinary hydrogen peroxide detected in healthy human volunteers.

Freshly-voided human urine contains significant concentrations of hydrogen peroxide (H2O2). This H2O2 appears to arise in whole or in part by superoxide-dependent autoxidation of urinary biomolecules. Since instant coffee also contains high levels of H2O2, we examined the effect of coffee drinking on urinary levels of H2O2. Studies on healthy human volunteers showed that coffee drinking is rapidly and reproducibly followed by increased levels of H2O2 detectable in the urine for up to 2 h after drinking the coffee. The levels of H2O2 detected in urine suggest that exposure of human tissues to H2O2 may be greater than is commonly supposed. It is possible that H2O2 in urine could act as an antibacterial agent, and that H2O2 is involved in the regulation of glomerular function.