Simultaneous Determination of Glucose and L‐Glutamate Using a Capillary Enzyme Reactor with Electrochemical Detection

An electrophoresis capillary that incorporates two enzymes for the simultaneous determination of glucose and L ‐glutamate is described. The enzymes deposited along the separation capillary walls react with their respective substrate as they are separated during the electrophoresis to produce hydrogen peroxide that is detected by amperometry as the hydrogen peroxide zone emerges from the end of the capillary. Even though both enzyme reactions produce a common product, hydrogen peroxide, the hydrogen peroxide produced by each enzyme reaction stays in narrow zones that migrate the length of the capillary at different rates. The rate of migration for the individual H₂O₂ zones is consistent with the expected mobility of neutral glucose and of anionic L ‐glutamate, respectively. This property allows each enzyme substrate to be characterized in a single experiment and in the presence of other electroactive substances.     

Protective Roles of Honey in Reproductive Health: A Review

Nowadays, most people who lead healthy lifestyles tend to use natural products as supplements, complementary medicine or alternative treatments. Honey is God’s precious gift to mankind. Honey has been highly appreciated and extensively used since ancient history due to its high nutritional and therapeutic values. It is also known to enhance fertility. In the last few decades, the important role of honey in modern medicine has been acknowledged due to the large body of convincing evidence derived from extensive laboratory studies and clinical investigations. Honey has a highly complex chemical and biological composition that consists of various essential bioactive compounds, enzymes, amino and organic acids, acid phosphorylase, phytochemicals, carotenoid-like substances, vitamins and minerals. Reproductive health and fertility rates have declined in the last 30 years. Therefore, this review aimed to highlight the protective role of honey as a potential therapeutic in maintaining reproductive health. The main role of honey is to enhance fertility and treat infertility problems by acting as an alternative to hormone replacement therapy for protecting the vagina and uterus from atrophy, protecting against the toxic effects of xeno-oestrogenic agents on female reproductive functions and helping in the treatment of gynaecological disorders, such as vulvovaginal candidiasis infection, that affect women’s lives.     

Simultaneous microchip enzymatic measurements of blood lactate and glucose

A miniaturized capillary electrophoretic (CE) microchip device for the simultaneous measurements of lactate and glucose is described. The new microchip bioassay protocol integrates an electrophoretic separation of lactate and glucose, post-column enzymatic reactions of these metabolites with their respective oxidase enzymes, and an amperometric (anodic) detection of enzymatically-liberated hydrogen peroxide at a gold-coated thick-film carbon detector. Factors influencing the response have been examined and optimized, and the analytical performance has been characterized. Applicability of the microchip assay to clinical samples, such as serum and blood, is demonstrated. The microchip protocol obviates cross enzymatic reactions and interferences from major oxidizable constituents common to dual glucose-lactate enzyme electrodes. Such ability to rapidly separate and quantitate lactate and glucose on a small microchip platform should find important clinical and biotechnological applications.     

Recoverable solution reaction of HiPco carbon nanotubes with hydrogen peroxide

There is increasing interest in developing single-walled carbon nanotubes (SWNTs)-based optical biosensors for remote or in vitro and in vivo sensing because the near-IR optical properties of SWNTs are very sensitive to surrounding environmental changes. Many enzyme-catalyzed reactions yield hydrogen peroxide (H(2)O(2)) as a product. To our knowledge, there is no report on the interaction of H(2)O(2) with SWNTs from the optical sensing point of view. Here, we study the reaction of H(2)O(2) with an aqueous suspension of water-soluble (ws) HiPco SWNTs encased in the surfactant sodium dodecyl sulfate (SDS). The SWNTs are optically sensitive to hydrogen peroxide in pH 6.0 buffer solutions through suppression of the near-IR absorption band intensity. Interestingly, the suppressed spectral intensity of the nanotubes recovers by increasing the pH, by decomposing the H(2)O(2) into H(2)O and O(2) with the enzyme catalase, and by dialytically removing H(2)O(2). Preliminary studies on the mechanisms suggest that H(2)O(2) withdraws electrons from the SWNT valence band by charge transfer, which suppresses the nanotube spectral intensity. The findings suggest possible enzyme-assisted molecular recognition applications by selective optical detection of biological species whose enzyme-catalyzed products include hydrogen peroxide.     

Protein release from interpenetrating polymer network hydrogels triggered by endogenous biomarkers

A biocompatible drug delivery system with a high-sensitive stimuli-responsive behavior is reported. Calcium alginate hydrogels interpenetrated with polyvinyl alcohol–diboronate polymer network (IPN) effectively respond to the presence of hydrogen peroxide through oxidative degradation of boronate esters. The degradation of the IPN entails the reopening of the original alginate pores, resulting in a 5–9 times increase in release rates of encapsulated proteins with molecular masses ranging from 16.7 to 66 kDa. The release can be triggered by hydrogen peroxide concentrations as low as 50 μM in the bulk solution. Alternatively, hydrogen peroxide can also be generated inside the hydrogels by incorporation of oxidase enzymes in the presence of their substrates, such as lactate, glucose, or hypoxanthine, which can serve as biomarkers of certain physiological disorders.     

Biological Efficacy of Compounds from Stingless Honey and Sting Honey against Two Pathogenic Bacteria: An In Vitro and In Silico Study

Honey inhibits bacterial growth due to the high sugar concentration, hydrogen peroxide generation, and proteinaceous compounds present in it. In this study, the antibacterial activity of stingless and sting honey against foodborne pathogenic bacteria isolated from spoiled milk samples was examined. The isolated bacterial strains were confirmed as Bacillus cereus and Listeria monocytogenes through morphological, biochemical, and 16 s RNA analysis. Physiochemical characterizations of the honey samples revealed that both of the honey samples had an acidic pH, low water content, moderate reducing sugar content, and higher proline content. Through the disc diffusion method, the antibacterial activities of the samples were assayed and better results were observed for the 50 mg/disc honey. Both stingless and sting honey showed the most positive efficacy against Bacillus cereus. Therefore, an in silico study was conducted against this bacterium with some common compounds of honey. From several retrieved constituents of stingless and sting honey, 2,4-dihydroxy-2,5-dimethyl 3(2H)-furan-3-one (furan) and 4H-pyran-4-one,2,3-dihydro of both samples and beta.-D-glucopyranose from the stingless revealed high ligand-protein binding efficiencies for the target protein (6d5z, hemolysin II). The root-mean-square deviation, solvent-accessible surface area, the radius of gyration, root-mean-square fluctuations, and hydrogen bonds were used to ensure the binding stability of the docked complexes in the atomistic simulation and confirmed their stability. The combined effort of wet and dry lab-based work support, to some extent, that the antimicrobial properties of honey have great potential for application in medicine as well as in the food industries.     

Characterization of mediated and non-mediated oxidase enzyme based glassy carbon electrodes

The performance and analytical characteristics of a glassy carbon glutaraldehyde immobilized glucose oxidase electrode have been established with regard to the direct detection of hydrogen peroxide produced from the reaction of glucose with oxygen. Measurements were performed at + 1.1 V vs. SCE, and selectivity was obtained by casting the surface with a cellulose acetate membrane. Results compared favorably with the classical platinum-enzyme probe. The mechanism of ascorbic acid interference in hydrogen peroxide detection is reported. Mediated detection was also investigated for oxidase enzymes (glucose oxidase and xanthine oxidase) immobilized on the bare glassy carbon electrode. The probes were characterized using a specific enzyme mediator in solution (phenazine methosulfate or dichlorophenol-indophenol) plus hexacyanoferrate(III) as an electrochemical mediator. The electrode was poised at + 0.36 V vs. SCE for the detection of hexacyanoferrate(II). The advantages of this dual mediator configuration include high stability and sensitivity of the electrochemical signal and the ability to use less positive potentials for increased selectivity. Application to other enzymes, such as hydrogenases, using such a binary redox configuration is suggested.     

CuS/RGO hybrid by one-pot hydrothermal method for efficient electrochemical sensing of hydrogen peroxide

In this study,a non-enzymatic hydrogen peroxide sensor was successfully fabricated on the basis of copper sulfide nanoparticles/reduced graphene oxide(CuS/RGO) electrocatalyst.Using thiourea as reducing agent and sulfur donor,CuS/RGO hybrid was synthesized through a facile one-pot hydrothermal method,where the reduction of GO and deposition of CuS nanoparticles on RGO occur simultaneously.The results confirmed that the CuS/RGO hybrid helps to prevent the aggregation of CuS nanoparticles.Electrochemical investigation showed that the as-prepared hydrogen peroxide sensor exhibited a low detection limit of 0.18μmol/L(S/N = 3),a good reproducibility(relative standard deviation(RSD) of4.21%),a wide linear range(from 3 to 1215 μmol/L) with a sensitivity of 216.9 μA L/mmol/cm~2 under the optimal conditions.Moreover,the as-prepared sensor also showed excellent selectivity and stability for hydrogen peroxide detection.The excellent performance of CuS/RGO hybrid,especially the lower detection limit than certain enzymes and noble metal nanomaterials ever reported,makes it a promising candidate for non-enzymatic H_2O_2 sensors.     

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.     

Fructose might contribute to the hypoglycemic effect of honey

Honey is a natural substance with many medicinal properties, including antibacterial, hepatoprotective, hypoglycemic, antioxidant and antihypertensive effects. It reduces hyperglycemia in diabetic rats and humans. However, the mechanism(s) of its hypoglycemic effect remain(s) unknown. Honey comprises many constituents, making it difficult to ascertain which component(s) contribute(s) to its hypoglycemic effect. Nevertheless, available evidence indicates that honey consists of predominantly fructose and glucose. The objective of this review is to summarize findings which indicate that fructose exerts a hypoglycemic effect. The data show that glucose and fructose exert a synergistic effect in the gastrointestinal tract and pancreas. This synergistic effect might enhance intestinal fructose absorption and/or stimulate insulin secretion. The results indicate that fructose enhances hepatic glucose uptake and glycogen synthesis and storage via activation of hepatic glucokinase and glycogen synthase, respectively. The data also demonstrate the beneficial effects of fructose on glycemic control, glucose- and appetite-regulating hormones, body weight, food intake, oxidation of carbohydrate and energy expenditure. In view of the similarities of these effects of fructose with those of honey, the evidence may support the role of fructose in honey in mediating the hypoglycemic effect of honey.     

Comparison of palladium-magnesium nitrate and ammonium dihydrogenphosphate modifiers for cadmium determination in honey samples by electrothermal atomic absorption spectrometry

The occurrence of certain trace elements, such as cadmium, in honey bee can be considered as an indication of environmental pollution. In the present work, two methods for Cd determination in honey by electrothermal atomic absorption spectrometry, using palladium-magnesium nitrate and ammonium dihydrogenphosphate as chemical modifiers, have been developed. In none of these cases honey samples required pretreatment. Honey was diluted in water (20% w/v), and hydrogen peroxide, nitric acid, and Triton X-100 were added to minimize the matrix effect. For the first method, 21.6 μg of palladium and 4.5 μg of magnesium were added as chemical modifier; for the second method, 60 μg of ammonium dihydrogenphosphate were employed. The limits of detection obtained were 0.32 ng g⁻¹ for ammonium dihydrogenphosphate method and 0.54 ng g⁻¹ for palladium-magnesium nitrate method. The characteristic mass was 0.7 and 1.0 pg for ammonium dihydrogenphosphate and palladium-magnesium nitrate methods, respectively. The relative standard deviation (<10%) and the analytical recovery (98-105%) were comparable in both methods. The optimised methods were applied to the determination of the cadmium content in unpolluted Galician honey samples. Cd has been detected the analysed samples in the range (n.d.—4.3 ng g⁻¹).     

Rate-promoting vibrations and coupled hydrogen-electron transfer reactions in the condensed phase: a model for enzymatic catalysis

A model is presented for coupled hydrogen-electron transfer reactions in condensed phase in the presence of a rate promoting vibration. Large kinetic isotope effects (KIEs) are found when the hydrogen is substituted with deuterium. While these KIEs are essentially temperature independent, reaction rates do exhibit temperature dependence. These findings agree with recent experimental data for various enzyme-catalyzed reactions, such as the amine dehydrogenases and soybean lipoxygenase. Consistent with earlier results, turning off the promoting vibration results in an increased KIE. Increasing the barrier height increases the KIE, while increasing the rate of electron transfer decreases it. These results are discussed in light of other views of vibrationally enhanced tunneling in enzymes.     

Query generation to search for inhibitors of enzymatic reactions

A method for the generation of intermediates of enzyme-catalyzed reactions is presented. These intermediates can be used as three-dimensional structural queries for searching for inhibitors of enzymatic reactions. The intermediates can be considered as being structurally quite close to transition-state analogues. For this application, a database containing detailed chemical information on metabolic reactions is used. The likely three-dimensional structure of the intermediates of enzyme-catalyzed reactions can be generated from the information in the database. For three reactions catalyzed by the enzymes AMP deaminase (EC code 3.5.4.6), triose phosphate isomerase (EC code 5.3.1.1), and arginase II (EC code 3.5.3.1), we show how a 3D model of these intermediates can be superimposed onto known inhibitors of these enzymes by a program that uses a genetic algorithm. For this, we test different methods for the superimposition using information on the enzymatic binding site, using information on physicochemical properties calculated from the molecular structure, or without having any information in the superimposition process. We show that these inhibitors are most similar to the corresponding intermediates regarding the 3D structure.     

Thermodynamics of industrially-important,enzyme-catalyzed reactions

The thermodynamics of 10 industrially-important, enzyme-catalyzed reactions are examined. The reactions discussed are: the conversions of penicillin G to 6-amino-penicillinic acid using the enzyme penicillin acylase; starch to glucose using amylases; glucose to fructose using glucose (xylose) isomerase; cellulose to glucose using cellulase; fumaric acid and ammonia to L-aspartic acid using L-aspartase; transcinnamic acid and ammonia to L-phenylalanine using L-phenylalanine ammonia lyase; L-histidine to urocanic acid and ammonia using L-histidine ammonia lyase; lactose to glucose and galactose using lactase; and the reactions catalyzed by amino acylases and proteases. The selection of these processes was based on the economic value of the products and their intrinsic industrial importance. The available thermodynamic properties, such as equilibrium constants, Gibbs energies (delta G degrees), enthalphies (delta H degrees), and heat capacity changes (delta Cp degrees) of these enzyme-catalyzed reactions, are reviewed and summarized. Recommendations are made for future research in this area.     

Enzyme electrode for L-amino acids and glucose

New electrodes based on chemically bound enzymes are described for glucose and L-amino acids. The decrease in the dissolved oxygen content during the enzyme reaction is measured polarographically. The electrodes are stable for at least 4 months and give a response in less than 1 min. These electrodes are more sensitive than the hydrogen peroxide-based electrodes described previously, because the unstable peroxide can be consumed in several ways such as catalase decomposition or reaction with other products.     

Advances in enzyme-free electrochemical sensors for hydrogen peroxide,glucose, and uric acid

Enzyme-free (also called non-enzymatic or direct) electrochemical sensors have been widely used for the determination of hydrogen peroxide, glucose, and uric acid. This review covers the recent progress made in this field. We also discuss the respective sensor materials which have strong effect on the electro-catalytic properties of the electrodes and govern the performance of these sensors. In addition, perspectives and current challenges of enzyme-free electrochemical sensors are outlined. Contains 142 references.

Zn掺杂CuO修饰电极的制备及电化学催化检测H_2O_2

利用滴涂法将Zn掺杂CuO纳米颗粒修饰到玻碳电极表面,通过循环伏安法和计时电流法,研究了Zn掺杂CuO纳米颗粒对H_2O_2的电催化性质。结果表明,制备了粒径为80~100nm的纳米颗粒,且分散良好。Zn掺杂CuO纳米颗粒修饰电极对H_2O_2具有良好的电催化还原作用。在pH=7.0的磷酸盐缓冲液中,当扫描速度介于20~350mV/s时,其氧化峰和还原峰电流均与扫描速度呈线性关系,说明电化学过程受吸附控制。探讨了支持电解质及pH值的影响,当H_2O_2的浓度在1.0×10~(-6)~6.3×10~(-3) mol/L浓度范围内时,还原峰电流与浓度呈良好的线性关系,检出限为0.1μmol/L。该传感器具有选择性高、重现性和稳定性好等特点。     

Chemical Analyses and Antimicrobial Activity of Nine Kinds of Unifloral Chinese Honeys Compared to Manuka Honey (12+ and 20+)

Honey has good antimicrobial properties and can be used for medical treatment. The antimicrobial properties of unifloral honey varieties are different. In this study, we evaluated the antimicrobial and antioxidant activities of nine kinds of Chinese monofloral honeys. In addition, headspace gas chromatography-ion mobility spectrometry (HS-GC-IMS) technology was used to detect their volatile components. The relevant results are as follows: 1. The agar diffusion test showed that the diameter of inhibition zone against Staphylococcus aureus of Fennel honey (21.50 ± 0.41 mm), Agastache honey (20.74 ± 0.37 mm), and Pomegranate honey (18.16 ± 0.11 mm) was larger than that of Manuka 12+ honey (14.27 ± 0.10 mm) and Manuka 20+ honey (16.52 ± 0.12 mm). The antimicrobial activity of Chinese honey depends on hydrogen peroxide. 2. The total antioxidant capacity of Fennel honey, Agastache honey, and Pomegranate honey was higher than that of other Chinese honeys. There was a significant positive correlation between the total antioxidant capacity and the total phenol content of Chinese honey (r = 0.958). The correlation coefficient between the chroma value of Chinese honey and the total antioxidant and the diameter of inhibition zone was 0.940 and 0.746, respectively. The analyzed dark honeys had better antimicrobial and antioxidant activities. 3. There were significant differences in volatile components among Fennel honey, Agastache honey, Pomegranate honey, and Manuka honey. Hexanal-D and Heptanol were the characteristic components of Fennel honey and Pomegranate honey, respectively. Ethyl 2-methylbutyrate and 3-methylpentanoic acids were the unique compounds of Agastache honey. The flavor fingerprints of the honey samples from different plants can be successfully built using HS-GC-IMS and principal component analysis (PCA) based on their volatile compounds. Fennel honey, Agastache honey, and Pomegranate honey are Chinese honey varieties with excellent antimicrobial properties, and have the potential to be developed into medical grade honey.     

Peroxidase activity of new mixed‐valence cobalt complexes with ligands derived from pyridoxal

New mixed‐valence cobalt complexes with ligands derived from pyridoxal were synthesized and characterized, and their application as mimetics of the peroxidase enzyme was investigated. Single‐crystal X‐ray diffraction was used to analyze all complex structures in the solid state and their electrochemical behavior was investigated. A reactivity pattern was observed in the complex synthesis regarding the cobalt compounds from which analogous zwitterionic derivatives were obtained. The importance of these compounds lies in understanding their behavior in an oxidizing environment and evaluating whether they can activate hydrogen peroxide to oxidize phenolic compounds. In nature, enzymes called peroxidases, which efficiently oxidize phenolic compounds, trigger many reactions involving the activation of hydrogen peroxide to oxidize organic substrates. However, these enzymes present several disadvantages, including denaturation and elevated costs. Therefore, these limitations can be overcome by expanding research into the study of synthetic catalysts for the oxidation of phenolic compounds using hydrogen peroxide, which is a highly relevant field of bioinorganic chemistry.     

Fabrication of Prussian Blue modified ultramicroelectrode for GOD imaging using scanning electrochemical microscopy

A Prussian Blue (PB) film modified disk ultramicroelectrode (UME) was fabricated by electrochemical deposition technique on a Pt-disk UME. The electrocatalytical reductions of hydrogen peroxide derived from glucose oxidase (GOD) on this modified UME were investigated. The enzymatic biochemical reactivity was imaged by scanning electrochemical microscopy (SECM) utilizing the PB film modified UME. It is evident that sensitivity and spatial resolution for hydrogen peroxide measurement were improved obviously. SECM images obtained clearly revealed the concentration profile of the reaction products around the enzymes. The PB film modified microelectrode is in the nature of simple preparation, high catalytic activity on hydrogen peroxide and substrate selectivity for SECM etc.