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Abstract: Background: Circulating heterophilic antibodies interfere with immunological assays in laboratory examinations; however, their rate of incidence is currently questionable. We developed an enzyme-linked immunosorbent assay (ELISA) to detect human anti-mouse antibodies (HAMAs) in routine examinations. Methods: The study samples were comprised of serum samples obtained from 290 inpatients and outpatients at our hospital. Mouse immunoglobulin G1 (mIgG1), mIgG2a, and mIgG2b were used as the antigens and horseradish peroxidase (HRP)-conjugated anti-human IgG and IgM were used to identify the HAMA isotype. Results: HAMAs were detected in 11.7% (34/290) of the samples. We observed 18 and 20 samples positive for IgG- and IgM-type HAMAs, respectively. Four samples contained both IgG- and IgM-type HAMAs. HAMAs against mIgG1, mIgG2a, and mIgG2b were found in 21, 14, and 13 samples, respectively. Existence of HAMAs was confirmed by western blotting using mIgG''s as the antigens and HAMAs as the primary antibodies. Heterophilic blocking reagent (HBR) was also used to block the heterophilic interactions. Unexpectedly, a low HBR concentration rather enhanced the interactions instead of blocking them. Conclusions: A considerable number of HAMA-positive samples, reacting with the heavy chain of mIg, were found in routine examinations. A sufficient amount of HBR should be used for blocking the heterophilic interactions. [Copyright &y& Elsevier]

Abstract: Blood coagulation factor XIII (FXIII) is a tetramer (A2B2) consisting of catalytic FXIII-A and carrier/protective FXIII-B subunits. Besides stabilizing fibrin and protecting it from fibrinolysis, FXIII is also involved in wound healing and angiogenesis. The latter functions could be important in body fluids other than blood. In this study highly sensitive chemiluminescent ELISAs were developed to measure low concentrations of FXIII-A, FXIII-B and FXIII-A2B2 antigen levels in tiny volumes of tear. For all three assays the limit of quantitation was below 0.02µg/L, within laboratory imprecision did not exceed 12% and the recovery was close to 100%. The calibration curves showed an excellent fitting using second order polynomial equation. In non-stimulated tears from 40 healthy volunteers a low, but consistent amount of FXIII-A and FXIII-B (medians: 1.74µg/L and 5.73µg/L, respectively) was measured, mostly in free non-complexed form. The respective median values normalized for tear protein concentration were 0.23µg FXIII-A/g and 0.85µg FXIII-B/g. There was no gender and age difference and the values showed non-Gaussian distribution. The results prove that FXIII-A and FXIII-B are low concentration components of tear proteome, which in normal conditions might play a role in the repair of corneal micro-injuries. The developed methods provide a tool for monitoring FXIII subunit and complex levels in pathological conditions. [Copyright &y& Elsevier]

Abstract: Various types of microplates made of polystyrene have been widely used for immunoassays. A new microplate suitable for the covalent immobilization of proteins and peptides was developed by thermal deposition of amino-modified parylene (parylene-A) on the microplate. The primary amine groups of the parylene-A was exploited for the covalent coupling of proteins and peptides. The optical transmittance at the wavelength of 400–500nm was estimated to be suitable for the application to immunoassays. The immobilization efficiency of the parylene-A coated microplate was demonstrated to be far improved in comparison to the conventional microplate by using horseradish peroxidase (HRP), anti-HRP antibody and a peptide with 9-residues as model biomolecules. [Copyright &y& Elsevier]

Abstract: Horseradish peroxidase has been successfully immobilized in a polypyrrole matrix onto disposable screen-printed carbon electrodes for the selective detection of Ochratoxin A. The chronoamperometric determination of this mycotoxin has been optimized by experimental design methodology, which implied the join evaluation of pH of the buffer solution, applied potential and concentration of H2O2. The slopes of the calibration curves built under the optimum conditions of the experimental variables have been used to estimate the reproducibility and the repeatability of the developed biosensor for Ochratoxin A. Relative standard deviation values of 1.9% (n =5 and α =0.05) and 7.1% (n =4 and α =0.05) were obtained for reproducibility and repeatability, respectively. The capability of detection for this method was 0.1ngmL−1 (α =0.05 and β <0.05). The viability of the developed biosensor in the determination of Ochratoxin A in spiked beer and in roasted coffee has been shown, yielding average recoveries of 103% and 99%, in that order, with relative standard deviation values less than 5% (n =3 and α =0.05) in both cases. [Copyright &y& Elsevier]

Abstract: Based on the immobilization enzyme technology and the fluorescence capillary analysis method, the authors have developed a highly sensitive fluorescence reaction system and a novel immobilization multienzyme glucose fluorescence capillary biosensor for determining glucose contents. Reaction principle of the system is that under the catalysis of glucose oxidase (GOD) and horseradish peroxidase (HRP) immobilized on inner surface of a medical capillary, β-d-glucose reacts with dissolved oxygen to form gluconic acid-δ-lactone and hydrogen peroxide, and then the latter reacts with l-tyrosine to produce a tyrosine dimer, which has maximal excitation and emission wavelengths at 320nm and 410nm, respectively. Fluorescence of the dimer is proportional to the concentration of the β-d-glucose. Optimization conditions suitable for the reaction system and the biosensor were as follows. Concentration of the l-tyrosine used as fluorescence reagent was 0.15molL−1, the active concentrations of the GOD and the HRP for the immobilization were 15kUL−1 and 8kUL−1, respectively. Consumptions of the sample and reagents in one determination were 5.0μL and 15μL, respectively. Quantitative range of the biosensor for the glucose was in the range 1–10μmolL−1, its relative standard deviation was less than 4.9%, and its detection limit was 0.62μmolL−1. The biosensor''s recovery was in the range 96–105%. Results of some serum determined with the biosensor and with a commercial glucose-kit were well coincident to each other. Accordingly, the biosensor can be applied to the determination of serum glucose contents in the diagnosis of diabetes. [Copyright &y& Elsevier]

Abstract: Vertically aligned nanowire array electrodes (NAEs) were prepared by electrodeposition of gold into an anodic aluminium oxide membrane (AAM), providing an ordered three-dimensional (3D) matrix for immobilization of redox proteins. Third-generation H2O2 biosensors were prepared by covalent immobilization of horseradish peroxidase (HRP) on the self-assembled monolayer modified NAEs. Direct electron transfer and electrocatalytic performances of the HRP/NAEs with different nanowire lengths (deposition time of 2, 4 and 5h) were investigated. Results showed that with longer nanowires, better performances were achieved. The HRP/NAE5h (5h deposition time) exhibited remarkable sensitivity (45.86μAmM−1 cm−2) towards H2O2 with a detection limit of 0.42μM (S/N =3), linearity up to 15mM and a response time of 4s. The ordered 3D gold nanowire array with high conductivity, excellent electron transfer capability and good biocompatibility proved promising for fabricating sensitive, selective, stable and mediator-free enzymatic biosensors. [Copyright &y& Elsevier]

Abstract: The use of heterogeneous conditions involving Montmorillonite K10 clay was investigated as a mild alternative to homogeneous acidolysis of a lignin model. Guaiacyl Dehydrogenation Oligomers (DHOs) synthesized by horseradish peroxidase were selected as starting material. Hydrolysis products were analyzed by gel permeation chromatography and by HPLC/mass spectrometry. Several experimental parameters were studied such as catalyst and substrate concentration, as well as reaction solvent composition in order to minimize high molar mass product formation generated by recombination mechanisms. In both catalytic modes, the best hydrolysis conditions were similar in terms of solvent composition with dioxane/water (90/10, v/v) and catalyst H+ concentration of about 0.01mol/L. Although the homogeneous catalysis generated only 28% of low molecular weight (LMW) products (monomers and dimers), clay catalysis generated 35%. In light of the qualitative analysis, both catalytic methods gave the same products, thus supporting similar hydrolysis mechanisms. [Copyright &y& Elsevier]

Abstract: The interaction between lanthanum ion (La3+) and horseradish peroxidase (HRP) in vitro was investigated using a combination of biophysical and biochemical methods. When the molar ratio of La3+ and HRP is low, it was found that the interaction between La3+ and HRP mainly depends on the electrostatic attraction, van der waals force and hydrogen bond etc. Thus, the interaction is weak and the La–HRP complex cannot be formed in vitro. As expected, the interaction can change the conformation of HRP molecule, leading to the increase in the non-planarity of the porphyrin ring in the heme group of HRP molecule, and then in the exposure degree of the active center, Fe(III) of the porphyrin ring of HRP molecule. Therefore, the catalytic activity of HRP for the H2O2 reduction is improved. When the molar ratio of La3+ and HRP is high, La3+ can strongly coordinate with O and/or N in the amide group of the polypeptide chain of HRP molecule, forming the La–HRP complex. The formation of the La–HRP complex causes the change in the conformation of HRP molecule, leading to the decrease in the non-planarity of the porphyrin ring in the heme group of HRP molecule, and then in the exposure degree of the active center, Fe(III) of the porphyrin ring of HRP molecule. Thus, the catalytic activity of HRP for the H2O2 reduction is decreased comparing with that of HRP in the absence of La3+. The results can provide some references for understanding the interaction mechanism between trace elements ions and peroxidase in living organisms. [Copyright &y& Elsevier]

Abstract: We describe herein the combination of electrochemical immunoassay using nanoporous gold (NPG) electrode with horseradish peroxidase (HRP) labeled secondary antibody–gold nanoparticles (AuNPs) bioconjugates for highly sensitive detection of protein in serum. The electroactive product of o-phenylenediamine (OPD) oxidized with H2O2 catalyzed by HRP was reduced in the Britton–Robinson (BR) buffer and the peak current of which was used to determine the concentration of antigen (Ag) in the analyte. The active surface area of NPG electrode was larger than that of a bare flat one. The presence of AuNPs enhanced the immobilized amount of HRP labeled antibody (Ab), which improved the sensitivity of the immunoassay when used as the secondary antibodies. As a result of these two combined effects, the sensitivity of the immunoassay for the determination of target protein was increased significantly. Using hepatitis B surface antigen (HBsAg) as a model, we demonstrate a dose response in the range of 0.01–1.0ng/mL with a detection limit of 2.3pg/mL. Analytical results of several human serum samples obtained using the developing technique are in satisfactory agreement with those given by enzyme-linked immune-absorbent assays (ELISA). In addition, the technique was about 100 times more sensitive in the detection of HBsAg than ELISA. All these demonstrated the feasibility of the present immunoassay method for clinical diagnosis. [Copyright &y& Elsevier]

Abstract: Schiff base derivate 4-[(4-phenylazo-phenyimino)-methyl]-phenol (4-PPMP) monomer was synthesized by condensation reaction and the chemical structure of the monomer has been characterized by UV–vis, FT-IR, 1H NMR spectroscopies. 4-PPMP readily dissolves in 1,4-dioxane, THF, DMF, diethyl ether, chloroform and DMSO. Its solubility in methanol and ethanol is much lower. Enzymatic oxidative polymerization of azobenzene derivate 4-[(4-phenylazo-phenyimino)-methyl]-phenol using horseradish peroxidase (HRP) in the presence of hydrogen peroxide as catalyst and oxidizing agent was carried out in various solvents (acetone, methanol, ethanol, N,N-DMF, and 1,4-dioxane) and phosphate buffers (pH 6, 6.8, 7, and 7.2) at room temperature. Studies have shown that a black polymer having a melting point of 290°C was successfully produced in good yields by utilizing aqueous 1,4-dioxane as the solvent at pH 6. Poly(4-[(4-phenylazo-phenyimino)-methyl]-phenol) P(4-PPMP) shows good solubility in 1,4-dioxane, DMF and DMSO but it is only sparingly soluble in chloroform, THF, methanol and ethanol. P-(4-PPMP) is insoluble in diethyl ether. Characterization of P-(4-PPMP) was carried out via UV–vis, FT-IR, 1H NMR, elemental analysis and SEC measurements. The number-average molecular weight (M n ), weight-average molecular weight (M w ) and polydispersity index (PDI) of the polymer were determined to be 7970.4, 8146.2 and 1.02gmol−1, respectively. FT-IR and 1H NMR studies confirmed the presence of phenylene and oxyphenylene units with in the polymer backbone. The optical band gaps (E g) of 4-PPMP and P-(4-PPMP) were calculated as 3.69 and 3.36eV, respectively. [Copyright &y& Elsevier]

Abstract: Biosensor performance depends on the effective functionalisation of a transducer with suitable biorecognition elements. During functionalisation, surface blocking steps are normally carried out to avoid later binding of undesirable molecules and thus guarantee biosensor specificity. However, these blocking steps may be deleterious in electrochemical systems where transduction ultimately relies on electron transfer between the electrode and a redox species in solution. This work presents a novel approach to develop improved amperometric biosensing platforms using microfabricated disk microelectrode arrays, based on the functionalisation of the inert surface surrounding the active microdisks. These devices more than doubled assay sensitivity compared to conventional biosensors produced using the same arrays. This approach benefits from three advantages: the functionalisation of a broader surface, the possibility to activate the microelectrodes immediately before detection, and access to enhanced rates of mass transport to microelectrodes that improve device sensitivity. To demonstrate this, we first studied the electrochemical behaviour of tetramethylbenzidine (TMB) at gold disk microelectrode arrays, and then used TMB as the redox mediator for the amperometric biosensing of HRP/H2O2. Down to 0.54pM H2O2 or as little as 25pM HRP were detected within 5s of enzyme activity in just 10μl of enzyme substrate solution. We postulate that microelectrode arrays may be used to develop novel electrochemical biosensing platforms that are faster and more sensitive than conventional biosensors. [Copyright &y& Elsevier]

Abstract: In this paper, DNA functionalized SWCNTs were used to immobilize horseradish peroxidase (HRP) on glassy carbon (GC) electrode. Cyclic voltammetry showed that the direct electrochemistry of HRP immobilized on DNA-SWCNTs hybrids was achieved. The DNA interlayer between the SWCNTs and HRP could be used to keep the activity of HRP. Compared with HRP-SWCNTs/GC and HRP-DNA/GC electrodes, the prepared HRP-DNA-SWCNTs/GC electrode exhibited more excellent electrochemical properties. Thus, the prepared HRP-DNA-SWCNTs/GC electrode was proposed as a third-generation H2O2 biosensor. The effect of pH and applied potential on the performance of the biosensor was discussed in detail. Under the optimal conditions, a wide linear range of the propose biosensor for the detection of H2O2 was observed from 6.0×10−7 to 1.8×10−3 M. The detection limit was found to be 3.0×10−7 M at a signal-to-noise ratio of 3. Furthermore, the proposed biosensor displayed rapid response, high stability, very good reproducibility and high sensitivity for the detection of H2O2. Determination H2O2 concentration in disinfector sample by the proposed biosensor also showed satisfactory result. [Copyright &y& Elsevier]

Abstract: The HSD2 (11-β-hydroxysteroid dehydrogenase-type 2 enzyme) containing neurons of the nucleus tractus solitarius (NTS) become activated during low-sodium and high-aldosterone states such as hypovolemia. This response may be due to hormonal and/or neural signals. Hormonal signals may activate neurons in the area postrema that innervate the HSD2 neurons. The vagus nerve projects directly to the HSD2 neurons and this could be another route whereby these neurons receive information about systemic sodium/aldosterone status. The peripheral sites of origin that contribute to this vagal projection remain unknown, and in the present study, we injected the transganglionic tracer, cholera toxin β-subunit-horseradish peroxidase (CTb-HRP), into wall of various gastrointestinal organs (stomach, small and large intestine) or liver of rats. Confocal microscopy of brainstem sections stained by a double immunohistochemical procedure was used to analyze whether the HSD2 neurons received axonal contacts from specific gastrointestinal structures. The major source of afferents arose from the stomach, mainly from its pyloric antrum, but a weaker input originated from the fundus region. A trace amount originated from the duodenum. The terminal part of the small intestine and large intestine did not to contribute to this projection. Similarly, no afferent inputs from the liver or portal vein were found. In conclusion, HSD2 neurons receive an input mainly from the stomach and these results are considered as potential sites affecting sodium intake. [Copyright &y& Elsevier]

Abstract: In this work, the results of photoacoustic calorimetry (PAC) studies involving CO photodissociation from horseradish peroxidase (HRP) and soybean peroxidase (SBP) are discussed. Both proteins contain Fe-protoporphyrin IX active sites and relatively open distal heme pockets (i.e., direct solvent access). In addition, it has been shown previously that SBP binds a Tris molecule in the distal pocket near the heme group potentially regulating ligand binding to the heme iron. Results of PAC studies indicate a fast (<∼50 ns) relaxation for both HRP and SBP subsequent to CO photolysis in both phosphate and Tris buffers and with varying concentrations of Tris. However, the molar volume/enthalpy changes associated with CO release are distinct between the two proteins. In the case of HRP, CO photolysis results in an enthalpy change of ∼2 kcal mol−1 and volume change of ∼−12 mL mol−1 attributed to solvation/structural changes regardless of buffer conditions. In contrast, SBP exhibits buffer and ionic strength dependent enthalpy changes ranging from ∼−23 kcal mol−1 in 50 mM phosphate buffer to ∼6 kcal mol−1 in Tris buffer with volume changes similar to those observed in HRP. The results are consistent with a model in which photodissociation of CO from ferrous HRP or SBP leads to CO migration from the distal heme pocket to the bulk solvent with a corresponding input of a water molecule all occurring in <∼50 ns. The differences in enthalpies are attributed to variations in hydrogen bond formation between the incoming water molecule(s) and the protein matrix in both HRP and SBP. [Copyright &y& Elsevier]

Abstract: In order to understand the toxic effect of terbium (Tb(III)) on the plant photosynthesis, we investigated the photosynthesis, the ultrastructural structure of chloroplast, the subcellular distribution of horseradish peroxidase (HRP), the activity of HRP, and the content of malondialdehyde in horseradish by a portable gas exchange system, transmission electron microscopy and the other biochemical technologies. The results indicated that after horseradish treated with 5mgL−1 Tb(III), the subcellular distribution of HRP was not obviously changed. However, the activities of guaiacol and ascorbate HRP were decreased comparing with that of horseradish treated without Tb(III). It could cause the peroxidation of membrane lipid, the damage of chloroplast ultrastructure and the decrease in the photosynthesis and the content of chlorophyll. Moreover, after horseradish treated with 60mgL−1 of Tb(III), the distribution of HRP on the plasma membrane and tonoplast was decreased, while the distribution of HRP on the cell wall was increased comparing with that of horseradish treated without Tb(III). The change in the subcellular distribution of HRP could induce the excess accumulation of the reactive oxygen, leading to the damage of the chloroplast ultrastructure and then the decrease in the photosynthesis. Furthermore, the effects of Tb(III) on the indexes mentioned above were increased with prolonging the treating time of Tb(III). These results demonstrated that the function of HRP in horseradish treated with Tb(III) was decreased, leading to the damage of chloroplast ultrastructure and then the inhibition of photosynthesis. It was a possible toxic effect of Tb(III) on the plant photosynthesis. [Copyright &y& Elsevier]

Abstract: A convenient and effective strategy for fabrication of hydrogen peroxide biosensor based on sodium alginate (SA) and polyvinyl butyral (PVB) as matrices was reported in this paper. The horseradish peroxidase (HRP) and SA were electro-co-deposited onto the surface of gold electrode, and the HRP–SA/Au electrode was further coated with PVB. The interaction between HRP and SA was characterized by UV–vis absorption spectroscopy, and the fabricating process of biosensor was characterized by electrochemical impedance spectroscopy (EIS). The electrochemical characteristics of the biosensor were studied by cyclic voltammetry and chronoamperometry. Experimental conditions were investigated which influence the performance of the biosensor, such as pH, and applied potential. The biosensor showed a linear response to H2O2 over a concentration range from 7.0×10−6 to 4.1×10−3 M with a detection limit of 1.8×10−6 M based on a signal-to-noise ratio of 3 under optimum conditions. The value of HRP in the composite was evaluated to be 1.38mM. The biosensor obtained from this study possesses high sensitivity, good reproducibility, and long-term stability. [Copyright &y& Elsevier]

Abstract: A simple and sensitive method using lateral flow immunoassay (LF) using chemiluminescence reaction has been developed for the detection of antigen. In this work, horseradish peroxidase (HRP) samples were used to confirm the validity of this method and human chorionic gonadotropin (hCG) samples were used to evaluate the limit of detection (LOD) of this method. Through HRP test, this method was confirmed to be reliable for immunoassay, and the results of hCG test show that this method was very sensitive compared with conventional strip test using color of gold nanoparticles or latex beads. [Copyright &y& Elsevier]

Abstract: A new method for determining berberine has been established based on the principle of fluorescence quenching. The calibration curve was found to be linear between F 0/F and the concentration of berberine with the range of 3.00–20.0μgmL−1. The detection limit was 0.51μgmL−1 and the relative standard derivative was 0.18%. Effects of pH, foreign ions and the optimization of variables on the determination of berberine have been examined. The mechanism of the fluorescence quenching has been discussed. The binding constant and the number of binding sites were 1.70×106 Lmol−1 and 1.14, respectively. The data, ΔH =42.71kJmol−1, ΔS =264.3JK−1 mol−1 and the mean value ΔG =−39.65kJmol−1 were estimated which showed that the reaction was spontaneous and endothermic. The main binding force was hydrophobic force because both ΔH and ΔS were positive. [Copyright &y& Elsevier]

Abstract: Peroxidases are oxidoreductases produced by a number of microorganisms and plants. Peroxidases catalyse a variety of reactions in the presence of peroxides such as hydrogen peroxide. Oxidative polymerisation of phenols and aromatic amines, conducted by horseradish peroxidase (HRP) in water and water-miscible organic solvents, may lead to new types of aromatic polymers. Peroxidase has a potential for soil detoxification, while HRP, as well as soybean and turnip peroxidases have been applied to the bioremediation of waste waters contaminated with phenols, cresols and chlorinated phenols. Lignin peroxidase (LiP) and manganese peroxidase (MnP) may be successfully used for biopulping and biobleaching in the paper industry and can produce oxidative breakdown of synthetic azo dyes. Peroxidase-based biosensors have found use in analytical systems for determination of hydrogen peroxide and organic hydroperoxide, while, co-immobilized with hydrogen peroxide-producing enzyme, they can be used for determination of glucose, alcohols, glutamate and choline. Peroxidase has also been used for practical analytical applications in diagnostic kits, such as quantification of uric acid, glucose, cholesterol, lactose, etc. Enzyme-linked immunosorbent assay (ELISA) tests, in which peroxidase enzyme is the most common enzyme used for labelling an antibody, are a simple and reliable way of detecting toxins, pathogens, cancer risk in bladder and prostate, and many other analytes. [Copyright &y& Elsevier]

Considering the development of highly confined piggery and increasing complaints about livestock manure odors, it is pressing to develop a practical way to reduce the odors. Peroxidase, which has been proved to be capable of removing toxic phenolic compounds from wastewater, may also be effective in deodorizing the swine manures. Horseradish peroxidase (HRP) (0.1¿3.0U/mL) with hydrogen peroxide (H2O2, 0.5¿6%) or calcium peroxide (CaO2, 0.1¿3.0g) was examined for the efficiency of controlling the release of seven malodor compounds, including three volatile fatty acids (isobutyric acid, isocaproic acid and isovaleric acid), two phenolic compounds (phenol and p-cresol) and two indolic compounds (indole and skatole) from swine manure slurry. Odor intensity and total nitrogen content in swine manure were also measured. The results showed almost 100% reduction in p-cresol, 54¿84% reduction in odor intensity, 32¿54% reduction in indolic compounds and 28¿41% reduction of VFAs. The effect of deodorization can last for at least 48h. [Copyright &y& Elsevier]

Application of immobilized horseradish peroxidase (HRP) in porous calcium alginate (ca-alginate) for the purpose of phenol removal is reported. The optimal conditions for immobilization of HRP in ca-alginate were identified. Gelation (encapsulation) was optimized at 1.0% (w/v) sodium alginate in the presence of 5.5% (w/v) of calcium chloride. Upon immobilization, pH profile of enzyme activity changes as it shows higher value at basic and acidic solution. Increasing initial phenol concentration results in a decrease in % conversion. The highest conversion belongs to phenol concentration of 2mM. Investigation into time course of phenol removal for both encapsulated and free enzymes showed that encapsulated enzyme had lower efficiency in comparison with the same concentration of free enzyme; however the capsules were reusable up to four cycles without any changes in their retention activity. Increasing enzyme concentration from 0.15 to 0.8units/g alginate results in gradual increase in phenol removal. The ratio of hydrogen peroxide/phenol at which highest phenol removal obtained is found to be dependent on initial phenol concentration and in the solution of 2 and 8mM phenol it was 1.15 and 0.94 respectively. [Copyright &y& Elsevier]

Abstract: The present work reports on enzyme attachment on and photoinduced release from TiO2 surfaces. TiO2 layers (amorphous and anatase) were modified with 3-aminopropyltriethoxysilane (APTES), followed by attachment of vitamin C and horseradish peroxidase (HRP). Using step by step XPS characterization and vis-spectroscopy we show that upon UV illumination the linker chain to the protein can be cut, releasing active HRP into the environment. The head silane group remains attached to the TiO2 surface. The kinetics of this photoinduced release is significantly faster for the anatase form of TiO2 compared with amorphous material. The results indicate that UV induced chain scission represents a very versatile tool for payload release from TiO2 surfaces. [Copyright &y& Elsevier]

Abstract: We developed a novel immunomagnetic bead ELISA based on IgY (egg yolk immunoglobulin) for detection of circulating antigen (CA) in sera of mice infected with Schistosoma japonicum. The assay involved the use of chicken polyclonal antibodies IgY against soluble egg antigens (SEA) of S. japonicum as a capture antibody and anti-SEA mouse monoclonal antibody NP28-5B labeled horseradish peroxidase (HRP) as a detecting antibody. Two groups of BALB/c mice infected with S. japonicum cercariae were used: lightly infected mice (infected with 10 S. japonicum cercariae) and heavily infected mice (infected with 30 S. japonicum cercariae). The CA was detectable as early as 4 and 5 weeks after infection in the sera of heavily and lightly infected mice, respectively. The CA levels rose rapidly and reached a peak in 8 weeks after infection and then remained a plateau for at least another 6 weeks in both groups. Moreover, the effect of praziquantel on the CA levels was also investigated. The heavily infected mice were treated with praziquantel and the CA levels in sera increased dramatically in the first week post-treatment and then decreased to the control level by 6 weeks after treatment. The novel assay appears to be sensitive for detection of schistosomal antigenemia and valuable to judge the efficacy of chemotherapy in murine schistosomiasis. [Copyright &y& Elsevier]

Abstract: A sensitive, rapid and simple method for analysis of protein was proposed by introducing an electric field-driven technique to the incubation process of immunoassay and immobilizing horseradish peroxidase (HRP) labeled antibody in a newly designed gel matrix to construct an immunosensor. With an electric field-driven technique, the incubation for immuno-recognition could be completed within 2min. The immobilized HRP showed excellent direct electrochemistry in this new gel matrix, and the detection procedure was greatly simplified by directly monitoring the sensitive electrochemical signal of HRP upon the immunoreaction. Using α-fetoprotein as a model the linear detection range was from 0.02 to 2.0ngmL−1 with a detection limit down to 138amolmL−1. The immunosensor and the proposed detection method showed good specificity and acceptable stability and accuracy. [Copyright &y& Elsevier]

Abstract: We report on a novel amperometric biosensor for detecting phenolic compounds based on the co-immobilization of horseradish-peroxidase (HRP) and methylene blue (MB) with chitosan on Au-modified TiO2 nanotube arrays. The titania nanotube arrays were directly grown on a Ti substrate using anodic oxidation first; a gold thin film was then coated onto the TiO2 nanotubes by an argon plasma technique. The morphology and composition of the fabricated Au-modified TiO2 nanotube arrays were characterized by scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS). Cyclic voltammetry and amperometry were used to study the proposed electrochemical biosensor. The effect of pH, applied electrode potential and the concentration of H2O2 on the sensitivity of the biosensor have been systemically investigated. The performance of the proposed biosensor was tested using seven different phenolic compounds, showing very high sensitivity; in particular, the linearity of the biosensor for the detection of 3-nitrophenol was observed from 3×10−7 to 1.2×10−4 M with a detection limit of 9×10−8 M (based on the S/N=3). [Copyright &y& Elsevier]

Abstract: A novel amperometric biosensor for the analytical determination of hydrogen peroxide was developed. The fabrication of the biosensor was based on the coimmobilization of horseradish peroxidase (HRP), methylene green (MG) and multiwalled carbon nanotubes within ormosils; 3-aminopropyltrimethoxysilane (APTMOS), 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane (ETMOS) and phenyltrimethoxysilane (PHTMOS). APTMOS determined the hydrophilicity/hydrophobicity of the ormosils and PHTMOS and ETMOS increased the physical and mechanical strength of the ormosil matrix. The ormosil modified electrodes were characterized with SEM, UV–vis spectroscopy and electrochemical methods. Cyclic voltammetry and amperometric measurements demonstrated the MG coimmobilized with HRP in this way, displayed good stability and could efficiently shuttle electrons between immobilized enzyme and electrode, and MWCNTs facilitated the electrocatalytic reduction of H2O2 at reduced over potential. The Micheaelis constant of the immobilized HRP was 1.8mM, indicating a high affinity of the HRP to H2O2 without loss of enzymatic activity in ormosil matrix. The prepared biosensor had a fast response of H2O2, less than 10s, and excellent linear range of concentration from 5×10−7 to 2×10−5 M with the detection limit of 0.5μM (S/N=3) under the optimum conditions. At the same time, the influence of solution pH, effect of enzyme amount, steady-state applied potential and temperature on the biosensor were investigated. The enzyme electrode retained about 90% of its initial activity after 30 days of storage in a dry state at 4°C. The preparation of the developed biosensor was convenient and showed high sensitivity with good stability. [Copyright &y& Elsevier]

Abstract: Direct electrochemistry and bioelectrocatalytic ability toward the reduction of hydrogen peroxide of horseradish peroxidase (HRP) were found at the multiwalled carbon nanotube/alumina-coated silica (MWCNT/ACS) nanocomposite modified glassy carbon electrode. The surface morphology of the HRP/MWCNT/ACS nanobiocomposite modified glassy carbon electrode was characterized by scanning electron microscopy. In cyclic voltammetric response, the immobilized HRP at the MWCNT/ACS nanocomposite modified glassy carbon electrode displayed a pair of well-defined and quasi-reversible redox peaks with a formal potential of −0.328V versus Ag/AgCl at a scan rate of 200mVs−1. The formal potential of HRP varied linearly in the range of solution pH values from 6.0 to 8.0 with a slope of −56mVpH−1, which is close to the theoretical value of −59mVpH−1 for a reversible one-electron coupled with one-proton electrochemical reaction process. Several important experimental variables were optimized. The optimized hydrogen peroxide biosensor exhibited a sensitivity of 0.157AM−1 cm−2, a detection limit of 0.63μM, a linear range of 1–500μM, and an apparent Michaelis–Menten constant of 0.39mM. The performance of the prepared biosensor was evaluated with respect to four possible interferences. [Copyright &y& Elsevier]

Abstract: Nanostructured cerium oxide (NanoCeO2) film deposited onto indium-tin-oxide (ITO) glass substrate by solution casting has been used for immobilization of horseradish peroxidase (HRP) via physiosorption technique. X-ray diffraction (XRD), scanning electron microscopy (SEM), UV–vis and electrochemical techniques have been utilized for characterization of NanoCeO2/ITO electrode and HRP/NanoCeO2/ITO bioelectrode. The HRP/NanoCeO2/ITO electrode exhibits value of the apparent Michaelis–Menten constant (K m) as 2.21μM, linear regression coefficient as 0.998 and linearity for hydrogen peroxide as 1.0–170μM obtained using electrochemical response measurements. Besides this, HRP/NanoCeO2/ITO bioelectrode can be used about 20 times and is stable for 5 weeks at 4°C. The results of photo-response studies carried out on HRP/NanoCeO2/ITO bioelectrode indicate reasonable agreement with those obtained using amperometric technique. [Copyright &y& Elsevier]

Abstract: A simple and controllable electrodeposition approach was established for one-step construction of novel reagentless biosensors by in situ formation of chitosan-carbon nanotubes-nile blue-horseradish peroxidase (CS-CNTs-NB-HRP) biocomposite film on electrode surface. The mediator effect of NB, conducting performance of CNTs and the biocompatible microenvironment of CS were combined by such one-step non-manual process. NB could interact with CNTs and resulted in good dispersion of CNTs-NB nanocomposites in aqueous solution. Cyclic voltammetry measurements demonstrated that electrons were efficiently shuttled between HRP and the electrode mediated by NB. The developed reagentless biosensor exhibited a fast amperometric response for the determination of H2O2 and 95% of the steady-state current was obtained within 2s. The linear response of the reagentless biosensor for the determination of H2O2 ranged from 1.0×10−6 to 2.4×10−4 moll−1 with a detection limit of 1.2×10−7 moll−1. The biosensor exhibited high reproducibility and long-time storage stability. The as-prepared biosensor also showed effective anti-interference capability. The ease of the one-step non-manual technique and the promising feature of the biocomposite could serve as a versatile platform for fabricating electrochemical biosensors. [Copyright &y& Elsevier]

Abstract: Horseradish peroxidase was cross-linked with cysteamine-capped Au nanoparticles and further immobilized on sodium alginate-coated Au electrode through polyelectrostatic interactions. The electrode was employed for constructing a reagentless amperometric biosensor for H2O2. The electrode showed linear response (poised at −400mV vs. Ag/AgCl) toward H2O2 concentration between 20μM and 13.7mM at pH 7.0. The biosensor reached 95% of steady-state current in about 15s, and its sensitivity was 40.1mA/M cm2. The detection limit of the enzyme-based electrode was determined as 3μM, at a signal-to-noise ratio of three. The electrode retained 97% of its initial analytical response after 1month of storage at 4°C in 50mM sodium phosphate buffer, pH 7.0. The stability of the biosensor was significantly reduced when it was incubated in high ionic strength solutions, retaining only 44% of its initial response after 1month of storage at 4°C in 1M NaCl ionic strength in 50mM sodium phosphate buffer, pH 7.0. [Copyright &y& Elsevier]

Abstract: Molecular dynamics simulations on hydrogen peroxide complex with wild-type (WT) and Arg38Leu mutated (R38L) Horseradish Peroxidase (HRP) were carried out over nanoseconds timescale in water solution at 300 K. Comparison of the results provides interesting insights about the role of highly conserved Arg38 and His42 residues in the chemical features of HRP, underlying its biological activity which initiates with Compound0 (Cpd0). In the WT-HRP enzyme current molecular dynamics simulations show, for the first time, that Arg38 residue: i) prevents the entrance of water inside the reaction cavity, hence providing a hydrophobic reactive scenario, ii) it maintains the distance between His42 and heme–H2O2 complex suitable for the occurrence of proton transfer reaction leading, thereafter, to heme–H2O2 disruption according to Poulos-Kraut mechanism. On the other hand, R38L mutant can be considered as a “wet enzyme” where the presence of water solvent molecules in the heme reaction pocket, unfavoring the initial heme–H2O2 complex formation, decreases the catalytic efficiency in agreement with experimental kinetics measurements. Furthermore, we note that Arg38Leu mutation pushes the His42 residue far from the heme–H2O2 complex, making unlikely a direct proton transfer and suggesting that, in the mutant, a solvent water molecule could be involved in the first step of the Poulos-Kraut mechanism. [Copyright &y& Elsevier]

Abstract: Effects of immobilization pH and pore characters of mesoporous silicas (MPSs), MCM-41, SBA-15, and MCF, were simultaneously investigated for the immobilization of horseradish peroxidase (HRP; EC 1.11.1.7). MCM-41 and SBA-15 were rod-like with respective average pore diameters of 32, and 54Å, while that of MCF with spherical cell and frame structure was 148Å. Moreover, the MPSs synthesized were of identical surface functional groups and similar contents of free silanol groups. At immobilization pH 6 and 8 almost 100% HRP loadings were obtained and insignificant leaching were observed for all types of supports at pH 6. However, MCF was found to give both the highest enzyme loading and leaching at pH 10. Maximum and minimum HRP activities were obtained at respective immobilization pH 8, and 6. Activities of immobilized HRP increased with support pore diameters in the order: MCM-41

Abstract: A novel amperometric hydrogen peroxide biosensor based on the immobilization of horseradish peroxidase (HRP) on DNA–silver nanohybrids (DNA–Ag) and poly(diallyldimethylammonium chloride) (PDDA)-protected gold nanoparticles (PDDA–Au) was successfully fabricated by combining the self-assembly technique with an in situ electrochemical reduction of the DNA–Ag+ complex. The preparation process of modified electrode was characterized with UV–vis absorption spectroscopy, transmission electron microscopy (TEM) and atomic force microscope (AFM). The electrochemical characteristics of the biosensor were studied by cyclic voltammetry and chronoamperometry. Experimental conditions influencing the biosensor performance such as pH, potential were optimized. The resulting biosensor (HRP/DNA–Ag/PDDA–Au/DNA–Ag/Au electrode) showed a linear response to H2O2 over a concentration range from 7.0μM to 7.8mM with a detection limit of 2.0μM (S/N=3) under optimized conditions. The apparent Michaelis–Menten constant () was evaluated to be 1.3mM. The sensor exhibited high sensitivity, good accuracy and an acceptable stability. [Copyright &y& Elsevier]

Abstract: Three-dimensional macroporous TiO2 was synthesized by a sol–gel procedure using polystyrene colloidal crystals as templates. SEM showed that a face-centered cubic (FCC) 3D macroporous structure was obtained. Horseradish peroxidase (HRP) was successfully immobilized on the surface of an optically transparent electrode (OTE). Photoelectrochemical properties were characterized using a three electrodes system and an ultraviolet lamp. The HRP/TiO2/OTE displays a rapid photocurrent response, approximately 178.7nA, under UV illumination (380nm). The sensitivity of H2O2 detection was 70.04μA/mM without UV illumination, and it increased to 102.97μA/mM when illuminated by UV. The amperometric response was also enhanced. The high response was due to the good biocompatibility of TiO2 and excellent photoelectrical property and the large effective surface of the three-dimensionally ordered macroporous structure. [Copyright &y& Elsevier]

Abstract: Aims: To clarify the mechanism of the protective effect of non-steroidal anti-inflammatory drugs (NSAIDs) on Alzheimer''s disease, inactivation of cholinesterase (ChE) induced by NSAIDs was examined. Main methods: Equine ChE and rat brain homogenate were incubated with NSAIDs and horseradish peroxidase (HRP) and H2O2 (HRP–H2O2). ChE activity was measured by using 5,5''-dithiobis(nitrobenzoic acid). By using electron spin resonance, NSAID radicals induced by reaction with HRP–H2O2 were detected in the presence of spin trap agents. Key findings: Equine ChE was inactivated by mefenamic acid with HRP–H2O2. ChE activity in rat brain homogenate decreased dependent on the concentration of mefenamic acid in the presence of HRP–H2O2. NSAIDs diclofenac, indomethacin, phenylbutazone, piroxicam and salicylic acid inactivated ChE. Oxygen radical scavengers did not prevent inactivation of ChE induced by mefenamic acid with HRP–H2O2. However, spin trap agents 5,5-dimethyl-1-pyrroline-l-oxide and N-methyl-nitrosopropane, reduced glutathione and ascorbic acid strongly inhibited inactivation of ChE, indicating participation of mefenamic acid radicals. Fluorescent emission of ChE peaked at 400 nm, and the Vmax value of ChE changed during interaction of mefenamic acid with HRP–H2O2, indicating that ChE may be inactivated through modification of tyrosine residues by mefenamic radicals. Significance: The protective effect of NSAIDs on Alzheimer''s disease seems to occur through inactivation of ChE induced by NSAIDs radicals. [Copyright &y& Elsevier]

Abstract: The use of plants as production hosts for recombinant glycoproteins, which is rapidly developing, requires methods for fast and reliable analysis of plant N-linked glycans. This study describes a simple small-scale method for the preparation of N-linked glycans from soluble plant protein and analysis thereof by matrix assisted laser desorption ionisation time of flight mass spectrometry (MALDI-TOF MS). Concentration and protease digestion of plant protein as well as deglycosylation is carried out in a single concentrator unit without the need for intermittent purification to minimize adsorptive loss and to facilitate handling. Plant protein is concentrated in a unit with a 5kDa cutoff, and after buffer exchange, pepsin (EC 3.4.23.1) digestion is carried out in the concentrator overnight to obtain peptides as substrates for deglycosylation. Deglycosylation is carried out with peptide-N-glycosidase A (PNGase A; EC 3.5.1.52) for 24h. Released N-glycans are purified using reverse-phase and cation exchange chromatography micro-columns for removal of peptides and desalting. N-Glycans are directly analyzed by MALDI-TOF MS without derivatization. The method for isolation of N-glycans is compatible with secreted proteins from cell culture supernatant as well as with soluble protein extracts from leaf tissue. As little as 5μg of plant glycoprotein is sufficient for N-glycan preparation for MALDI-TOF MS analysis using this method. [Copyright &y& Elsevier]

Abstract: We developed a new flowerlike ZnO–gold nanoparticles (GNPs)–Nafion nanocomposite which can promote the direct electron transfer of horseradish peroxidase (HRP) immobilized in the film effectively. The cyclic voltammetric results show that HRP entrapped in the composite could undergo a direct quasi-reversible electrochemical reaction with a formal potential (E 0′) of −0.32V (versus Ag/AgCl) in phosphate buffer solution (PBS). The flowerlike ZnO and GNPs show synergistic effect and the ZnO–GNPs–Nafion–HRP modified GC electrode gives an enhanced electrocatalytic activity towards the reduction of hydrogen peroxide (H2O2). The calculated apparent Michaelis–Menten constant () is 1.76mM, which is much lower than that reported, indicating a high catalytic activity of HRP. The catalysis currents increase linearly to the H2O2 concentration in a wide range of 1.5×10−5 to 1.1×10−3 M, with a correlation coefficient of 0.998. The detection limit is 9.0×10−6 M (at the ratio of signal to noise, S/N=3), demonstrating that the formed film provides a favorable microenvironment for the enzyme to retain its activity. Moreover, the modified electrode displays rapid response to H2O2 and possesses good stability and reproducibility. [Copyright &y& Elsevier]

Abstract: In this work, novel mesoporous silica hollow spheres (MSHS) were chosen as an immobilization matrix, to construct a mediator-free third-generation HRP biosensor. UV–vis spectroscopy revealed that horseradish peroxidase (HRP) entrapped in MSHS could retain its native structure. FTIR spectroscopy and nitrogen adsorption–desorption isotherms indicated that HRP are intercalated into the mesopores. The direct electron transfer of HRP entrapped in MSHS was observed. A pair of stable and well-defined redox peaks of HRP with a formal potential of about −0.150V (vs. Ag/AgCl) in 0.1M pH 7.0 phosphate-buffered solution (PBS) were obtained. The biosensor exhibited a fast amperometric response to H2O2 with a linear range of 3.9×10−6 to 1.4×10−4 M (R =0.997, N =20). The detection limit was 1.2×10−6 M based S/N=3. [Copyright &y& Elsevier]

Abstract: Long term in vitro cultures of plant tissue spontaneously change at morphological, cellular and biochemical level. Horseradish tissues were cultivated in vitro for more than 16 years. Primary crown-gall tumours were induced with Agrobacterium tumefaciens on leaf explants excised from plantlets propagated in vitro. Subcultured on hormone-free MS medium, transformed tissue continued to grow in an unorganized way (TN tumour) or as teratogenous tumour (TM teratoma) forming malformed shoots which never rooted. The aim of this study was to establish the morphological and biochemical status of this long term cultures. The study was performed by verifying biochemical marker of transformation and morphogenesis, which had been investigated when the culture was established (peroxidase, PPX) as well as by introducing new markers which have not been analyzed so far in horseradish tissues (ascorbate peroxidase, APX; phenylalanine-ammonia lyase, PAL; and phenolics content). Morphological changes were observed only in teratoma tissue. Peroxidase activity and isoenzyme pattern indicated stability of horseradish tissue culture. The new biochemical markers were in a complete (PAL) and partial (APX) correlation with the PPX results and are therefore useful addition in biochemical characterisation of these tissues and can also be applied as markers of morphogenesis and tumorization. [Copyright &y& Elsevier]

Abstract: The behavior at low-dose exposure (0.033–0.4kGy) of horseradish peroxidase (HRP) and of two different purified fractions of apple (Jonathan cultivar) peroxidases (named APR1S and APR2S) was studied. The HRP solutions were added with either 0.32M fructose or glucose in order to study their effect on enzymes activity response under γ (137Cs, dose rate 0.4kGy/h) irradiation. The obtained results showed similar behavior between HRP-sugar-added solution and apple fraction with higher oligosaccharides content (APR2S) undergoing low-dose treatment. The same pattern was observed between unglycosylated HRP and APR1S with lower oligosaccharides content. These similarities gave us the possibility to conclude that the presence of oligosaccharides, in more or less quantities, influences in the same way the peroxidases activity, from different plant species, exposed to γ irradiation. [Copyright &y& Elsevier]

Abstract: A new immunoassay strategy for sensitive atrazine determination based on magnetic beads is reported. The immuno-method is a competitive solid-phase immunoassay where the anti-atrazine antibody is immobilized on the magnetic beads surface and fixed at the reaction cell bottom using a simple magnet, which generates a magnetic field. Analyte and HRP (horseradish peroxidase) tracer compete for active sites of antibody. After the immunointeractions antibody-analyte and antibody-tracer, atrazine quantification from the sample is performed by injection of the chemiluminescence substrate (luminol, hydrogen peroxide and p-iodophenol). Different antibodies (polyIgG anti-atrazine Ab I and affinity purified polyIgG anti-atrazine AbI) were tested in this configuration. Also, optimum concentration of antibody-covered magnetic beads was set up (8mg/l Ab II). Finally, the performance of magnetic beads-based immunoassay for atrazine determination was evaluated demonstrating that the magnetic beads-based immunoassay is one of the most sensitive method for atrazine determination (LoD=3pg/l, IC50 =37pg/l, DR=10–1000pg/l). [Copyright &y& Elsevier]

Abstract: Aqueous and micellar catalysis of horseradish peroxidase was studied in a sequential injection system through selecting the oxidation by hydrogen peroxide of two phenothiazines: the water-soluble chlorpromazine and perphenazine, a low water soluble, micellised in a Tween 80 medium. The coloured free-radical intermediate formed was monitored spectrophotometrically at 527nm. The system enables the determination of chlorpromazine in water and perphenazine in micellar medium up to 1.25×10−4 molL−1, with quantification limits of 2×10−5 and 1.25×10−5 molL−1, respectively. R.S.D. values were in both cases less than 1.6%. The SIA system optimized consumed 125μL of sample, 1.5μg of peroxidase and 5×10−8 mol of hydrogen peroxide per determination, which guarantees economy in the use of sample and reagents with reduced residue production, in good agreement with the current recommendations of green chemistry. The developed system was applied to the determination of chlorpromazine and perphenazine in pharmaceutical preparations. Results revealed different reaction rates for aqueous and micellar media, which yielded determination frequencies of 17 and 31 determinations per hour, respectively. The potential effect of several compounds commonly used as excipients on analytical signals was studied and no interfering effect was noticeable. Statistical comparison of the results obtained with the proposed methodology and with the reference methods showed good agreement and indicate no significant difference at the 95% confidence level. [Copyright &y& Elsevier]

Abstract: The structure and activity of native horseradish peroxidase C (HRP) is stabilized by two bound Ca2+ ions. Earlier studies suggested a critical role of one of the bound Ca2+ ions but with conflicting conclusions concerning their respective importance. In this work we compare the native and totally Ca2+-depleted forms of the enzyme using pH-, pressure-, viscosity- and temperature-dependent UV absorption, CD, H/D exchange-FTIR spectroscopy and by binding the substrate benzohydroxamic acid (BHA). We report that Ca2+-depletion does not change the alpha helical content of the protein, but strongly modifies the tertiary structure and dynamics to yield a homogeneously loosened molten globule-like structure. We relate observed tertiary changes in the heme pocket to changes in the dipole orientation and coordination of a distal water molecule. Deprotonation of distal His42, linked to Asp43, itself coordinated to the distal Ca2+, perturbs a H-bonding network connecting this Ca2+ to the heme crevice that involves the distal water. The measured effects of Ca2 + depletion can be interpreted as supporting a structural role for the distal Ca2+ and for its enhanced significance in finetuning the protein structure to optimize enzyme activity. [Copyright &y& Elsevier]

Abstract: The thermal stability of umecyanin, a stellacyanin from horseradish roots, has been investigated by differential scanning calorimetry, optical absorption and fluorescence spectroscopy at neutral and alkaline pH. Above pH 9 the Cu(II) protein experiences a blue shift of the main visible absorption band at ∼600 nm and changes colour from blue to violet. The thermal transition of the protein is irreversible and occurs between 61.4 and 68.8 °C at pH 7.5 and between 50.7 and 57.4 °C at pH 9.8. The calorimetric data indicates that at both pH values the thermally induced transition of the protein between the native and denaturated states can be described in terms of the classical Lumry–Eyring unfolding model Native↔Unfolded→Final. The analysis of the reversible step in the unfolding pathway demonstrates a significant reduction in conformational stability (ΔG) of the alkaline form of the protein. Such a reduction is consistent with an enhanced flexibility of UMC at high pH and has mainly entropic character. [Copyright &y& Elsevier]

Summary: The cAMP response element-binding protein (CREB) is a transcription factor that plays important roles in cellular growth, proliferation and survival. Here, we report that a homologue of CREB transcription factor, Ca-CREB, was identified and functionally characterized in oyster, Crassostrea ariakensis. The full-length cDNA consists of 1397bp with an ORF encoding a 39.3kDa protein. Amino acid sequence analysis revealed that Ca-CREB shares conserved signature motifs with other CREB proteins. Ca-CREB was ubiquitously and constitutively expressed in oyster, and the expression level in hemocytes was higher than that in other tissues. The expression level of Ca-CREB was not modified after RLO stimulation, while tumor necrosis factor-alpha (TNF-α) expression was increased obviously, which was revealed by real-time reverse-transcriptase polymerase chain reaction (RT-PCR). Electrophoretic mobility shift assay (EMSA) and Western blotting showed that recombinant CREB proteins specifically bind the consensus CREB binding site, and DNA-binding activity and phosphorylation of Ca-CREB were induced by RLO. These results suggest that Ca-CREB is a CREB homologue and may be involved in immune responses against RLO. [Copyright &y& Elsevier]

Abstract: In this work, a novel chemiluminescence (CL) flow biosensor for glucose was proposed. Glucose oxidase (GOD), horseradish peroxidase (HRP) and gold nanoparticles were immobilized with sol–gel method on the inside surface of the CL flow cell. The CL detection involved enzymatic oxidation of glucose to d-gluconic acid and H2O2, and then the generated H2O2 oxidizing luminol to produce CL emission in the presence of HRP. It was found that gold nanoparticles could remarkably enhance the CL respond of the glucose biosensor. The enhanced effect was closely related to the sizes of gold colloids, and the smaller the size of gold colloids had the higher CL respond. The immobilization condition and the CL condition were studied in detail. The CL emission intensity was linear with glucose concentration in the range of 1.0×10−5 molL−1 to 1.0×10−3 molL−1, and the detection limit was 5×10−6 molL−1 (3σ). The apparent Michaelis–Menten constant of GOD in gold nanoparticles/sol–gel matrix was evaluated to be 0.3mmolL−1, which was smaller than that of GOD immobilized in sol–gel matrix without gold nanoparticles. The proposed biosensor exhibited short response time, easy operation, low cost and simple assembly, and the proposed biosensor was successfully applied to the determination of glucose in human serum. [Copyright &y& Elsevier]

Abstract: In this study, we report on a promising H2O2 biosensor based on the co-immobilization of horseradish peroxidase (HRP) and chitosan onto Au-modified TiO2 nanotube arrays. The titania nanotube arrays were directly grown on a Ti substrate using anodic oxidation first; a gold thin film was then uniformly coated onto the TiO2 nanotube arrays by an argon plasma technique. The morphology and composition of the fabricated Au-modified TiO2 nanotube arrays were characterized by scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS). Cyclic voltammetry and chronoamperometry were used to study and to optimize the performance of the resulting electrochemical biosensor. The effect of pH, applied electrode potential, the presence of the electron-mediator methylene blue, and the anodic oxidation time of the Ti substrate on the electrochemical biosensor has been systemically studied. Our electrochemical measurements show that the Au-modified TiO2 nanotube arrays provide excellent matrices for the immobilization of HRP and that the optimized electrochemical biosensor exhibits long linearity, a low detection limit, high stability and very good reproducibility for the detection of H2O2. Under the optimized conditions the linearity of the developed biosensor for the detection of H2O2 is observed from 5×10−6 to 4×10−4 moll−1 with a detection limit of 2×10−6 moll−1 (based on the S/N=3). [Copyright &y& Elsevier]

Abstract: An absorption-based surface plasmon resonance (SPRAbs) biosensor probe has been developed for simple and reproducible measurements of hydrogen peroxide using a modified Trinder''s reagent (a chromogenic reagent). The reagent enabled the determination of the hydrogen peroxide concentration by the development of deep color dyes (λ max =630nm) through the oxidative coupling reaction with N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline sodium salt monohydrate (MAOS; C13H20NNaO4S·H2O) and 4-aminoantipyrine (4-AA) in the presence of hydrogen peroxide and horseradish peroxidase (HRP). In the present study, urea as an adduct of hydrogen peroxide for color development could be omitted from the measurement solution. The measurement solution containing 5mM hydrogen peroxide was deeply colored at a high absorbance value calculated as 46.7cm−1 and was directly applied to the SPRAbs biosensing without dilution. The measurement was simply performed by dropping the measurement solution onto the surface of the SPR sensor probe, and the SPRAbs biosensor response to hydrogen peroxide was obtained as a reflectivity change in the SPR spectrum. After investigation of the pH profiles in the SPRAbs biosensor probe, a linear calibration curve was obtained between 1.0 and 50mM hydrogen peroxide (r =0.991, six points, average of relative standard deviation; 0.152%, n =3) with a detection limit of 0.5mM. To examine the applicability of this SPRAbs biosensor probe, 20mM glucose detection using glucose oxidase was also confirmed without influence of the refractive index in the measurement solution. Thus, the SPRAbs biosensor probe employing the modified Trinder''s reagent demonstrated applicability to other analyte biosensing tools. [Copyright &y& Elsevier]

Abstract: Positively charged Ni–Al layered double hydroxide nanosheets (Ni–Al LDHNS) have been used for the first time as matrices for immobilization of horseradish peroxidase (HRP) in order to fabricate enzyme electrodes for the purpose of studying direct electron transfer between the redox centers of proteins and underlying electrodes. X-ray diffraction (XRD) and high-resolution transmission electron microscopy (HRTEM) revealed that the HRP–Ni–Al LDHNS film had an ordered structure and that HRP was intercalated into Ni–Al LDHNS with a monolayer arrangement. Field emission scanning electron microscopy (FESEM) showed that the HRP–Ni–Al LDHNS film had a uniform, porous morphology. UV–vis spectroscopy indicated that the intercalated HRP retained its native structure after incorporation in the Ni–Al LDHNS film. The immobilized HRP in Ni–Al LDHNS on the surface of a glassy carbon electrode (GCE) exhibited good direct electrochemical and electrocatalytic responses to the reduction of hydrogen peroxide (H2O2) and trichloroacetic acid (TCA). The resulting H2O2 biosensor showed a wide linear range from 6.00×10−7 M to 1.92×10−4 M, low detection limit (4.00×10−7 M) and good stability. The results show that Ni–Al LDHNS provide a novel and efficient platform for the immobilization of enzymes and realizing direct electrochemistry and that the materials have potential applications in the fabrication of third-generation biosensors. [Copyright &y& Elsevier]

Abstract: An efficient, sensitive and fast stopped-flow method has been developed to determine asulam in water, based on its inhibition effect on the horseradish peroxidase–luminol–hydrogen peroxide chemiluminescence reaction, (HRP–luminol–H2O2). Ultra fast data acquisition (0.20s) facilitates excellent selectivity because no interferences from concomitants in the matrix act in such short time scale. The precision as repeatability (expressed as relative standard deviation, n =10) was 0.4% at a 40pM level. The detection limit was 1.5pM (0.35ng/L) and 7.15pM in pure and raw water, respectively. The calibration data over the range 5–60pM present a correlation coefficient of r =0.9993. The proposed method has been applied to determine asulam in water samples by using solid-phase extraction (SPE). Mean recovery value was 98.1±2% at 50pM level. [Copyright &y& Elsevier]