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In this paper, a novel label-free G-quadruplex DNAzyme sensor has been proposed for colorimetric identification of GMO using CaMV 35S promoter sequence as the target. The binary probes can fold into G-quadruplex structure in the presence of DNA-T (Target DNA) and then combine with hemin to form a DNAzyme resembling horseradish peroxidase. The detection system consists of two G-rich probes with 2:2 split mode by using the absorbance and color of ABTS2- as signal reporter. Upon the addition of a target sequence, two probes both hybridize with target and then their G-rich sequences combine to form a G-quadruplex DNAzyme, and the DNAzyme can catalyze the reaction of ABTS2- with H2O2. Then the linear range is from 0.05 to 0.5 μM while detection limit is 5 nM. These results demonstrate that the proposed G-quadruplex DNAzyme method could be used as a simple, sensitive and cost-effective approach for assays of GMO. [ABSTRACT FROM AUTHOR]

Abstract: Using a full factorial design the optimization of experimental conditions of enhanced chemiluminescence reaction (ECR) catalyzed by horseradish peroxidase (HRP-C) in the presence of 3-(10′-phenothiazinyl)propane-1-sulfonate (SPTZ) and 4-morpholinopyridine (MORP) as enhancers was performed. The effect of concentrations of SPTZ, hydrogen peroxide, MORP, luminol, and Tris on a ratio of peroxidase-catalyzed CL to background was studied. The use of the full 25 factorial design instead of “one-variable-a time” method allowed to increase the sensitivity of HRP-C determination 2355 fold without a change of detection limit. The obtained results open up very promising perspectives for using HRP-C-catalyzed ECR to improve the sensitivity of chemiluminescent enzyme immunoassay. [Copyright &y& Elsevier]

Abstract: A simple competitive enzyme-linked immunosorbent assay (cELISA) was established for rapid measurement of secretory immunoglobulin A (sIgA) in saliva. The method was based on competitive reaction between the immobilized IgA and free IgA in the solution for the limited amount of horseradish peroxidase-conjugated rabbit anti-human IgA. In comparison with the conventionally used Sandwich ELISA, the cELISA is simpler, low-cost, and shows better reproducibility since it is not affected by the variation of capture antibodies from different batches. The assay time was also significantly reduced from more than 5h to less than 3h. Different curve-fitting models were compared, among which the fully specified logit-log model gave the best results. The linear working range and limit of detection were found to be 0.1–100μgmL−1 and 0.05μgmL−1, respectively. Matrix effects of saliva samples were investigated and a reasonable range of dilution factors were proposed. The developed method offers a very practical approach for high-throughput measurement of sIgA in saliva samples. [Copyright &y& Elsevier]

Abstract: We developed a highly sensitive colorimetric enzyme immunoassay for the detection of carcinoembryonic antigen (CEA). This method employed gold nanoparticles (AuNPs) as carriers of anti-CEA antibody labeled with biotin, which served as an affinity tag for streptavidin-horseradish peroxidase (streptavidin-HRP) binding. Using this strategy, about 12 HRP molecules were coated onto each AuNP. Through “sandwich-type” immunoreaction, the AuNP–anti-CEA–HRP complex was brought into the proximity of magnetic microparticle. As a result, HRP molecules confined at the surface of the “sandwich” immunocomplexes catalyzed the enzyme substrate and generated an optical signal. The spectrophotometric measurement confirmed effective signal amplification. The signals were linearly dependent on CEA concentrations from 0.05 to 50ngmL−1 in a logarithmic plot, with a detection limit of 48pgmL−1. Intra- and inter-assay coefficients of variation were <8.5%. The CEA concentrations of serum specimens assayed by the developed immunoassay showed consistent results in comparison with those obtained by a conventional enzyme-linked immunosorbent assay. The developed method thus proved its potential use in clinical immunoassay of CEA. [Copyright &y& Elsevier]

Abstract: Progesterone (P4) is a steroidal hormone with a vital role in the maintenance of human and animal health. This paper describes the development of an immunosensor coupled to glassy carbon (GC) electrode and integrated to a microfluidic system to quantify P4 from bovine serum samples in a fast and sensitive way. The serum samples spiked with a given P4 concentration and a given P4 concentration bound to horseradish peroxide (HPR) were simultaneously added and, therefore, they competed immunologically with sheep monoclonal anti-P4 antibodies that were immobilized at a rotating disk. HRP in the presence of hydrogen peroxide (H2O2) catalyzes the chatecol (H2Q) oxidation to benzoquinone (Q). Its reverse electrochemical reduction to H2Q can be detected at a GC electrode surface at −0.15V by chronoamperometric measurements. These current responses are proportional to the enzyme activity and inversely proportional to the P4 amount present in bovine serum samples. This P4 immunosensor showed a linear working range from 0.5 to 12.5ngmL−1. The detection (DL) and quantification (QL) limits were 0.2 and 0.5ngmL−1, respectively. The electrochemical immunosensor had a higher sensitivity than the ELISA method using conventional spectrophotometric detections. However, both methods allowed us to obtain similar detection limits. The immunosensor allowed us to make up to 100 determinations on different samples without any previous pre-treatment. This behavior proved to be suitable to detect P4 in routine veterinary, clinical, biological, physiological, and analytical assays. [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: 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: 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: 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: 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: 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]

Abstract: In the present study, three luminol signal enhancers {4-methoxyphenol, 4-hydroxybiphenyl and 4-(1H-pyrrol-1-yl)phenol} were utilized in the chemiluminescence (CL) substrate solution of horseradish peroxidase (HRP). The latter was applied in a heterogenous enzyme immunoassay that has been previously described. The employment of these molecules greatly affected important assay parameters, such as detection limit and the range of the calibration curve and the results were compared with those obtained from other two similar enhancers that have been described from our group. Practically, the use of a novel enhancer, even if this is a slightly changed 4-substituted phenol derivative, can affect assay properties so dramatically, one can assume that another substrate/enzyme system was applied. Furthermore, the use of different luminol signal enhancers in the luminol/HRP/H2O2 system affected not only the intensity of the obtained signal, which is well known, but also its kinetics. It was monitored that the stronger intensity was combined with a more rapid decrease of the CL signal. [Copyright &y& Elsevier]

Abstract: Peroxynitrite was one of the important reactive oxygen species (ROS) which have been focused for many years. Peroxynitrite is an extremely strong and reactive oxidant which can cause many diseases linked to inflammatory processes and autoimmune diabetes, etc. ROS can significantly react with peroxidase and nicotinamide adanine dinucleotide (NADH) which is linked to numerous biological processes. NADH, NAD and horseradish peroxidase (HRP) were included in peroxidase–oxidase (PO) oscillation, dissolved oxygen was concerned with the oscillation and many ROS intermediates came into being in PO oscillation which was sensitive to the perturbation of ROS. The influence of peroxynitrite (ONOO−) on this oscillation system was investigated. It was found that the oscillation amplitude increased when the system was perturbed with peroxynitrite. There was a linear relationship between the increment ratio in the oscillation amplitude and perturbing peroxynitrite concentration in the range 2.50×10−8 to 1.56×10−6 mol/L. And further experimental results revealed that amplitude increasing may be caused by the effect of peroxynitrite on HRP. Based on this phenomenon, a highly sensitive method for the determination of peroxynitrite was developed. [Copyright &y& Elsevier]

An amperometric biosensor was prepared by in situ deposition of horseradish peroxidase (HRP) enzyme on a polyaniline (PANI)-doped platinum disk electrode. The PANI film was electrochemically deposited on the electrode at 100 mV s-1/Ag–AgCl. Cyclic voltammetric characterization of the PANI film in 1 M HCl showed two distinct redox peaks, which prove that the PANI film was electroactive and exhibited fast reversible electrochemistry. The surface concentration and film thickness of the adsorbed electroactive species was estimated to be 1.85×10-7 mol cm-2 and approximately 16 nm, respectively. HRP was electrostatically immobilized onto the surface of the PANI film, and voltammetry was used to monitor the electrocatalytic reduction of hydrogen peroxide under diffusion-controlled conditions. Linear responses over the concentration range 2.5×10-4 to 5×10-3 M were observed. Spectroelectrochemistry was used to monitor the changes in UV–vis properties of HRP, before and after the catalysis of H2O2. The biosensor surface morphology was characterized by scanning electron microscopy (SEM) using PANI-doped screen-printed carbon electrodes (SPCEs) in the presence and absence of (i) peroxidase and (ii) peroxide. The SEM images showed clear modifications of the conducting film surface structure when doped with HRP, as well as the effect of hydrogen peroxide on the morphology of biosensor. [Copyright &y& Elsevier]

Abstract: Horseradish peroxidase (HRP) and glucose oxidase (GOD) were immobilized by sunlight onto the photoreactive cellulose membrane prepared by the reaction of cellulose membrane with 1-fluoro-2-nitro-4-azidobenzene (FNAB). A correlation between sunlight intensity and immobilization was studied. Sunlight intensity required for optimum immobilization was found to be 21,625lux beyond which no appreciable increase in immobilization was observed. Around 2.5-fold increase in absorbance value was observed when HRP immobilization was carried out by sunlight than in dark or on untreated surface. Sunlight exposure gave better immobilization compared to 365nm UV light. Thus, sunlight could be used as a potential alternative to UV light for immobilization of biomolecules such as carbohydrate, DNA or protein. [Copyright &y& Elsevier]