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Nanomaterial-based peroxidase-mimetics are an emerging research field that promises to produce alternatives to horseradish peroxidase for a variety of applications. Generally, some peroxidase-mimetics substrates are used in acidic condition (pH ≤ 7). Then, it is necessary to screen some peroxidase-mimetics substrates suitable for basic condition because that some peroxidase-mimetics leached ions in acidic solution. In this paper, using Ni/Co layered double hydroxides (LDHs) as a nano-peroxidase mimic model, we evaluated three fluorogenic substrates suitable for basic condition though experimental conditions, reaction kinetic and glucose detection assay. And the detection of glucose method based on homovanillic acid (HVA) as fluorescent substrate gave wide linear range (0.02–20 μM) and low detection limit (0.01 μM). We also developed a novel platform that could study the inhibitory effects of ascorbic acid and glutathione based on the system of Ni/Co LDHs-HVA-H 2 O 2 . [ABSTRACT FROM AUTHOR]

Abstract: A gel-based non-instrumental immuno-affinity assay was developed for the rapid screening of chloramphenicol (CAP) in food samples with the limit of detection (LOD) of 1μgL−1. The immuno-affinity test column (IATC) consisted of a test layer containing anti-CAP antibody coupled gel, and a control layer with anti-HRP antibody coupled gel. Based on the direct competitive immuno-reaction and the horseradish peroxidase enzymatic reaction, the test results could be evaluated visually. Basically, blue color development represented the negative results, while the absence of color development represented the positive results. In this study, CAP spiked samples of raw milk, pasteurized milk, UHT milk, skimmed milk powder, acacia honey, date honey, fish and shrimp were tested. Little or none sample pretreatment was required for this assay. The whole procedure was completed within 10min. In conclusion, the gel-based immuno-affinity test is a simple, rapid, and promising on-site screening method for CAP residues in food samples, with no instrumental requirement. [Copyright &y& Elsevier]

Abstract: This work summarizes the manufacturing procedure of Horseradish peroxidase (HRP) based biosensors for the determination of the mycotoxin Ochratoxin A (OTA). The biosensors have been fabricated using the single technology of screen-printing. That is to say, an HRP containing ink has been directly screen-printed onto carbon electrodes, which offers a higher rapidity and simplicity in the manufacturing process of biosensors for OTA determination. The formal redox potential of the Fe(III/II) moiety of HRP has been used to demonstrate the effective loading of enzyme into the ink. The chronoamperometric oxidation current registered has been successfully related to the concentration of OTA in solution from different samples, including beer ones. Under the optimum conditions of the experimental variables, precision in terms of reproducibility and repeatability has been calculated in the concentration range from 23.85 to 203.28nM. A relative standard deviation for the slopes of 10% (n =4) was obtained for reproducibility. In the case of repeatability, the biosensor retained a 30% of the initial sensitivity after the third calibration. The average capability of detection for 0.05% probabilities of false positive and negative was 26.77±3.61nM (α =0.05 and β =0.05, n =3). [Copyright &y& Elsevier]

Abstract: A new composite material consisted of hyaluronic acid (HA), ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM]BF4) and cadmium sulfide (CdS) nanorod was fabricated and used for the immobilization of horseradish peroxidase (HRP) on the surface of a carbon ionic liquid electrode (CILE), which was prepared with 1-ethyl-3-methyl-imidazolium ethylsulphate ([EMIM]EtOSO3) as modifier. Spectroscopic results indicated that HRP remained its native structure in the composite film. Based on the synergistic effect of the materials used, an obvious promotion to the direct electron transfer efficient between HRP and CILE was achieved with a pair of well-defined redox peaks appeared in 0.1molL−1 phosphate buffer solution, indicating the realization of the direct electrochemistry of HRP. The immobilized HRP showed good electrocatalytic activity towards the reduction of trichloroacetic acid and H2O2 with the electrochemical parameters calculated. Based on the fabricated electrode, a new third-generation electrochemical biosensor was constructed with good stability and reproducibility. [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: A competitive enzyme-linked immunosorbent assay for dopamine (DA) has been optimized and characterized. DA is sensitive to oxygen and light according to a function of the pH on the DA oxidation process. The phenolic groups in DA are readily oxidisable to a quinoid form and thus, free DA deteriorates in alkaline media. Thus, effect of factors such as pH, enzyme-label with substrate, ionic strength and reaction time was considered on performance of ELISA. Assay was performed with 5μgmL−1 of BSA–DA and 1/7500 dilution of anti-DA antibody. A dose–response curve was constructed, and a limit of detection and a dynamic range for DA were accomplished to 1.0×10−9 M (0.19μgL−1) and five orders (3.2×10−8 M to 3.2×10−3 M) of magnitude, respectively. The correlation diagram of the absorbance obtained both in buffer and in serum has shown good agreement with correlation coefficient (R 2 =0.9947): Abs. (in serum)=0.6128×Abs. (in buffer)+0.2926. The cross-reactivity was examined with the structurally similar compounds. And the results demonstrated that epinephrine and 3-methoxytyramine showed cross-reactivity (18.9% each), whereas 3,4-dihydroxyphenylacetic acid and homovanillic acid showed low cross-reactivity (<1%). And percent recoveries of DA in serum were quite satisfactory. This provides usefulness of the present assay to monitor DA in serum. [Copyright &y& Elsevier]

Abstract: An unmodified gold nanoparticle-based colorimetric assay system in homogeneous format has been developed using hydrogen peroxide (H2O2) as a model analyte. H2O2 is added to o-phenylenediamine/horseradish peroxidase solution, and allowed to react for 10min. Then, unmodified gold nanoparticles that serve as “reaction indicators” are added to the reaction solution. The resulting mixture color changes dramatically from red to blue. The reason is that azoaniline, a horseradish peroxidase-catalyzed oxidation product, induces the nanoparticle aggregation. Using this approach, H2O2 can be semiquantitatively determined over the concentration range of ∼4 orders of magnitude by the naked eye. If the observed peak intensity at 420nm is used for the construction of the calibration plot, hydrogen peroxide can be accurately determined down to concentration levels of 1.3×10−6 M. Compared with the conventional electrochemical protocol, this sensing system offers several important advantages: (1) ability to be monitored by the naked eye, (2) avoiding the need of surface modification of electrodes or gold nanoparticles and (3) detection in homogeneous solution. It is worthy of note that this efficient and convenient strategy is also suitable for the detection of other species, such as glucose and cholesterol. [Copyright &y& Elsevier]

Abstract: A direct competitive chemiluminescent immunosensor system that exploits the competition between chloramphenicol (CAP) as an analyte and CAP–horseradish peroxidase conjugate as a tracer for binding to an anti-CAP antibody on a solid support was devised by installing a flow-through cell which was connected to an injector and a peristaltic pump inside a dark box, followed by positioning a photomultiplier tube as light detector in front of it. The anti-CAP antibody was immobilized onto positively charged Biodyne B membrane pieces by a dipping procedure. The operating conditions for the immunosensor were selected with respect to substrate composition (0.25, 13.3 and 0.66mM for luminol, H2O2 and p-iodophenol, respectively), injection volume of the substrate solution (200μL) and the concentrations of antibody for immobilization (0.10mgmL−1) and tracer (0.030mgmL−1). At these conditions, sensor response according to analyte concentration was well fitted to a linear equation when plotted in semi-logarithmic scale, with the limit of detection for CAP of 10−8 M. By using the immunosensor, CAP measurement in the model samples prepared from five food materials was conducted. [Copyright &y& Elsevier]

Abstract: A screen-printed carbon electrode modified with both HRP and LOD (SPCE–HRP/LOD) has been developed for the determination of l-lactate concentration in real samples. The resulting SPCE–HRP/LOD was prepared in a one-step procedure, and was then optimised as an amperometric biosensor operating at [0, −100]mV versus Ag/AgCl for l-lactate determination in flow injection mode. A significant improvement in the reproducibility (coefficient variation of about 10%) of the preparation of the biosensors was obtained when graphite powder was modified with LOD in the presence of HRP previously oxidised by periodate ion (IO4 −). Optimisation studies were performed by examining the effects of LOD loading, periodation step and rate of the binder on analytical performances of SPCE–HRP/LOD. The sensitivity of the optimised SPCE–HRP/LOD to l-lactate was 0.84nALμmol−1 in a detection range between 10 and 180μMol. The possibility of using the developed biosensor to determine l-lactate concentrations in various dairy products was also evaluated. [Copyright &y& Elsevier]

Abstract: As the mycotoxin ochratoxin A (OTA) can occur in roasted coffee, regulations and maximum limits are necessary. The need for simple, rapid and inexpensive detection methods which require a minimum of equipment led to the development of a clean-up tandem assay column for the detection of OTA in roasted coffee. In this study the column comprised two superposed layers: a layer capable of adsorbing at least part of the interfering fraction of the sample and a detection layer containing antibodies that were able to capture the analyte. No expensive instruments were needed as results were visually evaluated. The cut-off level, i.e. the smallest mycotoxin concentration resulting in no colour development, was 6μgkg-1. Assay validation was performed using samples fortified with OTA. The method gave a low percentage of false negative results and no false positive results. Assay performance was evaluated by screening naturally contaminated samples using the clean-up tandem assay column. The described method offers a rapid, simple and cost-effective screening tool, contributing to more effective quality control procedures. [Copyright &y& Elsevier]

Abstract: 3-Indoxyl phosphate (3-IP) is the first common electrochemical substrate of the two most important label enzymes in immunoassays: alkaline phosphatase (AP) and horseradish peroxidase (HRP). The product of the enzymatic reactions is indigo blue, an aromatic heterocycle compound insoluble in aqueous solutions. Through the reduction of water insoluble indigo in an alkaline media and in presence of a dithionite salt (Na2S2O4), a water-soluble compound, called leucoindigo, is obtained. This solubilisation methodology, widely used in the textile dyeing industry, is applied in this work for the measurement of AP and HRP activity. Leucoindigo is electroactive and also develops a rich yellow color in solution with a maximum absorbance at 415nm, so 3-IP is shown also as the first common chromogenic substrate for HRP and AP. Through an electrochemical (alternating current adsorptive stripping voltammetry) and a spectrophotometric detection of leucoindigo, picomolar levels of AP and HRP in solution can be measured. Using this approach, two ELISA assays to quantify pneumolysin (PLY) and osteocalcin (BGP) were carried out using AP and HRP, respectively as enzymatic label. Therefore, the suitability of the detection of leucoindigo in ELISAs that employ 3-IP as substrate is demonstrated. This approach can unify solutions and procedures to develop AP and HRP based affinity devices. [Copyright &y& Elsevier]

The flow-injection analysis of uric acid was developed based on the enhanced effect of horseradish peroxidase (HRP) on the luminol–hydrogen peroxide chemiluminescence (CL) system. With the assistance of uricase and HRP-coupled Sepharose columns, a detection limit of 5.0×10−8 M (1.5 μg l−1) and a dynamic range of 5.0×10−8 to 2.5×10−4 M can be obtained. A repetitive injection of 1.0×10−5 M uric acid solutions gave satisfactory reproducibility with a relative standard deviation of 2.14% (n=20). Possible interferences from major bioactive molecules in human fluids and other related compounds were studied. Uric acid in a standard serum sample and a urine sample was analyzed. The uric acid content of a standard reference serum sample obtained from the developed CL analysis agrees well with the certified value and recoveries of 95.4 to 105.4% were obtained for spiking the standard uric acid to the urine samples. The feasibility of the developed method for uric acid analysis in human fluids was confirmed. [Copyright &y& Elsevier]