Peroxidase Substrate Research Articles

Identification, heterologous expression and characterization of a dye-decolorizing peroxidase of Pleurotus sapidus

The coding sequence of a peroxidase from the secretome of Pleurotus sapidus was cloned from a cDNA library. Bioinformatic analyses revealed an open reading frame of 1551 bp corresponding to a primary translation product of 516 amino acids. The DyP-type peroxidase was heterologously produced in Trichoderma reesei with an activity of 55,000 U L−1. The enzyme was purified from the culture supernatant, biochemically characterized and the kinetic parameters were determined. The enzyme has an N-terminal signal peptide composed of 62 amino acids. Analysis by Blue Native PAGE and activity staining with ABTS, as well as gel filtration chromatography showed the native dimeric state of the enzyme (115 kDa). Analysis of the substrate range revealed that the recombinant enzyme catalyzes, in addition to the conversion of some classic peroxidase substrates such as 2,2′-azino-bis(3-ethylthiazoline-6-sulfonate) and substituted phenols like 2,6–dimethoxyphenol, also the decolorization of the anthraquinonic dye Reactive Blue 5. The enzyme also catalyzes bleaching of natural colorants such as β-carotene and annatto. Surprisingly, β-carotene was transformed in the presence and absence of H2O2 by rPsaDyP, however enzyme activity was increased by the addition of H2O2. This indicates that the rPsaDyP has an oxidase function in addition to a peroxidase activity. As a consequence of the high affinity to the characteristic substrate Reactive Blue 5 the rPsaDyP belongs functionally to the dyp-type peroxidase family.

Enhanced sensitivity of lateral-flow test strip immunoassays using colloidal palladium nanoparticles and horseradish peroxidase

Although lateral-flow test strip (LFTS) immunoassays are rapid and require no specialized equipment, they are less sensitive than culture and polymerase chain reaction (PCR)-based methods for detecting bacteria. This study compared the sensitivity of LFTS assays based on colloidal palladium nanoparticles (PdNPs) and colloidal gold nanoparticles (AuNPs). PdNPs demonstrated a time and concentration dependent oxidation of the horseradish peroxidase (HRP) substrate 3,3′,5,5′-tetramethylbenzidine (TMB), whereas AuNPs did not. PdNPs labeled HRP-conjugated antibody lateral flow test strip (PdNPs-HRP LFTS) assays were tested with 3,3′-diaminobenzidine (DAB), a water insoluble substrate. The sensitivity of PdNPs-HRP LFTS assays in detecting Listeria monocytogenes, Escherichia coli O157:H7 and Yersinia enterocolitica was 5–10-fold higher than that of AuNPs-based LFTS (AuNPs LFTS) assays. PdNPs-HRP LFTS assays of reduced milk inoculated with L. monocytogenes were more than 10-fold more sensitive than conventional AuNPs LFTS assays. These results suggest that the PdNPs-HRP LFTS immunoassay is a promising tool for ensuring food safety.

Introducing Ratiometric Fluorescence to MnO2 Nanosheet-Based Biosensing: A Simple, Label-Free Ratiometric Fluorescent Sensor Programmed by Cascade Logic Circuit for Ultrasensitive GSH Detection.

Glutathione (GSH) plays crucial roles in various biological functions, the level alterations of which have been linked to varieties of diseases. Herein, we for the first time expanded the application of oxidase-like property of MnO2 nanosheet (MnO2 NS) to fluorescent substrates of peroxidase. Different from previously reported fluorescent quenching phenomena, we found that MnO2 NS could not only largely quench the fluorescence of highly fluorescent Scopoletin (SC) but also surprisingly enhance that of nonfluorescent Amplex Red (AR) via oxidation reaction. If MnO2 NS is premixed with GSH, it will be reduced to Mn2+ and lose the oxidase-like property, accompanied by subsequent increase in SC's fluorescence and decrease in AR's. On the basis of the above mechanism, we construct the first MnO2 NS-based ratiometric fluorescent sensor for ultrasensitive and selective detection of GSH. Notably, this ratiometric sensor is programmed by the cascade logic circuit (an INHIBIT gate cascade with a 1 to 2 decoder). And a linear relationship between ratiometric fluorescent intensities of the two substrates and logarithmic values of GSH's concentrations is obtained. The detection limit of GSH is as low as 6.7 nM, which is much lower than previous ratiometric fluorescent sensors, and the lowest MnO2 NS-based fluorescent GSH sensor reported so far. Furthermore, this sensor is simple, label-free, and low-cost; it also presents excellent applicability in human serum samples.

Green synthesized nickel nanoparticles for targeted detection and killing of S. typhimurium

Simple and sensitive colorimetric immunosensor based on peroxidase mimetic activity and photothermal effect of nickel oxide nanoparticle (NiOGs) has been developed to detect and kill food borne pathogen Salmonella typhimurium. NiOGs showed superior peroxidase mimetic activity for oxidation of peroxidase substrate 3, 3′, 5, 5′-tetramethylbenzidine (TMB). Oxidation of TMB by NiOGs followed Michaelis–Menten kinetics with Km and Vmax values of 0.25mM and 2.64×10−8M/s respectively. NiOGs was coated with citric acid (CA–NiOGs) followed by conjugation with antibody (anti-S. typhimurium) (Ab–CA–NiOGs) that effectively captured S. typhimurium. Colorimetric detection of S. typhimurium by Ab–CA–NiOGs showed a linear relationship between pathogen concentration (1×101 to 1×106cfu/mL) and color signal (652nm) with limit of detection (LOD) of 10cfu/mL. The proposed method showed no cross reactivity against other pathogens. Recovery of S. typhimurium in milk and juice samples was found to be 95 to 100% and 92 to 99% respectively. NiOGs exposed to laser irradiation showed dose dependent increase in temperature and singlet oxygen within 5min. Bacteria bound to Ab–CA–NiOGs after laser irradiation, induced membrane damage and reduced bacterial viability to 6%. The bifunctional peroxidase-mimetic activity and photothermal effect of NiOGs can be exploited in selective sensing and killing of target pathogens respectively in food products.

Enhanced peroxidase-like activity of CuWO4 nanoparticles for the detection of NADH and hydrogen peroxide

Herein, we demonstrate for the first time the peroxidase mimicking properties of CuWO4 nanoparticles (CuWO4 NPs) and a simple colorimetric method for the determination of nicotinamide adenine dinucleotide (NADH) based on the inhibition of peroxidase-like properties of CuWO4 NPs. A one-pot hydrothermal process was used to synthesize the CuWO4 NPs and XRD, SEM, HR-TEM and XPS were used to confirm the formation of CuWO4 NPs. The synthesized CuWO4 NPs could catalyze the oxidation of typical peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB) by H2O2 to produce a visible blue color reaction with absorption maxima centered at 652nm. The CuWO4 NPs exhibit superior kinetics over the natural enzyme horse radish peroxidase (HRP) with lower Km value. The CuWO4 NPs peroxidase mimicking system was optimized for NADH sensing in the concentration range of 0.2–380μM (R2=0.98) with a detection limit of 0.2μM. Moreover, the CuWO4 NPs modified glassy carbon electrode (GCE) reduced H2O2 at a lower onsite potential (−200mV) thereby, allowing us to electrochemically sense hydrogen peroxide with a linear range of 24.87μM–3.6mM (R2=0.99) and a sensitivity of 207.64μAmM−1.

Colorimetric determination of copper ions based on regulation of the enzyme-mimicking activity of covalent triazine frameworks

Covalent triazine frameworks (CTFs) have garnered increasing interest recently because of their unique structures and properties. However, their application as enzyme mimics for sensing remains unexplored. In this study, an efficient strategy for selective colorimetric detection of Cu2+ ions was developed based on CTFs. This strategy relies on the peroxidase-like catalytic activity of CTFs being rationally modulated by Cu2+ ions. In the presence of Cu2+ ions, the peroxidase-like catalytic activity of the CTF is significantly stimulated and enhanced; this enables the CTF to catalyze the oxidation of the peroxidase substrate 3,3',5,5'-tetramethylbenzidine, which produces a color change from colorless to blue in the presence of H2O2. On the basis of the colorimetric method, a good linear relationship for Cu2+ ions can be obtained from 1.0μg/L to 80.0μg/L, with a limit of detection of 0.05μg/L. When this strategy was assessed for the detection of Cu2+ ions in real food samples, recoveries of 96.0–105.0% were attained. The proposed CTF material and colorimetric method constitute a promising approach for the rapid detection of Cu2+ ions that is selective, sensitive, and low cost.

β‐AgVO3 Nanorods as Peroxidase Mimetic for Colorimetric Determination of Glucose

β‐AgVO3 nanorods have been demonstrated to exhibit intrinsic peroxidase‐like activity. The oxidation of glucose can be catalyzed by glucose oxidase (GOx) to generate H2O2 in the presence of O2. The β‐AgVO3 nanorods can catalytically oxidize peroxidase substrates including o‐phenylenediamine (OPD), 3,3′,5,5′‐tetramethylbenzidine (TMB), and diammonium 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonate) (ABTS) by H2O2 to produce typical color reactions: OPD from colorless to orange, TMB from colorless to blue, and ABTS from colorless to green. The catalyzed reaction by the β‐AgVO3 nanorods was found to follow the characteristic Michaelis–Menten kinetics. Compared with horseradish peroxidase and AgVO3 nanobelts, β‐AgVO3 nanorods showed a higher affinity for TMB with a lower Michaelis–Menten constant (Km) value (0.04118 mM) at the optimal condition. Taking advantage of their high catalytic activity, the as‐synthesized β‐AgVO3 nanorods were utilized to develop a colorimetric sensor for the determination of glucose. The linear range for glucose was 1.25–60 μM with the lower detection limit of 0.5 μM. The simple and sensitive GOx‐β–AgVO3 nanorods–TMB sensing system shows great promise for applications in the pharmaceutical, clinical, and biosensor detection of glucose.

Fabrication of Ag-Cu2O/Reduced Graphene Oxide Nanocomposites as Surface-Enhanced Raman Scattering Substrates for in Situ Monitoring of Peroxidase-Like Catalytic Reaction and Biosensing.

Highly sensitive biosensors are essential in medical diagnostics, especially for monitoring the state of an individual's disease. An ideal way to achieve this objective is to analyze human sweat secretions by noninvasive monitoring. Due to low concentrations of target analytes in human secretions, fabrication of ultrasensitive detection devices is a great challenge. In this work, Ag-Cu2O/reduced graphene oxide (rGO) nanocomposites were prepared by a facile two-step in situ reduction procedure at room temperature. Ag-Cu2O/rGO nanocomposites possess intrinsic peroxidase-like activity and rapidly catalyze oxidation of the peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H2O2. On the basis of the excellent SERS properties and high peroxidase-like activity of the Ag-Cu2O/rGO nanocomposites, the catalytic oxidation of TMB can be monitored by SERS. This approach can detect H2O2 and glucose with high sensitivity and distinguish between diabetic and normal individuals using glucose levels in fingerprints. Our work provides direction for designing other SERS substrates with high catalytic activity and the potential for application in biosensing, forensic investigation, and medical diagnostics.

Application of enzyme-like activity of Fe-doped ZnS QDs for colorimetric determination of hydrogen peroxide

ABSTRACTFe-doped ZnS quantum dots (QDs) were synthesised using a hydrothermal method. Then, the peroxidase-like catalytic activity of these materials was investigated using the study of the oxidation of colourless peroxidase substrate, 3,3′,5,5′-tetramethylbenzidine, in the presence of H2O2 to produce a blue product. Finally, these new materials were used for determination of H2O2 aqueous samples. The obtained linear range and limit of detection for determination of H2O2 were 4.1–98 and 3.06 µg L−1, respectively. As a result, Fe-doped ZnS QDs could be used for inexpensive and highly sensitive and simple determination of H2O2 in aqueous samples.

Detection of Parathyroid Hormone Receptor 1 (PTH1R): A Novel Approach Using Enzymatic Ligands Applicable to Class B GPCRs

BACKGROUNDClass B GPCRs (receptors of the secretin family) are characterised by long C‐ and N‐terminal domains as well as by their binding of large peptide hormones. One of those, parathyroid hormone (PTH), has been reported to support C‐terminal extension without significant affinity loss for PTH1R. Indeed, the N‐terminal domain of PTH that interacts with the transmembrane domains of PTH1R leading to the activation of this receptor whereas the C‐terminal domain of PTH is involved in affinity interactions. Accordingly, we previously described a fusion protein based on the enhanced green fluorescent protein (EGFP), PTH‐EGFP. This fusion protein supported the detection of recombinant PTH1Rs, but failed to detect endogenous populations of this receptor. The objective of this study is to develop new fusion protein ligands with enzymatic activity which would allow the detection of endogenously expressed PTH1R.METHODSTo detect endogenous populations of PTH1R, we generated two new fusion proteins by linking PTH to the N‐terminal of a peroxidase. These peroxidases were either the horseradish peroxidase (HRP; PTH‐HRP) or the genetically modified soybean peroxidase APEX2 (PTH‐APEX2). Reaction of receptor‐bound enzymatic PTH‐fusion protein with widely available substrates allowed a significant signal amplification resulting in the detection of PTH1R. Alternatively, we also used the previously reported ligand PTH‐myc. The myc epitope (EQKLISEEDL) reacts with a number of monoclonal antibodies, some of them conjugated to a peroxidase. The three PTH‐fusion proteins were produced as conditioned medium (CM) following transfection of producer HEK 293a cells with the appropriate vector.RESULTSThe stimulation of recipient HEK 293a expressing PTH1R with a trimolecular complex composed of PTH‐myc, a monoclonal mouse anti‐myc antibody and an anti‐mouse HRP‐conjugated antibody led to the specific detection of this receptor either using a colorimetric peroxidase substrate (TrueBlue™) or a luminol‐based luminescent peroxidase substrate. These schemes also supported the detection of endogenously expressed receptors in the osteoblastic cell line HOS. The PTH‐APEX2 CM also supported the detection of recombinant populations of PTH1R using the colorimetric or luminescent approach but failed to detect endogenous populations of PTH1R. However, the PTH‐HRP construction was more sensitive and allowed the detection of endogenously expressed PTH1R in HOS and evidenced recombinant PTH1R as well. The specificity of the PTH1R labeling was confirmed by competing the binding of the PTH fusion proteins by an excess of PTH1R ligand (PTH1‐34 1 μM).CONCLUSIONSGiven that anti‐GPCRs antibodies are rarely valid and adapted to detect cell surface receptors, we proposed to detect intact receptors using the high affinity and specificity interaction between the receptor and its ligand. We illustrated this by generating PTH analogs prolonged at their C‐terminal by peroxidases or by an epitope recognized by widely available monoclonal antibodies that support enzymatic detection of PTH1R. Since all class B GPCRs ligands bind their receptor according to the two domain model, it could be possible to generalize the enzymatic ligand strategy to all the class B GPCRs.Support or Funding InformationCHU de Québec ‐ Université Laval, Quebec City, QC, Canada G1V 4G2

Peroxidase-like activity of nanocrystalline cobalt selenide and its application for uric acid detection.

Dendrite-like cobalt selenide nanostructures were synthesized from cobalt and selenium powder precursors by a solvothermal method in anhydrous ethylenediamine. The as-prepared nanocrystalline cobalt selenide was found to possess peroxidase-like activity that could catalyze the reaction of peroxidase substrates in the presence of H2O2. A spectrophotometric method for uric acid (UA) determination was developed based on the nanocrystalline cobalt selenide-catalyzed coupling reaction between N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt and 4-aminoantipyrine (4-AAP) in the presence of H2O2. Under optimum conditions, the absorbance was proportional to the concentration of UA over the range of 2.0–40 μM with a detection limit of 0.5 μM. The applicability of the proposed method has been validated by determination of UA in human serum samples with satisfactory results.

A new peroxidase from leaves of guinea grass (Panicum maximum): A potential biocatalyst to build amperometric biosensors

A new plant peroxidase was isolated from the leaves of guinea grass (Panicum maximum) and partially purified using a biphasic polymer system (poly(ethylene glycol) - ammonium sulfate) followed by size-exclusion chromatography and ultracentrifugation until obtaining a homogeneous extract containing a high peroxidase activity. The novel peroxidase was characterized as having a specific activity of 408U/mg and a molecular weight of 30kDa. The pH for its optimum activity was 8.0 and exhibited a high thermostability at 66°C with a kinact of 8.0×10−3min−1. The best substrates for peroxidase from guinea grass are o-dianisidine and 2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid). POD from guinea grass was directly immobilized on the surface of a graphene screen printed electrode and cyclic voltammograms in the presence of potassium ferrocyanide ([Fe(CN)6]3−/4−) as a redox species demonstrated an increase in the electron transfer process. The graphene- modified electrode exhibits excellent electrocatalytic activity to the reduction of H2O2, with a linear response in the 100μM to 3.5mM concentration range and a detection limit of 150μM. The new peroxidase from guinea grass allowed the modification of a graphene electrode providing a potential sensor detection system for determination of H2O2 in real samples with some biomedical or environmental importance.

Reusable, 3D-printed, peroxidase mimic–incorporating multi-well plate for high-throughput glucose determination

Three-dimensional (3D) printing technologies enable rapid laboratory-scale customization of experimental devices to fit the user’s requirements. To demonstrate how 3D-printed objects can be functionalized through incorporation of reactive substances into the raw materials prior to printing, this paper describes a multi-well plate fabricated using polylactic acid (PLA) filaments that had been treated in advance with iron oxide (Fe2O3, Fe3O4) nanoparticles that possess intrinsic peroxidase activities. The fabricated multi-well plate efficiently catalyzed the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) by the peroxidase substrate hydrogen peroxide and allowed measurements of the resulting absorbance through direct loading of the plate into a plate reader. The applicability of this 3D-printed peroxidase mimic–incorporating multi-well plate has been examined in terms of the device’s reusability and analytical performance, analyses of reaction kinetics, and the high-throughput determination of glucose concentrations in clinical and food samples. The results suggest that this functionalization scheme can diversify the utility of current 3D printing technologies in the fabrication of experimental devices with properties comparable with or superior to those of conventional systems.

Glutathione in plants: biosynthesis and physiological role in environmental stress tolerance.

Glutathione (GSH; γ-glutamyl-cysteinyl-glycine) is a small intracellular thiol molecule which is considered as a strong non-enzymatic antioxidant. Glutathione regulates multiple metabolic functions; for example, it protects membranes by maintaining the reduced state of both α-tocopherol and zeaxanthin, it prevents the oxidative denaturation of proteins under stress conditions by protecting their thiol groups, and it serves as a substrate for both glutathione peroxidase and glutathione S-transferase. By acting as a precursor of phytochelatins, GSH helps in the chelating of toxic metals/metalloids which are then transported and sequestered in the vacuole. The glyoxalase pathway (consisting of glyoxalase I and glyoxalase II enzymes) for detoxification of methylglyoxal, a cytotoxic molecule, also requires GSH in the first reaction step. For these reasons, much attention has recently been directed to elucidation of the role of this molecule in conferring tolerance to abiotic stress. Recently, this molecule has drawn much attention because of its interaction with other signaling molecules and phytohormones. In this review, we have discussed the recent progress in GSH biosynthesis, metabolism and its role in abiotic stress tolerance.

Facile Colorimetric Detection of Silver Ions with Picomolar Sensitivity.

Although various colorimetric methods have been actively developed for the detection of Ag+ ions because of their simplicity and reliability, the limits of detection of these methods are confined to the nanomolar (nM) level. Here, we demonstrate a novel strategy for colorimetric Ag+ detection with picomolar (pM) sensitivity. This strategy involves the use of poly(vinylpyrrolidone)- (PVP-) capped Pt nanocubes as artificial peroxidases that can effectively generate a colored signal by catalyzing the oxidation of peroxidase substrates. In the presence of Ag+ ions, the colored signal generated by these Pt cubes is greatly diminished because of the specific and efficient inhibition of Ag+ toward the peroxidase-like activity of the Pt cubes. This colorimetric method can achieve an ultralow detection limit of 80 pM and a wide dynamic range of 10-2-104 nM. To the best of our knowledge, the method presented in this work shows the highest sensitivity for Ag+ detection among all reported colorimetric methods. Moreover, this method also features simplicity and rapidness as it can be conducted at room temperature, in aqueous solution, and requires only ∼6 min.

Facile synthesis of enzyme functional metal-organic framework for colorimetric detecting H2O2 and ascorbic acid

In this work, a metal-organic frameworks material MIL-88 was prepared easily using solvent-thermal method, and was first found to have catalytic activities similar to those of biological enzymes such as catalase and peroxidase. The material was characterized by XRD, SEM, TEM, EDX, FT-IR techniques and an N2 adsorption method. It exhibited peroxidase-like activity through catalytic oxidation of the peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of H2O2, producing a blue-colored solution. Under optimal conditions, the absorbance at 652nm is linearly correlated with the concentration of H2O2 from 2.0×10−6mol/L to 2.03×10−5mol/L (R2=0.981) with a detection limit of 5.62×10−7mol/L (S/N=3). More importantly, a sensitive and selective method for ascorbic acid detection was developed using this material as a catalyst. The analytical method for ascorbic acid detection was observed to have a linear range from 2.57×10−6mol/L to 1.01×10−5mol/L (R2=0.989) with a detection limit of 1.03×10−6mol/L (S/N=3). This work suggests MOFs have advantages of preparing biomimetic catalysts and extends applications of the functional MOFs in the field of biosensor.

Colorimetric detection of glutathione in cells based on peroxidase-like activity of gold nanoclusters: A promising powerful tool for identifying cancer cells

Glutathione (GSH), the most abundant biothiol in cells, not only plays a pivotal role in protective and detoxifying functions of the cell, but also serves as a very important mediator in many cellular functions. Especially, the difference of GSH level between cancer cells and normal cells is regarded as one of most important physiological parameters for cancer diagnosis. It is thereby extremely necessary to develop a simple, sensitive, and reliable analytical method for detection of GSH in cells. On the basis of the inhibition effect of GSH on the peroxidase-like activity of GSH stabilized gold nanoclusters, here a novel and facile strategy for colorimetric detection of cellular GSH level was well established. In this sensing system, GSH can effectively inhibit the oxidation of peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB) to produce a blue colored product. Under the optimized conditions, the absorbance at 652 nm against GSH concentration shows a linear relationship within a range from 2 to 25 μM with detection limit of 420 nM. This excellent property allows our approach to be used to accurately evaluate the cellular GSH levels, and it is revealed that the overall GSH level in cancer cells was much higher than that in normal cells. The presented assay will enable a powerful tool for identifying cancer cells in a simple manner for biomedical diagnosis associated with GSH.

Facile Synthesis of Co<sub>3</sub>O<sub>4</sub> Nanoparticles and their Biomimetic Activity

Co3O4 nanoparticles with biomimetic activity have been synthesized using inorganic precursor by a simple liquid precipitation method without use of any organic and assisted agents. XRD and IR results showed that the as-prepared product was well-crystalline cubic Co3O4 phase with a lattice constant of a=8.079Å. The TEM image showed that the obtained Co3O4 product possesses a spherical and rectangular shape with a diameter ranging from 10 to 20nm. The Co3O4 nanoparticles catalyzed oxidation of typical peroxidase substrate (ABTS) and 1,3,5-trimethylbenzene (TMB) to give the same color changes as microperoxidase -11( MP-11), respectively. And the catalysis by Co3O4 nanoparticles shows typical Michaelis-Menten curves similar to that of MP-11. These results demonstrate that the Co3O4 nanoparticles possess a biomimetic catalysis activity similar to that found in natural enzyme. The kinetic parameters showed Co3O4 nanoparticles had efficient catalytic activity in comparison to MP-11.The easy preparation, good stability and special properties of Co3O4nanoparticles make them the promising enzyme mimics.

Intrinsic Peroxidase-like Activity of Ficin

Ficin is classified as a sulfhydryl protease isolated from the latex of fig trees. In most cases, a particular enzyme fits a few types of substrate and catalyzes one type of reaction. In this investigation, we found sufficient proofs for the intrinsic peroxidase-like activity of ficin and designed experiments to examine its effectiveness in a variety of scenarios. Ficin can transform peroxidase substrates to colored products in the existence of H2O2. Our results also indicate that the active sites of peroxidase-like activity of ficin are different from that of protease, which reveals that one enzyme may catalyze more than one kind of substrate to perform different types of reactions. On the basis of these findings, H2O2 releasing from MCF-7 cells was detected successfully. Our findings support a wider application of ficin in biochemistry and open up the possibility of utilizing ficin as enzymatic mimics in biotechnology and environmental monitoring.

Detection of antibodies against hepatitis B virus surface antigen and hepatitis C virus core antigen in plasma with a waveguide-mode sensor

In large-scale disasters, such as huge significant earthquakes, on-site examination for blood typing and infectious disease screening will be very helpful to save lives of victims who need surgical treatment and/or blood transfusion. However, physical damage, such as building collapse, electric power failure and traffic blockage, disrupts the capacity of the medical system. Portable diagnostic devices are useful in such cases of emergency. In this study, we evaluated a waveguide-mode sensor for detection of anti-hepatitis virus antibodies. First, we examined whether we can detect antigen-antibody interaction on a sensor chip immobilized hepatitis B virus surface (HBs) antigen and hepatitis C virus (HCV) core antigen using monoclonal mouse antibodies for HBs antigen and HCV core antigen. We obtained significant changes in the reflectance spectra, which indicate specific antigen-antibody interaction for anti-HBs antibody and anti-HCV antibody. Next, we examined the effect of horseradish peroxidase-conjugated secondary antibody using aminoethyl carbazole as the peroxidase substrate and found that the colorimetric reaction increases detection sensitivity for anti-HBs antibody more than 300 times. Finally, we successfully detected anti-HBs antibody in human blood samples with an enhancing method using a peroxidase reaction. Thus, a portable device utilizing a waveguide-mode sensor may be applied to on-site blood testing in emergency settings.