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Ascorbic acid plays a significant role in regulation of various bodily functions with high concentrations in immune cells and being involved in connective tissue maintenance. Commonly it is detected through various colorimetric methods. In this study, we propose a one-step simple method based on the inhibitory activity of ascorbic acid on horseradish peroxidase and hydrogen peroxide. The detection is observed by colorimetric changes to TMB (3,3′,5,5′ tetramethylbenzidine). The enzyme inhibition unit was optimized with a high level of linearity (r2 = 0.9999) and the level of detection and level of quantification were found to be 1.35 nM and 4.08 nM, respectively with higher sensitive compared to the HPLC method (11 μM). Both intra and inter-assays showed high correlations at different AA concentrations. (r2 > 0.9999). Similar results were also observed for vitamin C tablets, ascorbate salts, fruits, and market products (r2 = 0.999). There was negligible effect of interference by citric acid, lactic acid, tartaric acids, and glucose with high recoveries (>98%) at 1 mg/mL to 0.0078 mg/mL concentration ranges. The recovery error (RE%) was found to be less than 10%. Our detection method is distinguished by its simplicity, nano-level of detection, reproducibility, and potential application and adaptability as a point-of-use test.

Background: The AA9 (auxiliary activities) family of lytic polysaccharide monooxygenases (AA9 LPMOs) is a ubiquitous and diverse group of enzymes in the fungal kingdom. They catalyse the oxidative cleavage of glycosidic bonds in lignocellulose and exhibit great potential for biorefinery applications. Robust, high-throughput and direct methods for assaying AA9 LPMO activity, which are prerequisites for screening LPMOs with excellent properties, are still lacking. Here, we present a gluco-oligosaccharide oxidase (GOOX)-based horseradish peroxidase (HRP) colorimetric method for assaying AA9 LPMO activity. Results: We cloned and expressed a GOOX gene from Sarocladium strictum in Trichoderma reesei, purified the recombinant SsGOOX, validated its properties, and developed an SsGOOX-based HRP colorimetric method for assaying cellobiose concentrations. Then, we expressed two AA9 LPMOs from Thielavia terrestris, TtAA9F and TtAA9G, in T. reesei, purified the recombinant proteins, and analysed their product profiles and regioselectivity towards phosphoric acid swollen cellulose (PASC). TtAA9F was characterized as a C1-type (class 1) LPMO, while TtAA9G was characterized as a C4-type (class 2) LPMO. Finally, the SsGOOX-based HRP colorimetric method was used to quantify the total concentration of reducing lytic products from the LPMO reaction, and the activities of both the C1- and C4-type LPMOs were analysed. These LPMOs could be effectively analysed with limits of detection (LoDs) less than 30 nmol/L, and standard curves between the A515 and LPMO concentrations with determination coefficients greater than 0.994 were obtained. Conclusions: A novel, sensitive and accurate assay method that directly targets the main activity of both C1- and C4-type AA9 LPMOs was established. This method is easy to use and could be performed on a microtiter plate for high-throughput screening of AA9 LPMOs with desirable properties. [ABSTRACT FROM AUTHOR]

Thermo-responsive textiles with moisture/thermal management characteristics have attracted extensive attention for the purpose of improving human comfort or special job requirements. In this study, a thermo-responsive cotton (TR-cotton) fabric for moisture/thermal management was prepared by horseradish peroxidase/acetylacetone (ACAC)/hydrogen peroxide (H2O2)-initiated "graft from" polymerization on the fiber surface. Enzymatic graft polymerization is a new environmentally friendly method to prepare thermo-responsive textiles. Diethylene glycol monomethyl ether methacrylate (MEO2MA) and poly(ethylene glycol) methyl ether methacrylate (OEGMA500) were selected as thermosensitive monomers for graft copolymerization. The lower critical solution temperature (LCST) of P(MEO2MA-co-OEGMA500) was 29 °C. TR-cotton was hydrophilic at ambient temperature below the LCST and hydrophobic at a temperature above the LCST. The air/moisture permeability of TR-cotton at a high temperature (> LCST) was better than that at a low temperature (< LCST). In a cold environment, the fabric exhibited a heat preservation effect (~ 1.5 °C warmer than that observed for unmodified cotton fabric). This study could provide an enzyme catalysis method for the development of "smart textiles". [ABSTRACT FROM AUTHOR]

Ascorbic acid plays a significant role in regulation of various bodily functions with high concentrations in immune cells and being involved in connective tissue maintenance. Commonly it is detected through various colorimetric methods. In this study, we propose a one-step simple method based on the inhibitory activity of ascorbic acid on horseradish peroxidase and hydrogen peroxide. The detection is observed by colorimetric changes to TMB (3,3',5,5' tetramethylbenzidine). The enzyme inhibition unit was optimized with a high level of linearity (r 2  = 0.9999) and the level of detection and level of quantification were found to be 1.35 nM and 4.08 nM, respectively with higher sensitive compared to the HPLC method (11 μM). Both intra and inter-assays showed high correlations at different AA concentrations. (r 2  > 0.9999). Similar results were also observed for vitamin C tablets, ascorbate salts, fruits, and market products (r 2  = 0.999). There was negligible effect of interference by citric acid, lactic acid, tartaric acids, and glucose with high recoveries (>98%) at 1 mg/mL to 0.0078 mg/mL concentration ranges. The recovery error (RE%) was found to be less than 10%. Our detection method is distinguished by its simplicity, nano-level of detection, reproducibility, and potential application and adaptability as a point-of-use test., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Teerapol Srichana has patent #10202302127Y pending to Intellectual Property Office of Singapore. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

We have developed and tested a novel ELISA‐like approach for bacterial detection based upon selective adhesion of targeted bacteria to microwells with prefabricated bacterial bioimprints. Bioimprints were produced from three bacterial species; Escherichia coli (Gram‐negative), Rhodococcus rhodochrous (Gram‐positive) and Sarcina aurantiaca (Gram‐negative), by using molding with curable silicone from dense layers of bacterial cells deposited on a glass substrate. We demonstrated that the surface functionalized whole cell bioimprints were able to selectively recognize and bind their own bacterial cell type. In order to detect target bacteria that are bound to the matching bioimprint, we also developed silica nanoparticles dual‐functionalized with (3‐glycidyloxypropyl)trimethoxysilane (GLYMO) coupled with 4‐hydroxyphenylboronic acid (4‐HPBA), SiO2NPs/GLYMO/4‐HPBA, which were further conjugated with horseradish peroxidase (HRP). Bacterial detection was demonstrated to work in the established ELISA‐like protocol using the colorimetric reaction of the conjugated HRP with 3,3′,5,5′‐tetramethylbenzidine (TMB). The bioimprints were used instead of capture antibodies and HRP‐coated dual functionalized silica nanoparticles instead of a secondary antibody with TMB as the enzyme‐converted reagent, producing a colored byproduct. This bacterial bioimprint‐based detection method does not rely on any antibodies, uses stable and inexpensive reagents, and could potentially find application for rapid diagnostics of bacterial pathogens in clinical samples at the point of care. [ABSTRACT FROM AUTHOR]

Through the present work, anti-FMD IgY immunoglobulins for serotypes O; A, and SAT2 were prepared in chicken egg yolk through immunization of laying hens with locally prepared trivalent FMD vaccine. The antibody titres against three serotypes (O; A, and SAT2) measured by SNT were 2.58; 2.8 and 2.6 respectively and where 2.81; 2.81 and 2.8 respectively when measured by ELISA. Evaluation of Anti-FMD IgY conjugated with Horse radish peroxidase for detection of FMD virus in 100 tongue epithelium; 50 OP fluid samples; 20 nasal swabs from naturally infected cattle, and 100 tissue culture infected fluids revealed sensitivity of 85% indicating the success of preparation of anti-FMD immunoglobulins conjugated with horse radish peroxidase as local product available on request saving cost and time.

A boronate affinity monolith with improved affinity and selectivity for glycoproteins was prepared starting from two monomers. The first is 3-aminopropyltriethoxysilane-methacrylic acid (APTES-MAA), and the other is a polyhedral oligomeric silsesquioxane (POSS) monomer. In the next step, 3-(acrylamido)benzeneboronic acid was adopted as boronate affinity ligand, and ethylene glycol dimethacrylate as the crosslinker, and iso-propanol and octanol as binary porogens. The synergistic effect of APTES-MAA and POSS warrants good affinity and selectivity for glycoproteins, which results in a number of attractive features including (a) a wide operation pH range (from 5 to 8); (b) higher enrichment factors ranging from 19.3 to 20.6; (c) greater recoveries of glycoproteins between 95.8 and 107.1%; (d) lower relative standard deviations of ≤4.2%. Compared to the corresponding APTES-MAA/POSS-free monolith, the new boronate material had 1.7-fold increased glycoprotein recovery from complex samples. Glycoproteins in 500-fold diluted serum samples can be enriched by the boronate monolith. [ABSTRACT FROM AUTHOR]

We report the enhancement of chemiluminescence response of horseradish peroxidase (HRP) in bioassays by plasmonic surfaces, which are comprised of (i) silver island films (SIFs) and (ii) metal thin films (silver, gold, copper, and nickel, 1 nm thick) deposited onto glass slides. A model bioassay, based on the interactions of avidin-modified HRP with a monolayer of biotinylated poly(ethylene-glycol)-amine, was employed to evaluate the ability of plasmonic surfaces to enhance chemiluminescence response of HRP. Chemiluminescence response of HRP in model bioassays were increased up to ~3.7-fold as compared to the control samples (i.e. glass slides without plasmonic nanoparticles), where the largest enhancement of the chemiluminescence response was observed on SIFs with high loading. These findings allowed us to demonstrate the use of SIFs (high loading) for the detection of a biologically relevant target protein (glial fibrillary acidic protein or GFAP), where the chemiluminescence response of the standard bioassay for GFAP was enhanced up to ~50% as compared to bioassay on glass slides.

Abstract: A new generic concept for polymeric enzyme inhibitors is presented using the example of poly(2‐methyl‐2‐oxazoline) (PMOx) terminated with an iminodiacetate (IDA) function. These polymers are shown to be non‐competitive inhibitors for horseradish peroxidase (HRP). Mechanistic investigations revealed that the polymer is directed to the protein by its end group and collapses at the surface in an entropy‐driven process as shown by isothermal titration calorimetry. The dissociation constant of the complex was determined as the inhibition constant Ki using HRP kinetic activity measurements. Additional experiments suggest that the polymer does not form a diffusion layer around the protein, but might inhibit by inducing minor conformational changes in the protein. This kind of inhibitor offers new avenues towards designing bioactive compounds. [ABSTRACT FROM AUTHOR]

Shrimp allergy is a common cause of allergenic diseases. The authors describe an aptamer-based assay for tropomyosin, one of the major allergens of shrimp. After six rounds of SELEX based on nitrocellulose membrane, several aptamers with high affinity (K = 109.6 nM) were identified that can selectively recognize tropomyosin but not other proteins over a large range of concentrations. By using a fluorescent and graphene oxide based platform, a tropomyosin assay was worked out that has a 4.2 nM detection limit and work in the 0.5 to 50 μg·mL concentration range. The aptamers selected hold a great potential for tropomyosin detection in practical biosensor assays. [ABSTRACT FROM AUTHOR]

Surface modification of silver island films (SIFs) was carried out with Biotin-Poly (Ethylene-glycol)-Amine (BEA), which acts as a cross-linker between the silver surface and horse radish peroxidase (HRP) enzyme for optimum plasmon-enhanced enzymatic activity. SIFs-deposited blank glass slides and SIFs-deposited 3-Aminopropyltriethoxysilane(APTES)-coated glass slides were used as our plasmonic surfaces.In this regard, three different extent of loading of SIFs were also prepared (low, medium and high) on APTES-coated glass slides. Streptavidin-linked HRP enzyme was attached to SIFs-deposited blank glass slides and SIFs-deposited APTES-coated glass slides through the well-known biotin-streptavidin interactions. The characterization of these surfaces was done using optical absorption spectroscopy. The loading of SIFs on glass slides was observed to have significant effect on the efficiency of plasmon-enhanced enzymatic activity, where an enhancement of 200% in the enzymatic activity was observed when compared to our previously used strategies for enzyme immobilization in our preceding work[1]. In addition, SIFs-deposited on APTES-coated glass slides were found to be re-usable for plasmon-enhanced enzymatic reactions unlike SIFs deposited on to blank glass slides.

A detailed investigation of the dependence of the efficiency of plasmon-enhanced enzymatic reactions on the distance between silver island films (SIFs) and horse radish peroxidase (HRP) enzyme and on the loading of SIFs on glass surfaces is presented. Three different extent of loading of SIFs on glass slides were used: 1) low, 2) medium and 3) high, which was characterized by using optical absorption spectroscopy and scanning electron microscopy. Streptavidin-linked HRP enzyme was deposited onto SIFs and glass slides by using three different strategies: strategy 1: biotin-avidin protein assay (distance between SIFs and HRP = 4-8 nm), strategy 2: self assembled monolayers (SAMs) (1-5 nm), strategy 3: polymer layer (1-5 nm). The efficiency of enzymatic conversion of O-phenylenediamine dihydrochloride (OPD) to a colored product by HRP on SIFs and glass surfaces was assessed by optical absorption spectroscopy. The distance between SIFs and HRP and the extent of loading of SIFs on the glass surfaces were shown to have significant effect on the efficiency of plasmon-enhanced enzymatic reactions. In this regard, up to an %250 increase in enzymatic conversion of OPD was observed from SIFs with high loading using strategy 1. In addition, we have studied the potential of repeated use of SIFs in plasmon-enhanced enzymatic reactions.

ABESTRACT Introduction & Objective: Neuron tracing is a novel method that is used for detecting communications between cells, tracing nervous pathways, and recognizing the nuclei relating to different pathways. One of the most common tracing methods is putting cutting end of nerve inside the tracer solution. This method is so sensitive that improper insertion of the nerve inside a solution or histological preparation, would lead not to observing the tracers. We evaluated methods for observing tracers. Material & Methods: 10 adult dogs were assigned into two groups. In the first group, the superior laryngeal nerve was cut and its proximal end, after washing and des-heating, was put inside the horse radish peroxidase (HRP) solution. In the second group, after washing the proximal cutting end of superior laryngeal nerve, we separated tiny vessels supplying the nerve to prevent hemorrhage. Then, by using the needle (gauge 22), 2-3 distal end of nerve sheath was precisely removed and nerve fibers were separated and kept inside the HRP solution. In both groups, nerve end was kept inside the HRP solution for 2 hours. Three to five days later, after perfusion and fixation their head and neck, their pons, medulla oblongata, three upper spinal segments and vagal ganglions were removed, sectioned and prepared histochemically and stained with thionin. Results: The number of labeled cells in the first group was less (2-4 cells per section) than the second group (4-8 cells). This difference was statistically significant (P < 0.05). Conclusion: This study showed that separating nerve fibers and fixation of nerve end inside the solution increased the absorption of tracer.

We report the preparation and characterization of tungsten disulfide-multi-walled carbon nanotubes (WS 2 -MWCNTs) composite via a simple hydrothermal process and demonstrate its application as a biosensing platform for ultrasensitive determination of hepatitis B virus genomic DNA coupled with hybridization chain reaction (HCR) signal amplification. The sensing platform includes capture DNA probe (cDNA), auxiliary DNA, two DNA hairpins, and detection signal is amplified by horse radish peroxidase (HRP) on the DNA duplex. The biosensor is fabricated with the cDNA labeled at 3′ end using thiol immobilized on the WS 2 -MWCNTs/Au nanoparticels modified electrode. cDNA fixed onto electrode surface then reacts with target DNA through the 3′ end of auxiliary DNA. The remaining segment on 5′ end of auxiliary DNA then takes turns to open two alternating DNA hairpins. This leads to a HCR events to produce a nicked double-helix. A large amount of HRP is then combined on DNA helix by affinity interaction of biotin-avidin, each of which amplifies the final electrochemical signal through the catalytic reaction of H 2 O 2 and hydroquinol. Under optimal conditions, a dynamic range of 10 fM–0.1 nM is obtained with a detection limit of 2.5 fM. Synergistic effect of WS 2 -MWCNTs composite and HCR have proven to greatly improve sensitivity for DNA detection. [ABSTRACT FROM AUTHOR]

Graphene quantum dots (GQDs) have received extensive concern in many fields such as optical probe, bioimaging and biosensor. However, few reports refer on the influence of GQDs on enzyme performance. The paper reports two kinds of graphene quantum dots (termed as GO-GQDs and N,S-GQDs) that were prepared by cutting of graphene oxide and pyrolysis of citric acid and l -cysteine, and their use for the horse radish peroxidase (HRP) modification. The study reveals that GO-GQDs and N,S-GQDs exhibit an opposite effect on the HRP performance. Only HRP modified with GO-GQDs offers an enhanced activity (more than 1.9 times of pristine enzyme) and thermo-stability. This is because GO-GQDs offer a larger conjugate rigid plane and fewer hydrophilic groups compared to N,S-GQDs. The characteristics can make GO-GQDs induce a proper conformational change in the HRP for the catalytic performance, improving the enzyme activity and thermo-stability. The HRP modified with green luminescent GO-GQDs was also employed as a biocatalyst for sensing of H 2 O 2 by a fluorometric sensor. The colorless tetramethylbenzidine (TMB) is oxidized into blue oxidized TMB in the presence of H 2 O 2 by the assistance of HRP/GO-GQDs, leading to an obvious fluorescence quenching. The fluorescence intensity linearly decreases with the increase of H 2 O 2 concentration in the range from 2 × 10 − 9 to 2 × 10 − 4 M with the detection limit of 6.8 × 10 − 10 M. The analytical method provides the advantage of sensitivity, stability and accuracy compared with present H 2 O 2 sensors based on the pristine HRP. It has been successfully applied in the determination of H 2 O 2 in real water samples. The study also opens a new avenue for modification of enzyme activity and stability that offers great promise in applications such as biological catalysis, biosensing and enzyme engineering. [ABSTRACT FROM AUTHOR]

This work describes the development of an easy to fabricate biosensor for determination of oxytetracycline (OTC) in basis of electrocatalytically oxidation of OTC by horse radish peroxidase (HRP) in the presence of H 2 O 2 . This work is first report on determination of OTC by enzymatic modified electrode. Developed electrode was fabricated by modifying of CPE with HRP and MWCNTs. This electrode provides sensitive and selective determination for OTC. Detection limit, linear range and sensitivity for OTC determination were completely satisfactory and were about 35 nM, 15 μM–1.5 mM and 18.74 μA/mM, respectively. Applicability of developed electrode was verified with successfully determination of OTC in pharmaceutical and biologic samples. Also developed electrode was showed the selective determination of OTC in presence of uric and folic acid. [ABSTRACT FROM AUTHOR]

Herein, we describe a method for immobilization of biomolecules onto polyvinyl chloride activated by 1-fluoro-2-nitro-4-azidobenzene (FNAB) for a photochemical reaction. Immobilizing biomolecules onto the polyvinyl chloride surface were either very tedious to carry out or they require some specialized conditions and techniques for immobilization. Herein, we report a mild procedure for a covalent immobilization of enzymes using FNAB. HRP (Horse Radish Peroxidase) and invertase were chosen for demonstrating its efficacy onto activated polyvinyl chloride. [ABSTRACT FROM AUTHOR]

This article describes a microfluidic electrochemical immunoassay that features two strategies, viz. (a), the incorporation of magnetic nanoparticles (MNPs) into the central microfluidic channel and acting as a bioaffinity support for the immobilization of the antibody against the immunoreactive trypsin (anti-IRT), and (b), the electrodeposition of copper nanoparticles (CuNPs) on a gold electrode. IRT, a marker for cystic fibrosis, is extracted from blood samples onto a disk using ultrasonication, eluted, and then injected into the detection system where it is captured by anti-IRT-loaded nanoparticles (anti-IRT-Ab-MNPs). Bound IRT is electrochemically quantified after addition of HRP-labeled anti-IRT-Ab which, in the presence of HO, catalyzes the oxidation of catechol to form o-benzoquinone which is detected at a working potential of −150 mV (vs. Ag/AgCl). The electrochemical response to benzoquinone is proportional to the concentration of IRT in the range from 0 to 580 ng⋅mL. The coefficients of variation are <5 % for within-day assays, and <6.4 % for between-day assays. The method was compared to a commercial ELISA for IRT where is showed a correlation coefficient of close to 1. In our perception, this approach represents an attractive alternative to existing methods for screening newborns for cystic fibrosis. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

We report the enhancement of chemiluminescence response of horseradish peroxidase (HRP) in bioassays by plasmonic surfaces, which are comprised of (i) silver island films (SIFs) and (ii) metal thin films (silver, gold, copper, and nickel, 1 nm thick) deposited onto glass slides. A model bioassay, based on the interactions of avidin-modified HRP with a monolayer of biotinylated poly(ethylene-glycol)-amine, was employed to evaluate the ability of plasmonic surfaces to enhance chemiluminescence response of HRP. Chemiluminescence response of HRP in model bioassays were increased up to ~3.7-fold as compared to the control samples (i.e. glass slides without plasmonic nanoparticles), where the largest enhancement of the chemiluminescence response was observed on SIFs with high loading. These findings allowed us to demonstrate the use of SIFs (high loading) for the detection of a biologically relevant target protein (glial fibrillary acidic protein or GFAP), where the chemiluminescence response of the standard bioassay for GFAP was enhanced up to ~50% as compared to bioassay on glass slides. [ABSTRACT FROM AUTHOR]

A novel bionanocomposite, horse radish peroxidase- gold-nanoparticle-Calcium carbonate (HRP-AuNPs-CaCO), hybrid material was encapsulated by silica sol on a glassy carbon electrode (GCE). The fabricated modified electrode was used as a novel voltammetric sensor for electrochemical sensing of anti-HIV replication drug i.e. deferiprone. The surface morphology of the modified electrode was characterized by scanning electron microscopy (SEM). Results obtained from the voltammetric measurements show that HRP-AuNPs-CaCO modified GCE offers a selective and sensitive electrochemical sensor for the determination of deferiprone. Under experimental conditions, the proposed voltammetric sensor has a linear response range from 0.01 to 10,000 μM with a detection limit of 0.01 μM. Furthermore, the fabricated sensor was successfully applied to determine deferiprone level in spiked urine and serum samples. [ABSTRACT FROM AUTHOR]

In present work, gold nanorods were used for amplification of electrochemical sensing of anti-HIV replication drug i.e. deferiprone. Gold nanorods (nano Au) deposited onto pencil graphite electrode (PGE) has been utilized for covalent immobilization of horse radish peroxidase (HRP), via glutaraldehyde (Glu), for deferiprone detection using impedimetric technique. Gold nanorods (nano Au) prepared were characterized by TEM and XRD. The resulting nano Au sensor exhibited a good response to deferiprone with a wide linear range (0.005–1000 µM) and a low detection limit 0.005 µM. The biosensor also showed a short response time (within 15 s). In addition, the biosensor exhibited high reproducibility, good storage stability and anti-interference ability. The applicability of the nano Au sensor is to determine deferiprone level in spiked urine and serum samples. [ABSTRACT FROM AUTHOR]

Abstract: Gold nanoparticles (Au nps) have been synthesized in aqueous solution of polyvinyl pyrrolidone (PVP) by gamma radiolysis from HAuCl4·3H2O precursor and in presence of small concentrations of Ag+, 2-propanol and acetone. The effect of different experimental parameters, such as concentration of reactant, molecular weight of PVP on nanoparticle formation was studied. TEM image confirmed that spherical Au nps were formed when PVP of molecular weight 360,000Da was used as capping agent. H2O2 is a reactant in the enzyme catalyzed reaction of o-phenylene diamine (o-PDA). The reaction product has a weak absorption in the yellow region of the spectrum. When this product interacts with Au nps, it leads to enhancement of the absorption peak. The nanoparticles synthesized by radiation method were used for estimation of H2O2. The absorbance value of this peak at λ max was observed to change with H2O2 concentration, which was monitored for estimation of H2O2. The response is linear in the range of 2.5×10−6 moldm−3 to 2×10−4 moldm−3 and 1×10−7 moldm−3 to 3×10−6 moldm−3 H2O2 in two separate sets of experimental parameters with detection limit 1×10−7 moldm−3. [Copyright &y& Elsevier]

Surface modification of silver island films (SIFs) was carried out with Biotin-Poly (Ethylene-glycol)-Amine (BEA), which acts as a cross-linker between the silver surface and horse radish peroxidase (HRP) enzyme for optimum plasmon-enhanced enzymatic activity. SIFs-deposited blank glass slides and SIFs-deposited 3-Aminopropyltriethoxysilane(APTES)-coated glass slides were used as our plasmonic surfaces.In this regard, three different extent of loading of SIFs were also prepared (low, medium and high) on APTES-coated glass slides. Streptavidin-linked HRP enzyme was attached to SIFs-deposited blank glass slides and SIFs-deposited APTES-coated glass slides through the well-known biotin-streptavidin interactions. The characterization of these surfaces was done using optical absorption spectroscopy. The loading of SIFs on glass slides was observed to have significant effect on the efficiency of plasmon-enhanced enzymatic activity, where an enhancement of 200% in the enzymatic activity was observed when compared to our previously used strategies for enzyme immobilization in our preceding work[1]. In addition, SIFs-deposited on APTES-coated glass slides were found to be re-usable for plasmon-enhanced enzymatic reactions unlike SIFs deposited on to blank glass slides. [ABSTRACT FROM AUTHOR]

Horse radish peroxidase (HRP) has been electrochemically entrapped into perchlorate (ClO) doped polyaniline (PANI) film deposited onto indium-tin-oxide (ITO) coated glass plate. This HRP-PANI-ClO/ITO bioelectrode characterized using Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), UV-Visible spectroscopy, cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) techniques has been utilized for estimation of hydrogen peroxide (HO). This HO sensor exhibits response time of 5 s, linearity from 3 to 136 mM, sensitivity as 0.5638 µA mM cm with linear regression of 0.985. The value of the Michaelis-Menten constant ( K) has been obtained as 1.984 mM. Copyright © 2009 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Important biological events associated with plasma membranes, such as signal transduction, cell adhesion, and protein trafficking, are mediated through the membrane microdomains. However, it is difficult to assess the issue of how they assemble under physiological conditions. We developed a new approach to identify partners of a given molecule on the cell surface in living cells. The important feature of this system, termed as enzyme-mediated activation of radical source, is that activation of cross-linking reagent arylazide-biotin tag can be accomplished not by ultraviolet light, but by an enzyme, horseradish peroxidase. By using this method, we found that many kinds of receptor tyrosine kinases are associated with b1 integrin whereas a few receptor tyrosine kinases are associated with ganglioside GM1 in HeLa S3 cells. This system is a comprehensive approach to identify interactions between cell surface molecules under living conditions. The advantages of this approach are as follows: (i) easy, high throughput, and without the need for special equipment, (ii) applicable to systematic approaches such as proteomic analysis, (iii) applicable to studies on the interactions among not only proteins but also glycans and lipids. The biochemical approach although the enzyme-mediated activation of radical source reaction will provide a new insight into a wide range of research concerning cis interaction between biomolecules on the cell surface in living cells. [ABSTRACT FROM AUTHOR]

A new length detection-micro fluidic device was developed using a sodium sulfite/hydrogen peroxide autocatalytic reaction system as a model reaction, and sub-ppm levels of horse radish peroxidase were determined with this device. Sodium sulfite solution containing bromo thymol blue as an indicator and hydrogen peroxide solution were pumped into a T-shaped mixer with the help of a microliter syringe pump and then mixed. The blue solution had changed to yellow beyond the midpoint of the fine pathway (micro channel). Furthermore, when horse radish peroxidase (POx) was pumped, a part of the blue solution in the micro channel (blue band) was shortened. Therefore, it was possible to determine the POx concentration by via the length of the blue band. The relationship between the blue band length ( y, mm) and the common logarithm of the POx concentration ( x, M) gave a straight line in the concentration range of 0.05–1 ppm. The equation for the straight line was as follows: y = −3.78 log( x) + 16.4. The correlation coefficient was γ = 0.995, the relative standard deviation was 5.7% ( n = 10), and the detection limits of the POx concentration was 0.029 ppm. [ABSTRACT FROM AUTHOR]

Interstitial cells of Cajal (ICC) within the gastrointestinal (GI) tract play a critical role in the generation of electrical slow waves and as mediators of enteric motor neurotransmission. Kit immunohistochemistry has proven to be a reliable method to identify the location of these cells within the tunica muscularis and to provide information on how the distribution and density of these cells change in a variety of GI motility disorders. Because of the labile nature of Kit or its detection, ultrastructural immunocytochemistry using conventional chemical fixation methods has been difficult. We describe a novel in vivo technique to label ICC within GI tissues. Using antibodies directed against the extracellular domain of the Kit receptor, we have been able to live-label the stomach with Kit while the animal is under anaesthesia and the organ is still receiving normal blood supply. This approach provided optimum maintenance of ultrastructural features with significant binding of antibody to the Kit receptor. The loss of ICC in many human motility disorders suggests exciting new hypotheses for their aetiology. This method will prove useful to investigate the ultrastructural changes that occur in ICC networks in animal models of motility disorders that are associated with the loss of these cells. [ABSTRACT FROM AUTHOR]