A method to construct a third-generation horseradish peroxidase biosensor: Self-assembling gold nanoparticles to three-dimensional sol-gel network

A novel method for fabrication of horseradish peroxidase biosensor has been developed by self-assembling gold nanoparticles to a thiol-containing sol-gel network. A cleaned gold electrode was first immersed in a hydrolyzed (3-mercaptopropyl)-trimethoxysilane (MPS) sol-gel solution to assemble three-dimensional silica gel, and then gold nanoparticles were chemisorbed onto the thiol groups of the sol-gel network. Finally, horseradish peroxidase (HRP) was adsorbed onto the surface of the gold nanoparticles. The distribution of gold nanoparticles and HRP was examined by atomic force microscopy (AFM). The immobilized horseradish peroxidase exhibited direct electrochemical behavior toward the reduction of hydrogen peroxide. The performance and factors influencing the performance of the resulting biosensor were studied in detail. The resulting biosensor exhibited fast amperometric response (2.5 s) to H2O2. The detection limit of the biosensor was 2.0 mumol L-1, and the linear range was from 5.0 mumol L-1 to 10.0 mmol L-1. Moreover, the studied biosensor exhibited high sensitivity, good reproducibility, and long-term stability.

Sol-gel-derived amperometric biosensor for hydrogen peroxide based on methylene green incorporated in Nafion film

A hydrogen peroxide biosensor was fabricated by coating a sol-gel-peroxidase layer onto a Nafion-methylene green modified electrode. Immobilization of methylene green (MG) was attributed to the electrostatic force between MG(+) and the negatively charged sulfonic acid groups in Nafion polymer, whereas immobilization of horseradish peroxidase was attributed to the encapsulation function of the silica sol-gel network. Cyclic voltammetry and chronoamperometry were employed to demonstrate the feasibility of electron transfer between sol-gel-immobilized peroxidase and a glassy carbon electrode. Performance of the sensor was evaluated with respect to response time, sensitivity as well as operational stability. The enzyme electrode has a sensitivity of 13.5 mu A mM(-1) with a detection limit of 1.0 x 10(-7) M H2O2, and the sensor achieved 95% of the steady-state current within 20 s. (C) 2000 Elsevier Science B.V. All rights reserved.

In-situ FTIR study on adsorption and oxidation of native and thermally denatured calf thymus DNA at glassy carbon electrodes

In-situ Fourier transform infra-red (FTIR) spectra of native and thermally denatured calf thymus DNA (CT DNA) adsorbed and/or oxidized at a glassy carbon (GC) electrode surface are reported. The adsorption of native DNA occurs throughout the potential range (-0.2 similar to 1.3 V) studied, and the adsorbing state of DNA at electrode surface is changed from through the C=O band of bases and pyrimidine rings to through the C=O of cytosine and imidazole rings while the potential shifts negatively from 1.3 V to -0.2 V. An in-situ FTIR spectrum of native CT DNA adsorbed at GC electrode surface is similar to that of the dissolved DNA, indicating that the structure of CT DNA is not distorted while it is adsorbed at the GC electrode surface. In the potential range of -0.2 similar to 1.30 V, the temperature-denatured CT DNA is adsorbed at the electrode surface first, then undergoes electrochemical oxidation reaction and following that, diffuses away from the electrode surface. (C) 2001 Elsevier Science B.V. All rights reserved.

Self-gelatinizable graft copolymer of poly(vinyl alcohol) with 4-vinylpyridine as an immobilization matrix for the construction of a tyrosinase-based amperometric biosensor

A tyrosinase-based amperometric biosensor using a self-gelatinizable graft copolymer of poly(vinyl alcohol) with 4-vinylpyridine (PVA-g-PVP) as an immobilization matrix was constructed. The 4-vinylpyridine component of PVA-g-PVP enhances the adherence to a glassy carbon electrode surface. The content of 4-vinylpyridine in this immobilization matrix plays a key role in retaining the activity of tyrosinase. A simple, milder method was adopted by simply syringing the copolymer-tyrosinase aqueous solution on to the electrode surface and allowing water to evaporate at 4 degrees C in a refrigerator. Several parameters, including copolymer composition; pH, applied potential and enzyme membrane composition, ware optimized. The enzyme membrane composition can be varied to obtain higher sensitivity or a wider linear detection range. The biosensor was used for the determination of phenol, p-cresol and catechol. The biosensor exhibited excellent reproducibility, stability and sensitive response and can be used in flow injection analysis. The biosensor showed an extended linear range in hydrophilic organic solvents and it can be used in monitoring organic reaction processes. The analytical performance demonstrated this immobilization matrix is suitable for the immobilization of tyrosinase.

Hydrazines probe of interaction of horseradish peroxidase with cryo-hydrogel

The interaction between horseradish peroxidase (HRP) and the cryo-hydrogel was probed by using hydrazines which show high specificity of the reaction of the edge in the prosthetic heme of horseradish peroxidase. For comparison, the interaction of hydrazine with the horseradish peroxidase adsorbed on graphite electrode was also carried out by using steady-state response of the enzyme electrode and cyclic voltammetry. In order to obtain a proper explanation of the kinetic parameters for the enzymatic reaction, the theoretical expressions of I-max and K-M' in the Michaelis-Menten equation for the experimental system were provided. (C) 1997 Elsevier Science B.V.

Electrochemical studies on vitamin B-12 and its derivatives

The electrochemical studies on vitamin B-12 and its derivatives were reviewed in this paper. The importance of electrochemical studies for explaining the mechanism of B-12 coenzyme in body was discussed. The latest results of electrochemical studies on vitamin B-12 and its derivatives was reviewed. A prospect for the electrochemical studies in vitamin B-12 and its derivatives was suggested.

AMPEROMETRIC GLUCOSE SENSOR WITH FERROCENE AS AN ELECTRON-TRANSFER MEDIATOR

A glucose oxidase (GOD) electrode with ferrocene (Fc) used as an electron transfer mediator has been described. Using Nafion, Fc was modified on a glassy carbon (GC) electrode surface, and glucose oxidase was then immobilized on the Fc-Nafion film, forming a GOD-Fc-Nafion enzyme electrode. The preparation method was quite simple and rapid. The enzyme electrode showed a reversible reaction of the redox couple (Fc+/Fc), used in a biosensor system, displayed a sensitive catalytic current response (response time was less than 20 s) on variation of the glucose concentration, with a wide linear range up to 16 mM and with good repeatability. The enzyme electrode showed almost no deterioration over the course of three weeks. There was little or no interference from electro-active anions, such as ascorbic acid, to the determination of glucose based on Nafion film and lower oxidizing potentials of the enzyme electrode.

Bioelectrochemically-assisted reductive dechlorination of 1,2-dichloroethane by a Dehalococcoides-enriched microbial culture

The aim of this study was to verify the possibility to use a polarized graphite electrode as an electron donor for the reductive dechlorination of 1,2-dichloroethane, an ubiquitous groundwater contaminant. The rate of 1,2-DCA dechlorination almost linearly increased by decreasing the set cathode potential over a broad range of set cathode potentials (i.e., from −300 mV to −900 mV vs. the standard hydrogen electrode). This process was primarily dependent on electrolytic H2 generation. On the other hand, reductive dechlorination proceeded (although quite slowly) with a very high Coulombic efficiency (near 70%) at a set cathode potential of −300 mV, where no H2 production occurred. Under this condition, reductive dechlorination was likely driven by direct electron uptake from the surface of the polarized electrode. Taken as a whole, this study further extends the range of chlorinated contaminants which can be treated with bioelectrochemical systems.

On the pH dependence of electroactivity of poly(methylene blue) films

The electrochemical behavior of poly(methylene blue) on different electrodes has been investigated by electrochemical quartz crystal microbalance and in situ spectrophotometric measurements coupled to cyclic voltammetry. Polymeric films were obtained potentiodynamically and the charge transport mechanism was analyzed. The electrochemical results show that polymer electroactivity depends not only on pH but also on the substrate. Charge compensation changes with both pH and the size of the anions showing a transition in the pH range of polymer pKa. It was demonstrated by spectroelectrochemical experiments that the electroactivity of the film depends on the radical/radical cation equilibrium. The potentials where the most electroactive species are formed have been determined. (C) 2009 Elsevier Ltd. All rights reserved.

Immobilization of streptavidin in sol-gel films: Application on the diagnosis of hepatitis C virus

Recent advances have accelerated the development of biosensors for the analysis of specific gene sequences. In this kind of biosensor, a DNA probe is immobilized on a transducer and the hybridization with the target DNA is monitored by suitable methodology. In the present work, the streptavidin (STA) was encapsulated in thin films siloxane-poly(propylene oxide) hybrids prepared by sol-gel method and deposited on the graphite electrode surface by dip-coating process. Biotinylated 18-mer probes were immobilized through STA and a novel amperometric DNA biosensor for the detection and genotyping of the hepatitis C virus (genotypes 1, 2A/C, 2B and 3) is described. The HCV RNA from serum was submitted to reverse transcriptase-linked polymerase chain reaction (RT-PCR) and biotin-labeled cDNA was obtained. Thus, the cDNA was hybridized to the target-specific oligonucleotide probe immobilized on the graphite electrode surface and following the avidin-peroxidase conjugate was added. The enzymatic response was investigated by constant potential amperometry at -0.45 V versus Ag/AgCl using H2O2 and KI solutions. HCV RNA negative and positive controls and positive samples of sera patients were analyzed and the results were compared to commercial kit. The proposed methodology appeared to be suitable and convenient tool for streptavidin immobilization and diagnose of HCV disease. (c) 2006 Elsevier B.V. All rights reserved.

Estudo do comportamento eletroquímico da enzima peroxidase na presença de peróxido de hidrogênio e ácido 5-aminossalicílico

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Direct determination of phosphite in fertilizers by flow-injection amperometry

A sensor based on graphite electrode modified with palladium-platinum-palladium film is proposed for phosphite determination by flow-injection amperometry. The modified electrode was prepared by a sequential cathodic deposition of Pd, Pt and Pd on a graphite electrode from 0.5% m/v PdCl2 + 28% m/v NH4OH and 2% m/v H2PtCl6 + 10% v/v H2SO4 solutions. After suitable conditioning, the electrode showed catalytic activity for phosphite oxidation when 0. 15 V was applied. The proposed system handles approximately 50 samples per hour (0.0.1 - 0.05 mol L-1 Na-2 HPO3; R-2 = 0.9997), consuming ca. 70 mu L of sample per determination. The limit of detection and amperometric sensibility were 5 X 10(-4) mol L-1 and 1.5 mA L mol(-1), respectively. The proposed method was applied to analysis of fertilizer samples without pre-treatment. Results are in agreement with those obtained by spectrophotometry and titrimetry at 95% confidence level and good recoveries (96-109%) of spiked samples were found. Relative standard deviation (n=12) of a 0.01 mol L-1 Na2HPO3 sample was 2%. The useful lifetime of modified electrode was around 220 determinations. For routine purposes it means that this electrode can be continuously used for 5 hours.

Strategies for developing NADH detector based on meldola blue in different immobilization methods: A comparative study

A comparative study was accomplished between two immobilization methodologies for the meldola's blue (MB), used to decrease the oxidation potential of NADH. The study was performed with glassy carbon electrode by electropolymerization of pyrrole and MB through the technique of cyclic voltammetry being evaluated the effect of the number of cycles to define the thickness and the stability of the film. With 25 cycles it was obtained the best result. They were also investigated the modification of the graphite electrode with film formed by the system sol-gel PPO 300 or PPO 4000 for the dip-coating, through two methods: occlusion and adsorption. The best method was it of adsorption of MB during 10 min, following by deposit of the film PPO 300, favored for the time of drying (24 hours). The adsorption of MB followed by PPO 300 deposition presented broader linear range than PPy methodology. ©2006 Sociedade Brasileira de Química.

Evaluation of the interactions of DNA with the textile dyes Disperse Orange 1 and Disperse Red 1 and their electrolysis products using an electrochemical biosensor

A disposable pencil graphite electrode modified with dsDNA was used in combination with square wave voltammetry in order to evaluate the interaction of DNA with the textile dyes Disperse Orange 1 (DO1) and Disperse Red 1 (DR1), and with the products of their electrolysis. Significant changes in the characteristic oxidation peaks of the guanine and adenine moieties of immobilized dsDNA were observed after incubation of the modified electrode for 180 s in solutions of the dyes in their original forms. The same was observed using the electrolysis products obtained by oxidation and reduction conversions. The oxidation peak currents of the guanine and adenine moieties decreased when the concentrations of DO1 and DR1 were increased up to 5.0 × 10 -6 and 1.0 × 10-6 mol L-1, respectively; the signal decreases were more pronounced after interaction with the oxidized dyes, compared to the reduced compounds. The interactions between DNA and DO1, DR1, and the electrolyzed dyes were further investigated by UV-vis spectrophotometry in solution, and different effects such as hypochromism and hyperchromism were observed in the resulting DNA spectra. The investigated interactions showed clear evidence of changes in the DNA structure, and suggested a predominant intercalation mode leading to damage in the biomolecule. © 2013 Elsevier B.V.

Estudo da microestrutura e da resistência à corrosão integranular em chapas soldadas de aços inoxidáveis austeníticos série 304L após goivagem ao arco elétrico

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)