Optimization of an amperometric biosensor for the detection of hepatitis C virus using fractional factorial designs

Fractional factorial design and factorial with center point design were applied to the development of an amperometric biosensor for the detection of the hepatitis C virus. Biomolecules were immobilized by adsorption on graphite electrodes modified with siloxane-poly(propyleneoxide) hybrid matrix prepared using the sol-gel method. Several parameters were optimized, such as the streptavidin concentration at 0.01 mg mL(-1) and 1.0% bovine serum albumin, the incubation time of the electrodes in the complementary DNA solution for 30 minutes and a 1: 1500 dilution of the avidin-peroxidase conjugate, among others. The application of chemometric studies has been efficient, since the best conditions have been established with a restricted number of experiments, indicating the influence of different factors on the system.

Amperometric biosensor for ascorbic acid

Desenvolveu-se um biossensor para ácido L-ascórbico empregando ascorbato oxidase. A enzima foi extraída do mesocarpo de pepino com solução tampão fosfato 0,05 mol L-1, pH 5,8 contendo NaCl 0,5 mol L-1. Após diálise versus solução tampão fosfato 0,05 mol L-1, pH 5,8 a enzima foi imobilizada em rede de nylon através de ligação covalente com glutaraldeído. A membrana foi acoplada em eletrodo de O2 e a reação monitorada pelo consumo de oxigênio a -600 mV em análise em fluxo (solução tampão fosfato 0,05 mol L-1, pH 5,8 como carregador e vazão 0,5 mL min-1). A curva analítica apresentou-se linear entre 1,2x10-4 a 1,0x10-3 mol L-1. O tempo de vida do biossensor foi de 500 análises. Amostras de medicamentos foram analisadas com a metodologia proposta e os resultados comparados com os obtidos com HPLC.

A new amperometric biosensor for salicylate based on salicylate hydroxylase immobilized on polipyrrole film doped with hexacyanoferrate

An amperometric biosensor for salicylate detection was developed by immobilizing salicylate hydroxylase via glutaraldehyde onto a polypyrrole film doped with hexacyanoferrate, supported on a glassy carbon electrode surface. The sensor monitors the catechol produced in the enzymatic reaction on the film surface, at an applied potential of 150 mV vs. SCE. A [NADH]/[salicylate] ratio between 2 and 4 gave the best response. The biosensor presented the best performance in a solution with pH=7.4. The response time was about 40 s. A linear range of response was observed for salicylate concentrations between 1.0x10(-5) and 1.0x10(-4) mol l(-1) and the equation adjusted for this curve was I=(-0.04+/-0.01)+(11.4+/-0.2)[salicylate] with a correlation coefficient of 0.999 for n=6. The biosensor retains its activity for at least 10 days despite daily use. The results obtained using the biosensor for salicylate determination, in three different samples of antithermic drugs, presented a good correlation with the standard colorimetric method.

Amperometric differential determination of ascorbic acid in beverages and vitamin C tablets using a flow cell containing an array of gold microelectrodes modified with palladium

A simple and attractive method for quantification of ascorbic acid (AA) in beers, soda, natural juices and commercial vitamin C tablets was achieved by combining Bow injection analysis and amperometric detection. An array of gold microelectrodes electrochemically modified by deposition of palladium was employed as working electrode which was almost unaffected by fouling effects. Ascorbic acid was quantified in beverages and vitamin tablets using amperometric differential measurements. This method is based on three steps involving the flow injection of: 1) the sample plus a standard addition of AA, 2) the pure sample, and 3) the enzymatically-treated sample. The enzymatic treatment was carried out with Cucumis sativus tissue, which is a rich source of ascorbate oxidase, at pH 7. The calibration plots for freshly prepared ascorbic acid standards were very linear in the concentration range of 0.18-1.8 mg L-1 with a relative standard deviation (RSD) < 1%, while for real samples the deviations were between 2.7% to 8.9%.

Evaluation of a highly sensitive amperometric biosensor with low cholinesterase charge immobilized on a chemically modified carbon paste electrode for trace determination of carbamates in fruit, vegetable and water samples

A highly sensitive amperometric biosensor for determination of carbamate pesticides directly in water, fruit and vegetable samples has been evaluated, electrochemically characterized and optimized. The biosensor strip was fabricated in screen printed technique on a ceramic support using silver-based paste for reference electrode, and platinum-based paste for working and auxiliary electrodes. The working electrode was modified by a layer of carbon paste mixed with cobalt(II) phthalocyanine and acetylcellulose. Cholinesterase (ChE) enzymes with low enzymatic charge were immobilized on this layer. The operational simplicity of the biosensor consists in that a small drop (similar to 50 mu l) of substrate or sample is deposited on a horizontally positioned biosensor strip representing the microelectrochemical cell. The working potential of the biosensor was 370 mV versus Ag/AgI on a ship reference electrode preventing the interference of electroactive species which are oxidable at more positive potentials. The biosensor was applied to investigate the degradation of two reference ChE inhibitors in freeze dried water under different storage conditions and for direct determination of some N-methylcarbamates (NMCs) in fruit and vegetable samples at ppb concentration levels without any sample pretreatment. A comparison of the obtained results for the total carbamate concentration was done against those obtained using HPLC measurements. (C) 1999 Elsevier B.V. B.V. All rights reserved.

Relation between ambient humidity and ionic conductivity of poly(thionaphtheneindole): Behaviour as an amperometric humidity sensor

The electrical characteristics of oxidized poly(thionaphtheneindole) were investigated as a function of ambient relative humidity (r.h.). The current flowing through a pressed pellet of material between two massive gold electrodes plotted against voltage gives a wave-shaped curve with a halfwave potential at V = similar to 3 V. The current recorded at 4 V (plateau of the wave) is a sigmoidal function of r.h, with the inflexion point at similar to 60%. An interpretation of these findings is given, based on the influence of water on the dielectric constant of the material and on acid-base equilibrium between poly(thionaphtheneindole) and water, from which protons are produced. The behaviour of poly (thionaphtheneindole) as the active component of an amperometric humidity sensor is also reported.

Flow injection amperometric detection of ascorbic acid using a Prussian Blue film-modified electrode

The PB film-modified electrode was used as an amperometric detector for flow injection analysis of ascorbic acid. The modified electrode detector showed good sensitivity, stability and reproducibility. The calibration curve for ascorbic acid was linear over the concentration range from 5.0 x 10(-6) to 1.0 x 10(-3) mol l(-1) with a slope of 19.9 mA mol(-1) per litre and a correlation coefficient of 0.999. The detection limit of this method was 2.49 x 10(-6) mol l(-1). The relative standard deviation of six replicate injections of 2.5 x 10(-4) mol l(-1) ascorbic acid was 2.5%. The results obtained for ascorbic acid determination in pharmaceutical products are in good agreement with those obtained by using the procedure involving the reaction between triiodide and ascorbic acid. (C) 2001 Elsevier B.V. B.V. All rights reserved.

Determination of malic acid in real samples by using enzyme immobilized reactors and amperometric detection

The malate dehydrogenase (MDH) and ascorbate oxidase were immobilized independently, onto silanized controlled porous silica and packed in a tygon tube. The reactors were inserted in the flow system, and the malic acid was determined by measurement of NADH produced by enzymatic reaction. The NADH was reoxidized in a wall jet cell that consisted of spectrographic graphite, Ag/AgCl, KCl(sat), and steel needle as work, reference, and counter electrodes, respectively. The current intensities were measured at 390 mV. The malate calibration curve shows a linear range from 5.0 x 10(-6) to 1.0 x 10(-4) molL(-1), the lifetime was 40 analyses, after that a decrease of 20% on the response is observed. Three different citric juices were analyzed and a good correlation between the proposed method and spectrophotometric commercial kit were obtained.

Use of FIA systems for on-line dilution in multielement determination by ICP-MS

A flow-injection (FI) system to match concentrations was used as an auto-diluter in multielement determination by inductively coupled plasma-mass spectrometry (ICP-MS). The flow system comprised loop-based injection or a timed valve that introduced a variable sample volume info a spray chamber through a standard Meinhard nebulizer of an ICP-MS. Routinely analyzed samples such as water, plant, and steel were selected. The accuracy of multielement determination was checked against water standard reference material from the National Institute of Standards and Technology (1643d), plant standard reference material from the National Bureau of Standards (1572 citrus leaves), and steel standard reference material from the National Bureau of Standards (AISI 4340). The measuring system was calibrated with a multielement solution, yielding a linear plot with good precision [relative standard deviation (RSD) < 3%, n = 12]. The results were in agreement at a 95% confidence level with the certified values for the reference materials and also with those obtained by continuous aspiration and by (FI) with a discrete volume. (C) 1999 John Wiley & Sons, Inc.

A Chloro-Bridged Linear Chain Imine-Copper(II) Complex and Its Application as an Enzyme-Free Amperometric Biosensor for Hydrogen Peroxide

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

Manganese Phthalocyanine as a Biomimetic Electrocatalyst for Phenols in the Development of an Amperometric Sensor

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Determination of Oxalate in Grass by FIA with a Spectrophotometric Detection System Using a Two Enzyme Reactor

A new methodology for soluble oxalic acid determination in grass samples was developed using a two enzyme reactor in an FIA system. The reactor consisted of 3 U of oxalate oxidase and 100 U of peroxidase immobilized on Sorghum vulgare seeds activated with glutaraldehyde. The carbon dioxide was monitored spectrophotometrically, after reacting with an acid-base indicator (Bromocresol Purple) after it permeated through a PTFE membrane. A linear response range was observed between 0.25 and 1.00mmol l-1 of oxalic acid; the data was fit by the equation A=-0.8(±1.5)+ 57.2(±2.5)[oxalate], with a correlation coefficient of 0.9971 and a relative standard deviation of 2% for n=5. The variance for a 0.25 mmol l-1 oxalic acid standard solution was lower than 4% for 11 measurements. The FIA system allows analysis of 20 samples per hour without prior treatment. The proposed method showed a good correlation with that of the Sigma Kit.

Flow injection amperometric determination of persulfate in cosmetic products using a Prussian Blue film-modified electrode

A flow-injection system with a glassy carbon disk electrode modified with Prussian Blue film is proposed for the determination of persulfate in commercial samples of hair bleaching boosters by amperometry. The detection was obtained by chronoamperometric technique and the sample is injected into the electrochemical cell in a wall jet configuration. Potassium chloride at concentration of 0.1 mol L-1 acted as sample carrier at a flow rate of 4.0 mL min-1 and supporting-electrolyte. For 0.025 V (vs. Ag/AgCl) applied voltage, the proposed system handles ca. 160 samples per hour (1.0 10-4 - 1.0 10-3 mol L-1 of persulfate), consuming about 200 μL sample and 11 mg KCl per determination. Typical linear correlations between electrocatalytic current and persulfate concentration was ca. 0.9998. The detection limit is 9.0 10-5 mol L-1 and the calculated amperometric sensibility 3.6 103 μA L mol -1. Relative standard deviation (n =12) of a 1.0 10-4 mol L-1 sample is about 2.2%. The method was applied to persulfate determination in commercial hair-bleaching samples and results are in agreement with those obtained by titrimetry at 95% confidence level and good recoveries (95 - 112%) of spiked samples were found. © 2003 by MDPI.

Development and application of a highly selective biomimetic sensor for detection of captopril, an important ally in hypertension control

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

Use of hemoglobin as alternative to peroxidases in cholesterol amperometric biosensors

Sophisticated molecular architectures can be produced with the layer-by-layer (LbL) method, which may combine distinct materials on the same film. In this study, we take advantage of this capability to produce cholesterol amperometric biosensors from LbL films containing hemoglobin (Hb) and cholesterol oxidase in addition to the polyelectrolytes poly(allylamine hydrochloride) (PAH) and poly(ethylene imine) (PEI). Following an optimization procedure, we found that an LbL film deposited onto ITO substrates, with the architecture ITO(PEI/Hb)5(PEI/COx)10, yielded a sensitivity of 93.4 μA μmol L-1 cm-2 for cholesterol incorporated into phospholipid liposomes, comparable to state-of-the-art biosensors. Hb acted as efficient electron mediator and did not suffer interference from phospholipids. Significantly, cholesterol could also be detected in real samples from chicken egg yolk, with no effects from potential interferents, including phospholipids. Taken together these results demonstrate the possible fabrication of low cost, easy-to-use cholesterol amperometric biosensors, whose sensitivity can be enhanced by further optimizing the molecular architectures of the LbL films. © 2012 Elsevier B.V.