A novel method to construct a third-generation biosensor: self-assembling gold nanoparticles on thiol-functionalized poly(styrene-co-acrylic acid) nanospheres

A novel method for fabrication of horseradish peroxidase (HRP) biosensor has been developed by self-assembling gold nanoparticles on thiol-functionalized poly(styrene-co-acrylic acid) (St-co-AA) nanospheres. At first, a cleaned gold electrode was immersed in thiol-functionalized poly(St-co-AA) nanosphere latex prepared by emulsifier-free emulsion polymerization of St with AA and function with dithioglycol to assemble the nanospheres, then gold nanoparticles were chemisorbed onto the thiol groups. Finally, horseradish peroxi- dase was immobilized on the surface of the gold nanoparticles. The sensor displayed an excellent electrocatalytical response to reduction of H2O2 without the aid of an electron mediator. The sensor was highly sensitive to hydrogen peroxide with a detection limit of 4.0 mumol l(-1), and the linear range was from 10.0 mumol l(-1) to 7.0 mmol l(-1). The biosensor retained more than 97.8% of its original activity after 60 days of use. Moreover, the Studied biosensor exhibited good current repeatability and good fabrication reproducibility.

Self-assembling gold nanoparticles on thiol-functionalized poly(styrene-co-acrylic acid) nanospheres for fabrication of a mediatorless biosensor

A novel strategy to construct a sensitive mediatorless sensor of H2O2 was described. At first, a cleaned gold electrode was immersed in thiol-functionalized poly(styrene-co-acrylic acid) (St-co-AA) nanosphere latex prepared by emulsifier-free emulsion polymerization St with AA and function with dithioglycol to assemble the nanospheres, then gold nanoparticles were chemisorbed onto the thiol groups and formed monolayers on the surface of poly(St-co-AA) nanospheres. Finally, horseradish peroxidase (HRP) was immobilized on the surface of the gold nanoparticles. The sensor displayed an excellent electrocatalytical response to reduction of H2O2 without the aid of an electron mediator. The biosensor showed a linear range of 8.0 mu mol L-1-7.0 mmol L-1 with a detection limit of 4.0 mu mol L-1. The biosensor retained more than 97.8% of its original activity after 60 days' storage. Moreover, the studied biosensor exhibited good current reproducibility and good fabrication reproducibility.

Hydrogen peroxide biosensor based on microperoxidase-11 entrapped in lipid membrane

A highly catalytic activity microperoxidase-11 (MP-11) biosensor for H2O2 was developed to immobilizing the heme peptide in didodecyldimethylammonium bromide (DDAB) lipid membrane. The enzyme electrode thus obtained responded to H2O2 without electron mediator or promoter, at a potential of +0.10 V versus Ag \ AgCl. A linear calibration curve is obtained over the range from 2.0 x 10(-5) to 2.4 x 10(-3) M. The biosensor responds to hydrogen peroxide in 15 s and has a detection limit of 8 x 10(-7) M (S/N = 3) Providing a natural environment with lipid membrane for protein immobilization and maintenance of protein functions is a suitable option for the design of biosensors.

Biomimetic biosensor based on lipidic layers containing tyrosinase and lutetium bisphthalocyanine for the detection of antioxidants

This paper describes the preparation of a biomimetic Langmuir-Blodgett film of tyrosinase incorporated in a lipidic layer and the use of lutetium bisphthalocyanine as an electron mediator for the voltammetric detection of phenol derivatives, which include one monophenol (vanillic acid), two diphenols (catechol and caffeic acid) and two triphenols (gallic acid and pyrogallol). The first redox process of the voltammetric responses is associated with the reduction of the enzymatically formed o-quinone and is favoured by the lutetium bisphthalocyanine because significant signal amplification is observed, while the second is associated with the electrochemical oxidation of the antioxidant and occurs at lower potentials in the presence of an electron mediator. The biosensor shows low detection limit (1.98 x 10(-6)-27.49 x 10(-6) M), good reproducibility, and high affinity to antioxidants (Km in the range of 62.31-144.87 mu M). The excellent functionality of the enzyme obtained using a biomimetic immobilisation method, the selectivity afforded by enzyme catalysis, the signal enhancement caused by the lutetium bisphthalocyanine mediator and the increased selectivity of the curves due to the occurrence of two redox processes make these sensors exceptionally suitable for the detection of phenolic compounds. (C) 2010 Elsevier B.V. All rights reserved.

Biomimetic biosensor based on lipidic layers containing tyrosinase and lutetium bisphthalocyanine for the detection of antioxidants

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.

Desenvolvimento de um biosensor amperométrico para a detecção do vírus da Hepatite C

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Desenvolvimento de um biossensor para a detecção de antibióticos β-lactâmicos no leite crú

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

beta-Lactamase-based biosensor for the electrochemical determination of benzylpenicillin in milk

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

Monoamine oxidase B layer-by-layer film fabrication and characterization toward dopamine detection

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

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.

Effect of the mediator in feedback mode-based SECM interrogation of indium tin-oxide and boron-doped diamond electrodes

Indium tin-oxide (ITO) and polycrystalline boron-doped diamond (BDD) have been examined in detail using the scanning electrochemical microscopy technique in feedback mode. For the interrogation of electrodes made from these materials, the choice of mediator has been varied. Using Ru(CN) 4− 6 (aq), ferrocene methanol (FcMeOH), Fe(CN) 3− 6 (aq) and Ru(NH 3) 3+ 6 (aq), approach curve experiments have been performed, and for purposes of comparison, calculations of the apparent heterogeneous electron transfer rates (k app) have been made using these data. In general, it would appear that values of k app are affected mainly by the position of the mediator reversible potential relative to the relevant semiconductor band edge (associated with majority carriers). For both the ITO (n type) and BDD (p type) electrodes, charge transfer is impeded and values are very low when using FcMeOH and Fe(CN) 3− 6 (aq) as mediators, and the use of Ru(NH 3) 3+ 6(aq) results in the largest value of k app. With ITO, the surface is chemically homogeneous and no variation is observed for any given mediator. Data is also presented where the potential of the ITO electrode is fixed using a ratio of the mediators Fe(CN) 3− 6(aq) and Fe(CN) 4− 6(aq). In stark contrast, the BDD electrode is quite the opposite and a range of k app values are observed for all mediators depending on the position on the surface. Both electrode surfaces are very flat and very smooth, and hence, for BDD, variations in feedback current imply a variation in the electrochemical activity. A comparison of the feedback current where the substrate is biased and unbiased shows a surprising degree of proportionality.

Electrochemical and electron transfer investigations of copper proteins

A study of the pH and temperature dependence of the redox potentials of azurins from five species of bacteria has been performed. The variations in the potentials with pH have been interpreted in terms of electrostatic interactions between the copper site and titrating histidine residues, including the effects of substitutions in the amino acid sequences of the proteins on the electrostatic interactions. A comparison of the observed pH dependences with predictions based on histidine pK_a values known for Pseudomonas aeruginosa (Pae), Alcaligenes denitrificans (Ade), and Alcaligenes faecalis (Afa) azurins indicates that the Pae and Ade redox potentials exhibit pH dependences in line with electrostatic arguments, while Afa azurin exhibits more complex behavior. Redox enthalpies and entropies for four of the azurins at low and high pH values have also been obtained. Based on these results in conjuction with the variable pH experiments, it appears that Bordetella bronchiseptica azurin may undergo a more substantial conformational change with pH than has been observed for other species of azurin.

The temperature dependence of the redox potential of bovine erythrocyte superoxide dismutase (SOD) has been determined at pH 7.0, with potassium ferricyanide as the mediator. The following thermodynamic parameters have been obtained (T = 25°C): E°' = 403±5 mV vs. NHE, ΔG°' = -9.31 kcal/mol, ΔH°' = -21.4 kcal/mol, ΔS°' = -40.7 eu, ΔS°'_(rc) = -25.1 eu. It is apparent from these results that ΔH°', rather than ΔS°', is the dominant factor in establishing the high redox potential of SOD. The large negative enthalpy of reduction may also reflect the factors which give SOD its high specificity toward reduction and oxidation by superoxide.

Double proton transfer and one-electron oxidation behaviors in double H-bonded glycinamide-formamidine complex and comparison with biological base pair

The behaviors of double proton transfer (DPT) occurring in a representative glycinamide-formamidine complex have been investigated employing the B3LYP/6-311++G** level of theory. Computational results suggest that the participation of a formamidine molecule favors the proceeding of the proton transfer (PT) for glycinamide compared with that without mediator-assisted case. The DPT process proceeds with a concerted mechanism rather than a stepwise one since no zwitterionic complexes have been located during the DPT process. The barrier heights are 14.4 and 3.9 kcal/mol for the forward and reverse directions, respectively. However, both of them have been reduced by 3.1 and 2.9 kcal/mol to 11.3 and 1.0 kcal/mol with further inclusion of zero-point vibrational energy (ZPVE) corrections, where the lower reverse barrier height implies that the reverse reaction should proceed easily at any temperature of biological importance. Additionally, the one-electron oxidation process for the double H-bonded glycinamide-formamidine complex has also been investigated. The oxidated product is characterized by a distonic radical cation due to the fact that one-electron oxidation takes place on glycinamide fragment and a proton has been transferred from glycinamide to formamidine fragment spontaneously. As a result, the vertical and adiabatic ionization potentials for the neutral double H-bonded complex have been determined to be about 8.46 and 7.73 eV, respectively, where both of them have been reduced by about 0.79 and 0.87 eV relative to those of isolated glycinamide due to the formation of the intermolecular H-bond with formamidine. Finally, the differences between model system and adenine-thymine base pair have been discussed briefly.