Detection of a few DNA copies by real-time electrochemical polymerase chain reaction.

In the current work, accurate quantification over 10 to 108 DNA copies has been successfully achieved for the first time by real-time electrochemical PCR. This has been made possible thanks to the combined use of a fully automated house-built electrochemical qPCR device, optimized for parallel heating and electrochemical monitoring of up to 48 PCR solutions, and the appropriate selection of a DNA intercalating redox probe retaining a strong affinity binding to ds-DNA at the PCR measurement temperature of 72 °C (corresponding to the PCR elongation step). This has also been achieved through the identification of the key parameters governing the onset electrochemical signal decrease and amplitude signal decrease as a function of the PCR cycle for a given DNA intercalating redox probe, thus allowing us to predict the electrochemical PCR kinetic plots from the values of the DNA affinity binding constant determined as a function of temperature. To the best of our knowledge, the analytical performances of the current electrochemical qPCR outperform all of those previously published, in terms of detection limit, dynamic range, reproducibility and melting curve analysis compared to that achieved on a commercialized bench-top fluorescence-based qPCR instrument.

Cyclic voltammetry modeling of proton transport effects on redox charge storage in conductive materials: application to a TiO2 mesoporous film.

Cyclic voltammetry is a particularly useful tool for characterizing charge accumulation in conductive materials. A simple model is presented to evaluate proton transport effects on charge storage in conductive materials associated with a redox process coupled with proton insertion in the bulk material from an aqueous buffered solution, a situation frequently encountered in metal oxide materials. The interplay between proton transport inside and outside the materials is described using a formulation of the problem through introduction of dimensionless variables that allows defining the minimum number of parameters governing the cyclic voltammetry response with consideration of a simple description of the system geometry. This approach is illustrated by analysis of proton insertion in a mesoporous TiO2 film.

Tuning the reactivity of nanostructured indium tin oxide electrodes toward chemisorption.

This contribution highlights correlation between the surface concentration of a chemisorbed organophosphorous probe (flavin mononucleotide) and the relative hydroxyl surface coverage of nanostructured ITO electrodes, which can be tuned during post-deposition reductive annealing. The resulting modified electrodes are very stable in aqueous solution, highly hydrophilic and fully-accessible to the bulk solution.

Spectroelectrochemistry of Fe(III)- and Co(III)-mimochrome VI artificial enzymes immobilized on mesoporous ITO electrodes.

UV-visible absorption spectroelectrochemistry elucidated the different redox behaviours of Fe(III)- and Co(III)-mimochrome VI artificial enzymes, adsorbed on mesoporous conductive films of ITO. The reduction of the ferric complex was rapid and reversible, while the cobaltic complex exhibited irreversible processes probably related to multiple coordination states.

Molecular dynamics simulations of ferrocene-terminated self-assembled monolayers.

The present work describes our studies of the Fc(CH(2))(12)S-/C(10)S-Au monolayers to provide a more detailed molecular description. Molecular dynamics simulations of these mixed monolayers are carried out in conditions close to the electrochemical ones. For this purpose, a supporting electrolyte is added (NaClO(4) 1 M) and the electron transfer process is modeled through molecular simulations of ferrocene both in its neutral (initial state) and oxidized form (final state). The heterogeneity of the surface, that is, "clustered " or "isolated" ferrocene moieties, has been considered for the ferrocenylalkylthiolates using the same grafting densities. The structural properties (density profiles and angular distributions) are described in terms of redox induced orientation changes by comparison between the initial and final states. It is established that this orientation change due to the oxidation of the ferrocene to the ferrocenium is mainly observed in the random system, and it is less pronounced in the cluster system. Finally, the energy contributions underline the role played by the supporting electrolyte.

Description of ferrocenylalkylthiol SAMs on gold by molecular dynamics simulations.

Molecular dynamics simulations of mixed monolayers consisting of Fc(CH2)12S-/C10S-Au SAMs are carried out to calculate structural (density profiles, angular distributions, positions of atoms) and energetic properties. The purpose of this paper is to explore the possible inhomogeneity of the neutral ferrocene moieties within the monolayer. Five systems have been studied using different grafting densities for the ferrocenylalkylthiolates. The angular distributions are described in terms of the relative contributions from isolated and clustered ferrocene moieties in the binary SAMs. It is shown that the energetic contributions strongly depend on the state of the ferrocene. The ability of molecular dynamics simulations to enable better understanding the SAM structure is illustrated in this work.

Molecular dynamics description of grafted monolayers: effect of the surface coverage.

Molecular dynamics simulations of monolayers of metal-chelating ligands grafted onto a graphite surface in water are carried out to calculate structural (density profiles, radius of gyration, and asphericity coefficients), dynamical (diffusion coefficients), and energetical properties as a function of the surface coverage. The purpose is to provide a better understanding of the dependence of various properties of these monolayers on the surface coverage. A critical value of the surface coverage from which all structural properties derive a limiting value has been established. It also appears that the chains rather adopt an elongated conformation along the direction normal to the surface from this critical surface coverage. The hydrogen-bonding structure and dynamics of water molecules are reported. An ordered structure of water in the region close to the terminal groups of the grafted molecules is shown at a relatively high surface coverage. This ordering is similar to that observed in the case of water in interaction with a solid surface.

[Contribution of simultaneous multiple immunoassays to biology]. / Apport des immunoanalyses multiple simultanées á la biologie.

During the last years, research in the area of immunoanalysis has been oriented towards the marketing of more reliable, faster and low cost kits. In this field, simultaneous multiple immunoanalysis that offer long-time ignored original and specific advantages, has undergone tremendous improvements triggered by striking technological innovations and the advent of lanthanide chelate based markers. Simultaneous multiple immunoassays have turned from semi-quantitative methods restricted to pharmacology to quantitative methods for medical biology. We chose first to describe several commercial systems among the most representative then we stressed on the main emerging systems based on optical or electrochemical detection as well as some immunosensor devices. The array of simultaneous multiple immunoassays currently available appears attractive enough so as to take a significant area within the analytical arsenal at the biologist disposal.

Determination of horseradish peroxidase and a peroxidase-like iron porphyrin at a Nafion-modified electrode.

A catalytic coupling reaction between 4-amino antipyrine and a N,N-disubstituted aniline derivative has been exploited in the indirect electrochemical detection of horseradish peroxidase (HRP) and of a biomimetic catalyst, the iron(III) sulfonated tetraphenyl porphyrin. In the presence of hydrogen peroxide and one of the two catalysts a cationic electroactive quinone-iminium dye P+ was formed and detected by linear scan voltammetry using a screen-printed electrode coated with a Nafion film. Detection limits of 10(-12) M for HRP and 4 x 10(-10) M for the iron porphyrin have been achieved. In conclusion the iron porphyrin is considered to be a promising alternative to the HRP label in enzyme immunoassays with electrochemical detection.

Hybridization assay at a disposable electrochemical biosensor for the attomole detection of amplified human cytomegalovirus DNA.

A disposable electrochemical biosensor for the detection of DNA sequences related to the human cytomegalovirus (HCMV) is described. The sensor relies on the adsorption of an amplified human cytomegalovirus DNA strand onto the sensing surface of a screen-printed carbon electrode, and to its hybridization to a complementary single-stranded biotinylated DNA probe. The extent of hybrids formed was determined with streptavidin conjugated to horseradish peroxidase. The peroxidase label was indirectly quantified by measuring the amount of the chromophore and electroactive product 2,2'-diaminoazobenzene generated from the o-phenylenediamine substrate. The intensity of differential pulse voltammetric peak currents resulting from the reduction of the enzyme-generated product was related to the number of target HCMV-amplified DNA molecules present in the sample, and the results were compared to those obtained with standard methods, i.e., agarose gel electrophoresis quantification and colorimetric hybridization assay in a microtiter plate. A detection limit of 0.6 amol/ml of HCMV-amplified DNA fragment was obtained with the electrochemical DNA biosensor. The electrochemical method was 23,000-fold more sensitive than the gel electrophoresis technique and 83-fold more sensitive than the colorimetric hybridization assay in a microtiter plate.

An electrochemical metalloimmunoassay based on a colloidal gold label.

A novel, sensitive electrochemical immunoassay has been developed using a colloidal gold label that, after oxidative gold metal dissolution in an acidic solution, was indirectly determined by anodic stripping voltammetry (ASV) at a single-use carbon-based screen-printed electrode (SPE). The use of disposable electrodes allows for simplified measurement in 35 microL of solution. The method was evaluated for a noncompetitive heterogeneous immunoassay of an immunoglobulin G (IgG) and a concentration as low as 3 x 10(-12) M was determined, which is competitive with colorimetric ELISA or with immunoassays based on fluorescent europium chelate labels. The high performance of the method is related to the sensitive ASV determination of gold(III) at a SPE (detection limit of 5 x 10(-9) M) and to the release of a large number of gold(III) ions from each gold particle anchored on the immunocomplex (e.g., 1.7 x 10(5) gold atoms are theoretically contained in a 18-nm spherical gold particle).

An immunomagnetic electrochemical sensor based on a perfluorosulfonate-coated screen-printed electrode for the determination of 2,4-dichlorophenoxyacetic acid.

A disposable immunomagnetic electrochemical sensor involving a magnetic particle-based solid phase and a Nafion film-coated screen-printed electrode (Nafion-SPE) stuck at the bottom of a polystyrene cylinder (microwell of 300 microL) was developed and evaluated in a competitive immunoassay of the widely used herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The competitive binding of 2,4-D and 2,4-D labled with alkaline phosphatase (AP) for a limited amount of polyclonal anti-2,4-D antibody-coated magnetic beads was monitored electrochemically by measuring the AP labled++ activity bound to the beads. The phosphoric acid ester of [[(4-hydroxyphenyl)amino]-carbonyl]cobaltocenium hexafluorophosphate was used as the AP substrate. This anionic substrate (S-) is enzymatically transformed at pH 9.0 into a cationic phenol derivative (P+) which can be easily accumulated in the polyanionic Nafion coating and determined by cyclic voltammetry. During the enzyme reaction, the AP-associated beads were localized on the surface of the Nafion-SPE with the aid of a magnet, thus effectively increasing the concentration of P+ in the Nafion-modified electrode vicinity. The enzyme generation of P+ close to the electrode surface, and thereby to the Nafion film, resulted in a high amplification of the response. A detection limit of 0.01 microgram L-1 2,4-D was thus achieved. The performance of the sensor was successfully evaluated on river water samples spiked with 2,4-D, indicating that this convenient and sensitive technique offers great promise for decentralized environmental applications.

Simultaneous detection of three drugs labeled by cationic metal complexes at a nafion-loaded carbon paste electrode.

The synthesis of the pentaammine ruthenium(II) complex of N-isonicotinoyl-nortriptyline (NORPy-Ru(2+)) was performed and its electrochemical properties at a nafion-loaded carbon paste electrode were examined. The anodic oxidation of the positively charged labeled antidepressant proceeded at -0.06 V (versus Ag/AgCl, 0.05 M Cl(-)). A detection limit of 0.075 muM (S/N=3) was achieved at physiological pH by square-wave voltammetry after a 5-min preconcentration step, with a linear response over the range 0.075-5.0 muM. With a view to a future triple-analyte immunoassay, the detection of NORPy-Ru(2+) was also examined in the presence of two labeled antiepileptics previously synthesised, i.e. phenytoin labeled by a ferroceneammonium salt (oxidation potential at 0.26 V) and phenobarbital labeled by a cobaltocenium salt (reduction potential at -1.05 V). The simultaneous detection of the three labeled drugs proceeded with analytical performances similar to those corresponding to the separate accumulation of each tracer. However it was observed that the pentaammine ruthenium(II) complexes of pyridine and its derivatives were not stable in the presence of serum, which does not allow for their use as redox cationic labels in a multi-analyte immunoassay to be envisaged.

Ion-exchange voltammetry as a solid-phase microextraction analytical method: factors influencing the mass transfer to perfluorosulfonated ionomer film-coated electrodes and some of their consequences on the current responses.

A simple theoretical model of mass transfer kinetics at an electrode coated with an ion-exchange film polymer is proposed. The model takes into account the analyte depletion in solution and gives the relationship between the ion-exchange voltammetric (IEV) peak current and the initial analyte concentration in the sample matrix. The verification of the model is investigated at disposable Nafion film-coated screen-printed electrodes, using the redox cationic (ferrocenylmethyl)trimethylammonium salt. It is shown that the theoretical model and the experimental data fit satisfactorily insofar as the variation of the extraction and apparent diffusion coefficients of the salt with the film thickness are taken into account. Indeed, the film thickness plays a crucial role for the optimization of the IEV sensitivity, because the physicochemical properties of the recast Nafion polymer are dependent on the amount per unit area of Nafion deposited on the electrode surface.

Renewable perfluorosulfonated ionomer carbon paste electrode for competitive homogeneous electrochemical immunoassays using a redox cationic labeled hapten.

A Nafion-loaded carbon paste electrode (CPE) was prepared and its square wave voltammetric response at a fresh surface was optimized using (ferrocenylmethyl)-trimethylammonium iodide (1) as a model of a cation that accumulates irreversibly. Low current responses could be obtained reproducibly without preconditioning at a CPE loaded with 1-octanol and neutralized Nafion. Use of the former made the method more sensitive, and use of the latter gave lower background currents and stable analytical response over a long period of Nafion-loaded CPE use. Under optimized conditions and using a 5 min accumulation period, the modified electrode gave a linear current response over the range of 5 nM to 1.5 microM 1, a detection limit of 5 nM 1 and a relative standard deviation of 5% for 0.5 microM 1. Using these optimized conditions, the competitive homogeneous immunoassay of phenytoin as a model hapten was carried out with cobaltocenium-labeled phenytoin as tracer. Clinical serum samples (5 microL) of phenytoin at therapeutic concentrations (20-80 microM) could be analyzed using this method.

Ferrocenylethyl phosphate: an improved substrate for the detection of alkaline phosphatase by cathodic stripping ion-exchange voltammetry. Application to the electrochemical enzyme affinity assay of avidin.

The ferrocenylethyl phosphate disodium salt was synthesized and used as a new substrate for alkaline phosphatase (AP). The enzyme-generated ferroceneethanol was selectively and sensitively detected at a Nafion film-coated electrode by anodic preconcentration of the ferricinium salt, followed by cyclic voltammetry. The accumulated ferricinium units could be expelled from the polymer film in their neutral form by cathodic stripping, and so the Nafion-modified electrode could be reused for more than 10 measurements with a standard deviation less than 3%. Values of 0.75 mM for the Michaelis constant and 1.42 µmol s(-)(1) (mg of protein)(-)(1) for the maximal velocity were found. The regenerable Nafion-coated electrode was employed for the indirect detection of AP down to 2 × 10(-)(12) M and for the noncompetitive heterogeneous enzyme assay of avidin, whose detection limit was 5 × 10(-)(12) M.

Reduction of hyperlipidemia in the LA/N-corpulent rat by dietary fish oil containing n-3 fatty acids.

The LA/N rat, when homozygous for the corpulent gene (cp/cp), is obese, hyperphageous, hyperinsulinemic, hypertriglyceridemic and prone to the development of vascular and myocardial lesions. The hypertriglyceridemia, which in 3-month-old cp/cp males is 282 +/- 42 mg/dl and in females, 512 +/- 83 mg/dl, results from the presence of a large triacylglycerol-rich VLDL. The moderate hypercholesterolemia in these animals is largely due to markedly elevated HDL levels, which reach 172 +/- 21 mg total lipid/dl in males and 154 +/- 22 mg total lipid/dl in females. The LA/N-cp rat is thus an interesting animal model of endogenous hypertriglyceridemia in which to examine the hypolipidemic effects of pharmacological agents and also dietary oil supplements containing the n-3 fatty acids. In this study, 1-month-old male and female cp/cp rats were fed a normal low fat laboratory chow supplemented with either 10% olive oil or 10% redfish (Sebastes marinus) oil ad libitum for a period of 2 months. The redfish oil contained 4.9 +/- 0.1% of its total fatty acids as eicosapentaenoic (20:5(n-3)) and 2.3 +/- 0.5% as docosahexaenoic acid (22:6(n-3)), the predominant fatty acids being gondoic (20:1(n-3)), 21.9 +/- 0.9% and cetoleic acid (22:1(n-11)), 21.7 +/- 1.7%, which are of dietary origin. Daily caloric intake was similar to the oil-fed versus control rats. However, the oil-fed animals weighed significantly more than the controls after 2 months of oil supplementation. Redfish oil reduced serum triacylglycerols by 54% in males and 45% in females after 2 months. VLDL levels, after the same time period, were reduced by 44% in males and 39% in females. HDL lipid mass was significantly reduced in both sexes (by 27% in males and 49% in females). However, the levels remained above those of male LA/N +/+ rats of the same age and Long-Evens rats. Olive oil feeding significantly reduced serum cholesterol, triacyglycerols and phospholipids in male but only cholesterol and phospholipids in female animals. This oil had no significant effect upon VLDL total lipid levels in either sex, but significantly increased the particle diameter with a concomitant reduction in the cholesterol and phospholipid content. HDL total lipid levels were unaffected: However, HDL total cholesterol increased significantly in males only. Both oils markedly reduced serum LDL levels in both sexes.(ABSTRACT TRUNCATED AT 400 WORDS)