Electroanalysis of Naringin at Electroactivated Pencil Graphite Electrode for the Assessment of Polyphenolics with Intermediate Antioxidant Power.

A simple and rapid differential pulse voltammetric (DPV) method using a single-use electroactivated pencil graphite electrode (PGE*) is proposed for the rapid screening of the total content of polyphenolics (TCP) with intermediate antioxidant power (AOP) in grapefruit peel and fresh juice. The results were compared and correlated with those provided by the HPLC-DAD-MS method. NG voltammetric behavior at PGE* was studied by cyclic voltammetry and an oxidation mechanism was suggested. The experimental conditions (type of PGE, electroactivation procedure, pH, nature and concentration of supporting electrolyte) for NG DPV determination were optimized. The NG peak current varied linearly with the concentration in the ranges 1.40 × 10-6-2.00 × 10-5 and 2.00 × 10-5-1.40 × 10-4 mol/L NG and a limit of detection (LoD) of 6.02 × 10-7 mol/L NG was attained. The method repeatability expressed as relative standard deviation was 7.62% for the concentration level of 2.00 × 10-6 mol/L NG. After accumulation for 240 s of NG at PGE* the LoD was lowered to 1.35 × 10-7 mol/L NG, the linear range being 6.00 × 10-7-8.00 × 10-6 mol/L NG. The developed electrochemical system was successfully tested on real samples and proved to be a cost-effective tool for the simple estimation of the TCP with intermediate AOP in citrus fruits.

Determination of the antiradical properties of olive oils using an electrochemical method based on DPPH radical.

The present work describes the development of an electrochemical method based on the use of 2,2'-diphenyl-1-picrylhidrazyl free radical (DPPH) for the determination of the antiradical properties of several olive oils. Differential pulse voltammetry was used as measuring technique while the electrochemical process was recorded at a platinum screen-printed working electrode. The decrease in 2,2'-diphenyl-1-picrylhidrazyl peak current intensity was measured at a specific potential value of +160 mV vs. screen-printed pseudo-reference electrode, in the presence of α-, δ- and γ-tocopherol and olive oil samples, respectively. The obtained results using differential pulse voltammetry, as detection technique for real samples analysis, showed a satisfactory agreement with those obtained by high-performance liquid chromatography coupled with fluorescence detection. The reported electrochemical method is rapid and easy to use, feasible and accessible to be used as an alternative to 2,2'-diphenyl-1-picrylhidrazyl spectrophotometric based method.

Evaluation of Geranium spp., Helleborus spp. and Hyssopus spp. polyphenolic extracts inhibitory activity against urease and α-chymotrypsin.

This study was meant to determine the inhibitory activity of tannins and flavonoid compounds from Geranium robertianum, Helleborus purpurascens and Hyssopus officinale plant polyphenol rich extracts against urease and α-chymotrypsin. The G. robertianum, H. purpurascens and H. officinale extracts were purified and concentrated by microfiltration and ultrafiltration. Phenolic compounds including flavonoids and tannins have been linked to many pharmacological activities. Thus, the polyphenolic content of the extracts was assessed by UV-Vis spectroscopy and HPLC. The concentrated extracts enriched in polyphenolic compounds (flavonoids, tannins and phenolic acids) showed a significant inhibition against urease from jack bean (over 90%), whereas in case of the α-chymotrypsin, they proved to have an inhibition below 54%. The results of this support the use of G. robertianum, H. purpurascens and H. officinale polyphenolic extracts as potential sources of urease inhibitors. Among the three plant extracts tested, H. officinale polyphenolic extracts exhibited a high inhibitory activity (92.67%) against urease and low inhibition (19.6%) against α-chymotrypsin and could be considered as possible remedy in ulcer treatment.

Development of a label-free aptasensor for monitoring the self-association of lysozyme.

A novel aptamer and surface plasmon resonance (SPR)-based sensor was developed for the label-free detection of lysozyme. The aptasensor is characterised by a detection limit of 1 µg mL(-1) and a linear range of 5-50 µg mL(-1). As an application, we examined the usefulness of the aptasensor for monitoring the early stages of the aggregation of lysozyme. It was surprisingly found that, despite a significant decrease in monomer content during aggregation, the response of the aptasensor for protein solutions aged for 12 hours was similar to that for the fresh protein. To correlate the results obtained with the aptasensor with the composition of lysozyme solutions at various time points, we examined them in detail by atomic force microscopy (AFM), thioflavin T fluorescence, size-exclusion chromatography (SEC) and Matrix Assisted Laser Desorption Ionisation Time of Flight Mass Spectrometry (MALDI-TOF-MS). All methods together indicated that during the initial hours of aggregation, the protein solutions contained small lysozyme oligomers (mainly dimers) and decreasing amounts of monomers. Our results thus suggest that the aptamer also recognizes lysozyme dimers/oligomers. A higher non-specific binding was observed for the aggregated lysozyme at the surface of the aptasensor as compared to the native protein. This was attributed to the hydrophobic patches which are exposed by the unfolded lysozyme and/or oligomer species, allowing for different adsorption and organisation at the surface of the aptasensor. This hypothesis is supported by square wave voltammetry (SWV) studies using solutions of aggregated lysozyme. A higher electrochemical signal due to the direct oxidation of tyrosine/tryptophan residues was observed for aged protein solutions as compared to the fresh solution, indicative of an increased number of such exposed electroactive residues and of overall increased surface hydrophobicity of the protein. Our work presents a label-free lysozyme aptasensor that is useful not only for the detection of the protein monomer but also for observing the onset of aggregation. The approach can be extended to other proteins which are prone to aggregation.

Disposable biosensor based on platinum nanoparticles-reduced graphene oxide-laccase biocomposite for the determination of total polyphenolic content.

A disposable amperometric biosensor was developed for the detection of total polyphenolic compounds from tea infusions. The biosensor was designed by modifying the surface of a carbon screen-printed electrode with platinum nanoparticles and reduced graphene oxide, followed by the laccase drop-casting and stabilization in neutralised 1% Nafion solution. The obtained biosensor was investigated by scanning electron microscopy and electrochemical techniques. It was observed that platinum nanoparticles-reduced graphene oxide composite had synergistic effects on the electron transfer and increased the electroactive surface area of the carbon screen-printed electrode. The constructed analytical tool showed a good linearity in the range 0.2-2 µM for caffeic acid and a limit of detection of 0.09 µM. The value of Michaelis-Menten apparent constant was calculated from the electrochemical version of Lineweaver-Burk equation to be 2.75 µM. This disposable laccase biosensor could be a valuable tool for the estimation of total polyphenolic content from tea infusions.

Validated HPLC-Fl method for the analysis of S-adenosylmethionine and S-adenosylhomocysteine biomarkers in human blood.

The natural methyl donor group, S-adenosylmethionine and its product, S-adenosylhomocysteine play an important role in many biochemical reactions involving transmethylation reactions. These compounds can be used as biomarkers in incipient diagnosis of various pathological disorders therefore the validation of a suitable method to routinely analysis of these compounds is very important. In this paper, a high performance liquid chromatrography method for S-adenosylmethionine and S-adenosylhomocysteine measurement as fluorescent 1,N(6)-ethanoderivatives from biological samples was validated in terms of selectivity, linearity range of the response (R > 0.9993), detection limit (9 × 10(-9) and 4.4 × 10(-9) mol L(-1)), the limit of quantitation (9.7 × 10(-9) and 5.7 × 10(-9) mol L(-1)), precision, trueness and robustness. The method for quantification simultaneous of these compounds is rapid, sensitive and precise and appropriate for clinical analysis.

Monitoring of rosmarinic acid accumulation in sage cell cultures using laccase biosensor.

INTRODUCTION: A recently developed laccase based biosensor is used for polyphenols determination from in vitro Salvia cultures, the results being expressed as rosmarinic acid equivalent content. OBJECTIVE: The aim of this work was to use a previously developed laccase biosensor for the determination of total phenolic content from in vitro cultivated Salvia, and to support the biosensors further application for the assessment of polyphenols metabolites. METHODOLOGY: The biosensor was constructed by drop casting 3 µL of laccase solution and stabilisation with 0.1 % Nafion solution onto a DropSens carbon screen-printed electrode. Electrochemical measurements were carried out in a 0.1 mol/L phosphate buffer (pH 4.50), the applied working potential being -30 mV versus reference electrode. RESULTS: The response of the biosensor developed was characterised in terms of repeatability, accuracy and precision; the limit of detection was 7.5 × 10(-7) mol/L, the limit of determination was 9.5 × 10⁻7 mol/L, and linear response range for rosmarinic acid was 1 × 10⁻6-10⁻5 mol/L. CONCLUSION: A stable, sensitive and simple biosensor based on laccase-nafion was used for monitoring the total polyphenolic content from two in vitro cultivated plants. The biosensor response was free of electrochemical interferences and of possible interferences from growth media constituents, demonstrating a high sensitivity for rosmarinic acid determination in cell culture suspensions.

Biosensors for the determination of phenolic metabolites.

Antioxidants are groups of chemical substances, the most abundant being polyphenols, mainly found in plants, fruits and vegetables. They include flavonoids, flavonoid derivatives, polyphenols, carotenoids and anthocyanins. Currently, the nutritional quality of many foodstuffs is guaranteed by the presence of antioxidant compounds. The importance of these chemicals as indicators and preservatives of nutritional quality makes necessary the development of accurate, versatile and rapid analytical tools necessary to detect their presence in many foodstuffs and to assess their antioxidant efficacy. In this chapter, enzyme-based biosensors such as monophenol monooxygenase (tyrosinase), catechol oxidase (laccase) and horseradish peroxidase (HRP) are reviewed. Actually, these biosensors are the most commonly used for the detection of polyphenols and flavonoids content.

Methods for the determination of antioxidant capacity in food and raw materials.

A comprehensive description of the most frequently used methods to determine the antioxidant activity in food and raw materials is given. The methods are classified into two categories, depending on the type of the assessment carried out. Several methods for the assessment ofantioxidant efficacy using free radical scavenging such as oxygen radical absorbance capacity assay (ORAC), total radical trapping antioxidant parameter assay (TEAC), ferric reducing antioxidant power assay (FRAP) and 2,2'-diphenyl-11-picrylhydrazyl (DPPH) assay are described. An example of methods based on the assessment of antioxidant efficacy using significant biological substrates is also presented. Critical opinions concerning the proposed methods are presented.

Hydrogel-magnetic nanoparticles with immobilized L-asparaginase for biomedical applications.

The association of magnetic nanoparticles, which could be controlled by a magnetic field and have dimensions which facilitate their penetration in cells/tissues, with hydrogel type biopolymeric shells confer them compatibility and the capacity to retain and deliver bioactive substances. The main objective of this work is the development of a new system based on a biocompatible polymer with organic-inorganic structure capable of vectoring support for biologic active agents (L: -asparaginase, e.g.). Characterization of size and morphology of the hydrogel-magnetic nanoparticles with entrapped L: -asparaginase was made using Dynamic Light Scattering method, Transmission Electron Microscopy and Confocal Microscopy. The structure of magnetic nanoparticles coated with hydrogel was characterized by Fourier Transformed Infrared Spectroscopy. The cytotoxicity of nanoparticles was evaluated and also the interactions with microorganisms. We obtained hydrogel-magnetic nanoparticles with L: -asparaginase entrapped, with sizes below 30 nm in dried stage, capable to penetrate the cells and tissues.

Nanostructured Biomaterials with Controlled Properties Synthesis and Characterization.

Magnetic nanoparticles were obtained using an adjusted Massart method and were covered in a layer-by-layer technique with hydrogel-type biocompatible shells, from chitosan and hyaluronic acid. The synthesized nanocomposites were characterized using dynamic light scattering, transmission electron microscopy, and Fourier transformed infrared spectroscopy. Biocompatibility of magnetic nanostructures was determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) cell proliferation assay, swelling tests, and degradation tests. In addition, interaction of hydrogel-magnetic nanoparticles with microorganisms was studied. The possibility of precise nanoparticles size control, as long as the availability of bio-compatible covering, makes them suitable for biomedical applications.

Screen-printed electrodes with electropolymerized Meldola Blue as versatile detectors in biosensors.

Electropolymerization of Meldola Blue was carried out by cyclic voltammetry in the range from -0.6 to +1.4 V vs. Ag/AgCl, thus defining a new immobilization procedure of the phenoxazine mediator on screen-printed graphite electrodes. Evidence of polymer formation was provided by electrochemical and Fourier transform infrared spectroscopy (FTIR) data. Following polymerization, Meldola Blue preserved the ability to catalyze NADH oxidation allowing to achieve a detection limit of 2.5 x 10(-6) mol l(-1) and a sensitivity of 3713 microA l mol(-1) in amperometric determinations at 0 V vs. Ag/AgCl. In addition, the polymeric mediator was found to facilitate the reduction of hydrogen peroxide in the absence of peroxidase. Typical calibration at -0.1 V vs. Ag/AgCl shows a detection limit of 8.5 x 10(-5) mol l(-1), a sensitivity of 494 microA l mol(-1) and a linear range from 2.5 x 10(-4) to 5 x 10(-3) mol l(-1) hydrogen peroxide.