Catalytic electrooxidation of NADH for dehydrogenase amperometric biosensors

The developments in the techniques of NADH catalytic oxidation relevant for incorporation in amperometric biosensors with dehydrogenase enzymes are reviewed with special emphasis in the years following 1990. The review stresses the direct electro-catalytic methods of NAD⁺ recycling as opposed to enzymatic regeneration of the coenzyme. These developments are viewed and evaluated from a mechanistic perspective of recycling of NADH to enzymatically active NAD⁺, and from the point of view of development of technologically useful reagentless dehydrogenase biosensors. An effort is made to propose a method for the standardization of evaluation of new mediating and direct coenzyme recycling schemes. A perspective is given for the requirements that have to be met for successful biosensor development incorporating dehydrogenase enzymes that open the analytical possibilities to a number of new analytes. The intrinsic limitations of the system are finally discussed and a view of the future of the field is presented.     

Electrochemical identification of flavonoid dyes in solid work of art samples by abrasive voltammetry at paraffin-impregnated graphite electrodes

An electrochemical method for identifying flavonoid-type dyes in microsamples from works of art is reported. Square wave voltammograms of natural insoluble dyestuffs based on flavonoid structure dragoon's blood, weld, old fustic, gamboge, Brazilwood and logwood (Campeche wood) attached to paraffin-impregnated graphite electrodes in contact with 0.25 M HAc + 0.25 M NaAc aqueous buffer display characteristic peaks in the potential region between +0.85 and −0.85 V versus AgCl/Ag. Sequential experiments in contact with 0.05 M AlCl₃ and 0.05 M Na₂MoO₄ plus HAc/NaAc and 0.05 M H₃BO₃ + 0.10 M NaOH solutions also provide dye-characteristic signals allowing for an unambiguous identification of each one of the pigments. Individual pigments were satisfactorily identified in mixtures of pigments and real samples from textile pieces found in Castellfort (Valencian Region, Spain) attributed to the 15th century production of local textile workshops.     

Synthesis and biological evaluation of analogues of butyrolactone I and molecular model of its interaction with CDK2

A series of analogues of butyrolactone I, a natural product isolated from Aspergillus terreus that selectively inhibits the CDK2 and CDK1 kinases and that has been found to exhibit an interesting antiproliferative activity, have been synthesized. Its antitumor activity has been tested. Molecular models of the complex between butyrolactone I and the CDK2 active site have been built using a combination of conformational search and automated docking techniques. The stability of the resulting complexes has been assessed by molecular dynamics simulations and the experimental results obtained for the synthesized analogues are rationalized based on the molecular models.     

Determination of triazine herbicides in natural waters by solid-phase extraction and non-aqueous capillary zone electrophoresis

Capillary zone electrophoresis (CZE) in an organic medium was used to analyse triazines at sub-ppb concentration levels in natural waters after a preconcentration step using conventional C18 cartridges and new Oasis HLB devices. With both sorbents, satisfactory results were obtained on analysing deionized water. However, on analysing natural waters, both sorbents showed very different types of behaviour. The different variables affecting the elution of both sorbents were studied, resulting in the choice of Oasis HLB as the most suitable for later separation by CZE in non-aqueous medium. Combination of a preconcentration step with electrokinetic injection revealed that capillary electrophoresis with simple UV detection can also be used satisfactorily for the quantification of micropollutants in natural waters. The detection limits obtained varied between 0.01 and 0.05 microg l(-1), depending on the type of matrix analysed. The day-to-day precision varied between 0.9% and 2.3%, expressed as the relative standard deviation.     

Identification of lipid binders in old oil paintings by separation of 4-bromomethyl-7-methoxycoumarin derivatives of fatty acids by liquid chromatography with fluorescence detection

A HPLC-fluorescence method for identification of drying oils from binding media or protective film used in pictorial works of art prior to conservation or restoration is proposed. Fluorescence derivatization of fatty acids released by hydrolysis of structural drying oils is studied. The derivatization reagent was 4-(bromomethyl)-7-methoxycoumarin with 18-crown-6 as catalyst. Mobile phase was programmed from methanol-water (90:10 v/v) to methanol-water (100:0 v/v) in 25 min. The excitation and emission wavelengths were 325 and 395 nm, respectively. Under these chromatographic conditions, coumarin derivatives of myristic, palmitic, oleic and stearic acids were satisfactorily resolved. The method shows good sensitivity, with a detection limit of 6.0 x 10(-8) mmol, and good linearity between 1.0 x 10(-7) and 1.8 x 10(-4) mmol of each analyte. Peak area ratios among fatty acids derivatives, especially the stearic acid/palmitic acid peak area ratio, are useful to identify the drying oils. The proposed method has been successfully applied to artistic samples from items of the cultural heritage of Valencia (Spain).     

Surface thermodynamic properties of monolayers versus reconstitution of a membrane protein in solid-supported bilayers

Atomic force microscopy (AFM) was used to study the influence of a membrane protein, lactose permease of Escherichia coli (LacY), on the surface spreading behavior and the features of self-assembled phospholipids bilayers on mica. The miscibility of phospholipids used, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), was investigated by surface pressure area isotherm measurements at the air-water interface. A composition with an equimolar proportion of POPC and DMPC was used to form the liposomes. Surface layers formed with DMPC:POPC (0.5:0.5, mol/mol) or LacY reconstituted in proteoliposomes with the same phospholipid composition were imaged by using AFM. When lactose permease was reconstituted in DMPC:POPC (0.5:0.5, mol/mol), self-assembled structures that remained firmly adsorbed onto the mica surface were observed. These sheets had an irregular shape and their upper layer was more corrugated than that obtained for the phospholipid matrix.