Maria Isabel Viseu

Mechanism of the electrochemical reduction of tetrazolium blue in non-ionic micelles: Part I. Polarography

Abstract A mechanism is proposed for the electrochemical reduction of tetrazolium blue (TB 2+ ) on the surface of a dropping mercury electrode. The reaction was performed in a heterogeneous solvent: non-ionic micelles of Triton X-100, in an aqueous buffer solution, at pH 7.0. The mechanism, deduced from polarographic data, was found to consist of: (i) two irreversible diffusion-controlled processes, each one with two electrons transferred; and (ii) at least two more electron-transfer paths in which one or both of the oxidized and reduced species are adsorbed on the electrode. The oxidation-reduction potentials obtained for the two redox pairs involved (TB 2+ /TBH + and TBH + /TBH 2 ) are −0.134 and −0.709 V, respectively, versus a standard hydrogen electrode.

Time evolution of the thermotropic behavior of spontaneous liposomes and disks of the DMPC–DTAC aqueous system

In this work, solubilization of the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) by the cationic detergent n-dodecyltrimethylammonium chloride (DTAC) was studied in aqueous solution, at a fixed DMPC concentration and variable detergent:lipid (D:L) molar ratios. The colloidal nanostructures present in different stages of the solubilization process were characterized using micro-differential scanning calorimetry (DSC) and dynamic light scattering (DLS) techniques. For total (analytical) D:L molar ratios below approximately 1, DTAC monomers incorporate into the DMPC liposome bilayers, forming smaller and more fluid vesicles than pure DMPC liposomes. At D:L approximately 1-2, vesicles begin to rupture, coexisting with intact vesicles and bilayer fragments. At D:L approximately 2-12.5, discoidal and spherical micelles are formed and coexist with vesicles; a slow structural rearrangement of the system, monitored in successive DSC heating/cooling cycles, was observed, and is reported for the first time. Finally, for D:L above approximately 15-20, the bilayers are completely dissolved, and the main aggregates in solution become spherical micelles, which slowly evolve to cylindrical (threadlike) micelles. Based on the dependence of the temperature and enthalpy of transitions on the total D:L molar ratio, at constant DMPC concentration, a schematic model, showing the different colloidal nanostructures present in the solubilization process, is proposed.

Spontaneous vesicles, disks, threadlike and spherical micelles found in the solubilization of DMPC liposomes by the detergent DTAC.

The spontaneous colloidal nanostructures formed in water by the zwitterionic phospholipid DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) with the cationic detergent DTAC (n-dodecyltrimethylammonium chloride) were investigated at a fixed DMPC concentration and variable detergent:lipid total molar ratios (D:L). Apparent (neutral-sphere-equivalent) hydrodynamic diameters (Φ(e)) of liposomes and micelles were obtained by dynamic light scattering (DLS). Fluorescence lifetime imaging microscopy (FLIM), using chlorophyll-a as a probe, showed the morphology of giant vesicles and threadlike micelles. Micro-differential scanning calorimetry (micro-DSC) detected the presence of bilayers, in vesicles and discoidal micelles (disks). Pure DMPC liposomes are multilamellar and polydisperse (Φ(e)≈100-10,000 nm). As D:L increased, smaller vesicles were found, due to the bigger spontaneous curvature of the bilayer: at D:L=1, ULVs (unilamellar vesicles; Φ(e)≈100 nm) appeared and, at D:L=2-10, ULVs coexisted with disks (Φ(e)≈30 nm). Bilayers totally disappeared at D:L≥15, giving rise to spheroidal (Φ(e)≈2-16 nm) and threadlike (Φ(e)≈100-10,000 nm) micelles. A quasi-equilibrium structural diagram for the DMPC-DTAC-water system shows equivalent diameters of the scattering nanoparticles as a function of D:L. The results obtained herein for the system DMPC-DTAC show the role of electrostatic interactions in the formation of the mixed structures.

Effect of the spreading procedure on the formation of cationic–anionic mixed monolayers

Abstract Mixed monolayers of dioctadecyldimethylammonium bromide (DODAB) with sodium hexadecylsulfate (SHS) can be obtained by spreading both components at the interface, co-spread (1) or separately spread (2), or by incorporation of the water soluble surfactant SHS from the subphase (3). The most efficient procedure is co-spreading, when equimolar amounts are used. On the other hand, when the components are spread separately at the interface, or when SHS is adsorbed from the aqueous subphase, it is not possible to obtain an efficiency as high as that of co-spreading, even after 5 h. The equimolar composition at the interface can also be immediately obtained after solvent evaporation when SHS is added to the subphase in large excess to DODAB (9:1); alternatively, the same composition can be obtained from smaller excesses of SHS to DODAB, but only after larger time intervals. The composition of the mixed monolayers obtained by procedures 2 and 3 was estimated from the correlation compressibility–composition, obtained independently by co-spreading.

Mechanism of the electrochemical reduction of tetrazolium blue in non-ionic micelles: Part II. Cyclic voltammetry and controlled-potential coulometry

Abstract Combined cyclic voltammetric and controlled-potential coulometric data enabled the study of the electrochemical behaviour of tetrazolium blue (TB2+) on the surface of a glassy carbon electrode. A mechanism is proposed for the reduction and reoxidation of this dye in a heterogeneous solvent-non-ionic micelles of Triton X-100 - in a aqueous buffer solution at pH 7.0. The mechanism of reduction is analogous to the one proposed previously on a dropping mercury electrode (M.I. Viseu, M.L.S. Simoes Goncalves, S.M.B. Costa and M.I.C. Ferreira, J. Electroanal. Chem., 282 (1990) 201). The reoxidation reaction consists of an irreversible diffusion-controlled process, with four electrons transferred, with a superimposed adsorption process. Controlled-potential coulometry also permitted the separation of the two reduced forms of tetrazolium blue: TBH+ in solution and TBH2 adsorbed on the electrode.

Behaviour of the water-soluble meso-tetra(4-methylpyridyl)porphine in mixed monolayers and in Langmuir–Blodgett films

The water-soluble tetracationic meso-tetra(4-methylpyridyl)porphine (PO2) was incorporated in mixed Langmuir monolayers (LM) with the anionic surfactant sodium hexadecylsulfate (SHS) and stearic acid (SA). The stability of the monolayer and the molecular packing of PO2 vary with the surface pressure and amount of stearic acid in the monolayer. The aggregation or tilting of PO2, occurring at the long transition during the compression run at the air–water interface, is reversible for all the ternary systems studied (PO2/SHS/SA). The organization of PO2 in LM, deduced from the π–A isotherms, is confronted with the spectroscopic data of LB monolayers and multilayers on silica substrates. At surface pressures below the long transition only one layer transfers onto the solid substrate whatever the number of vertical transfer strokes performed, while at high surface pressures LB films with several layers were formed. All absorption spectra of LB films exhibit a Soret maximum at λ1 ≈ 433 nm, which is ascribed to a nearly flat conformation of the PO2 ring. LB monolayers and multilayers of systems PO2/4SHS/4SA and PO2/4SHS/8SA, formed at high π(50 mN m−1), show an additional absorption band centred at λ2 ≈ 405 nm, which may account for a nonplanar conformation of PO2 with the pyridinium rings out of the porphine plane. The results suggest that the planar form of PO2 is imposed in the first layer by direct contact in a parallel orientation to the solid substrate, while the nonplanar form may coexist or even prevail in upper layers of LB films formed at high π.

Evaluation of partition coefficients in micelles from combined steady-state and time-resolved fluorescence-quenching data

Abstract A method is proposed to evaluate the partition law of a quencher between the aqueous and the micellar pseudophases, using combined steady-state and time-resolved fluorescence-quenching data. This method was applied to the partition between non-ionic triton X-100 micelles and water of two quenchers of the chlorophyll a fluorescence: (i) tetrazolium blue (TB2+) and (ii) anthraquinone-2-sulfonate (AQS−). The partition law obtained indicates that the distribution statistics of both quenchers is not Poissonian, but, instead, has mainly a binomial character.

Mechanism of photosensitized reduction of tetrazolium blue I. homogeneous solvent: ethanol

Abstract The electron transfer from l -ascorbic acid to tetrazolium blue, photosensitized by the first triplet state of chlorophyll a, was studied in a homogeneous medium (ethanol) by steady state (continuous irradiation) and time-resolved (conventional flash photolysis) photochemical techniques. The complete photoreduction of tetrazolium blue (TB2+) in this medium leads to the consecutive formation of two products: TBH+ and TBH2 TBH+, which is in an intermediate reduction state, can be detected spectroscopically (absorption maximum, approximately 530 nm) but is not easily isolated as it undergoes further reduction to form TBH2 TBH2 is the completely reduced product, which can be detected (absorption maximum, approximately 580 nm) after long reaction times. The yield of one or both of these products depends mainly on the components of the system and their relative concentrations (especially the donor to acceptor ratio), on the presence and concentration of oxygen and on the duration of the experiment (time of irradiation), which determines the ratio of the two electron acceptors of the chlorophyll triplet, TB2+ and TBH+. A mechanistic model is proposed to rationalize the results obtained in both steady state and transient conditions for the different types of system analysed. This model considers mainly the processes of the chlorophyll triplet unimolecular decay, the triplet quenching by other triplet and ground state chlorophyll molecules and by the two electron acceptors TB2+ and TBH+ and the subsequent recombination reactions of the resulting radicals.

Mechanism of photosensitized reduction of tetrazolium blue II. Heterogeneous solvent: Triton X-100 micelles

Abstract The electron transfer from sodium L-ascorbate to tetrazolium blue, photosensitized by the first triplet state of chlorophyll a , was studied in a heterogeneous medium (Triton X-100 micelles) by steady state (continuous irradiation) and time-resolved (conventional flash photolysis) photochemical techniques. As in the solvent ethanol (see part I of this series), in Triton X-100 micelles the complete photoreduction of tetrazolium blue (TB 2+ ) leads to the consecutive formation of two products: TBH + (with an absorption maximum at 540 nm) which is in an intermediate reduction state and TBH 2 (with an absorption maximum at 580 nm) which is the completely reduced product. The yield and ratio of these products depend mainly on the components of the system and their relative concentrations (especially the donor to acceptor ratio), the duration of the experiment (number of flashes performed of time or irradiation), and the micellar concentration, which determines the mean occupation number of the reactant species in the micelles and their distribution law in these aggregates. A mechanistic model is proposed to rationalize the combined steady state and transient results for the different types of system analysed. This model considers mainly the processes of chlorophyll triplet unimolecular decay, quenching of the triplet by oxidative electron transfer mechanisms resulting in radical species and the subsequent radical recombinations. The rate constants of some of these processes were evaluated using two different approaches: (1) a formalism based on bimolecular processes for the quenching reactions, which enabled the rate constants to be evaluated in terms of the total or analytical concentrations of the reagents; (2) a formalism based on unimolecular processes within the confined reaction medium of a micelle, which enabled the rate constants to be evaluated in terms of the effective concentrations or mean occupation numbers of the reagents in the micelles.