Ana S. Viana

Redox induced orientational changes in a series of short chain ferrocenyl alkyl thiols self-assembled on gold(111) electrodes

Abstract A series of short chain alkyl thiols (n=3–10) with a ferrocene terminal group were allowed to form organised monolayers at gold(111) surfaces. The redox activity of the monolayers was monitored using cyclic voltammetry and subtractively normalised interfacial Fourier transform infrared spectroscopy (SNIFTIRS). The in situ infrared spectra show that the oxidation of the ferrocene groups in the monolayers studied is accompanied by a rotation of the ferrocene groups towards a position where the plane of the cyclopentadienyl rings moves to a position normal to the surface of the electrode. Furthermore, electrochemical and spectroscopic data indicate that monolayers with longer alkyl chains are more ordered than the shorter analogues. A trend was observed in which the rate of adsorption and the surface coverage vary between the length of the alkyl chains.

Ethanol effects on binary and ternary supported lipid bilayers with gel/fluid domains and lipid rafts.

Ethanol-lipid bilayer interactions have been a recurrent theme in membrane biophysics, due to their contribution to the understanding of membrane structure and dynamics. The main purpose of this study was to assess the interplay between membrane lateral heterogeneity and ethanol effects. This was achieved by in situ atomic force microscopy, following the changes induced by sequential ethanol additions on supported lipid bilayers formed in the absence of alcohol. Binary phospholipid mixtures with a single gel phase, dipalmitoylphosphatidylcholine (DPPC)/cholesterol, gel/fluid phase coexistence DPPC/dioleoylphosphatidylcholine (DOPC), and ternary lipid mixtures containing cholesterol, mimicking lipid rafts (DOPC/DPPC/cholesterol and DOPC/sphingomyelin/cholesterol), i.e., with liquid ordered/liquid disordered (ld/lo) phase separation, were investigated. For all compositions studied, and in two different solid supports, mica and silicon, domain formation or rearrangement accompanied by lipid bilayer thinning and expansion was observed. In the case of gel/fluid coexistence, low ethanol concentrations lead to a marked thinning of the fluid but not of the gel domains. In the case of ld/lo all the bilayer thins simultaneously by a similar extent. In both cases, only the more disordered phase expanded significantly, indicating that ethanol increases the proportion of disordered domains. Water/bilayer interfacial tension variation and freezing point depression, inducing acyl chain disordering (including opening and looping), tilting, and interdigitation, are probably the main cause for the observed changes. The results presented herein demonstrate that ethanol influences the bilayer properties according to membrane lateral organization.

Self-assembled monolayer of an iron(III) porphyrin disulphide derivative on gold

A novel iron(III) porphyrin disulphide derivative have been successfully immobilised on gold surfaces by self-assembly. The redox response of the modified electrodes was compared with the obtained for a similar iron porphyrin in solution, confirming the immobilisation of the metalloporphyrin. The gravimetric data obtained by electrochemical quartz crystal microbalance (EQCM) during adsorption allowed an estimation of the electrode coverage, providing further evidence for the formation of the porphyrin SAM. The modified electrodes were also measured by conventional and imaging ellipsometry. The electrocatalytic activity of the two modified electrodes was tested for the reduction of the molecular oxygen.

An efficient non-mediated amperometric biosensor for nitrite determination

In this paper we propose the construction of a new non-mediated electrochemical biosensor for nitrite determination in complex samples. The device is based on the stable and selective cytochrome c nitrite reductase (ccNiR) from Desulfovibrio desulfuricans, which has both high turnover and heterogeneous electron transfer rates. In opposition to previous efforts making use of several redox mediators, in this work we exploited the capacity of ccNiR to display a direct electrochemical response when interacting with pyrolytic graphite (PG) surfaces. To enable the analytical application of such bioelectrode the protein was successfully incorporated within a porous silica glass made by the sol-gel process. In the presence of nitrite, the ccNiR/sol-gel/PG electrode promptly displays catalytic currents indicating that the entrapped ccNiR molecules are reduced via direct electron transfer. This result is noteworthy since the protein molecules are caged inside a non-conductive silica network, in the absence of any mediator species or electron relay. At optimal conditions, the minimum detectable concentration is 120 nM. The biosensor sensitivity is 430 mA M(-1) cm(-2) within a linear range of 0.25-50 microM, keeping a stable response up to two weeks. The analysis of nitrites in freshwaters using the method of standard addition was highly accurated.

Biomimetic membrane rafts stably supported on unmodified gold

The formation of lipid bilayers on bare gold containing gel/fluid and liquid disordered/liquid ordered domains (lipid rafts), essential for the functioning of biological membranes, is reported here for the first time. Such binary and ternary lipid mixtures deposited on gold are improved biomimetic platforms. However, golds hydrophobic nature has been an obstacle for direct deposition, and most studies rely on previous modification of its surface. In this work, lipid mixtures were deposited under different experimental conditions, including those commonly used for other solid supports such as mica, which are known to yield planar and organized bilayers. Atomic force microscopy imaging was used to study the topography of the lipid films at the nanoscale. The coverage, continuity and packing were addressed by ellipsometry and cyclic voltammetry, taking advantage of gold optical/electrical properties. A high quality bilayer displaying well organized lipid rafts is obtained by small or large unilamellar vesicle fusion in 10 mM Hepes buffer without added salt, while the presence of NaCl inhibits the formation of a lipid bilayer and leads to tubular structures. The raft-containing bilayer is stable over a wide range of potential sweep, enabling the development of new lipid raft based biosensing interfaces.

Antibody oriented immobilization on gold using the reaction between carbon disulfide and amine groups and its application in immunosensing.

Carbon disulfide (CS(2)) can spontaneously react with amine groups to form dithiocarbamates on gold surface, providing the possibility to immobilize some compounds with primary or secondary amine groups in one step. Using this principle, an immunosensor interface prepared for immunoglobulin G (IgG) sensing surface toward anti-IgG has been fabricated for the first time by simply immersing gold slides into a mixed aqueous solution of CS(2) and protein A, followed by incubation in immunoglobulin G solution. The reaction between CS(2) and protein A has been followed by UV-vis spectroscopy, whereas cyclic voltammetry has been employed in the characterization of the modified gold surface with CS(2) and protein A, both methods indicating that protein A immobilization is implemented by CS(2). Conventional ellipsometry, atomic force microscopy (AFM), as well as surface plasmon resonance (SPR) have been used to evaluate the specific binding of protein A with IgG and IgG with anti-IgG, revealing that IgG is specifically captured to form the biosensing interface, maintaining its bioactivity. Compared to direct adsorption of IgG on the gold surface, the IgG sensing surface constructed of CS(2) and protein A is far more sensitive to capture anti-IgG as its target molecule. In addition, the modified surface is proven to have good capability to inhibit nonspecific adsorption, as supported by control experiments using lysozyme and BSA. To conclude, antibody immobilization using this one-step method has potential as a simple and convenient surface modification approach for immunosensor development.

Development of functionalized nanoparticles for vaccine delivery to dendritic cells: a mechanistic approach

AIM Produce biodegradable nanoparticles to target antigen-presenting cells (APCs) and evaluate their potential to be used as a vaccine delivery system. MATERIALS & METHODS Untargeted PEGylated poly(d,l-lactic-co-glycolide)-based nanoparticles and mannose-grafted nanoparticles were formulated and physicochemically characterized. Immortalized and primary APCs were used to study nanoparticle internalization patterns. The endocytic pathways and intracellular trafficking followed by nanoparticles were also investigated. RESULTS & DISCUSSION Nanoparticles displayed mannose residues available for binding at the nanoparticle surface. Different nanoparticle internalization patterns by immortalized and primary APCs were verified. Macropinocytosis, clathrin-mediated endocytosis, caveolin- and lipid raft-dependent endocytosis are involved in nanoparticles internalization. Nanoparticles demonstrate both endolysosomal and cytosolic localizations and a tendency to accumulate nearby the endoplasmic reticulum. CONCLUSION The developed nanoparticles might drive antigens to be presented through MHC class I and II molecules to both CD8(+) and CD4(+) T cells, favoring a complete and coordinated immune response.

Potentiostatic and AFM morphological studies of Zn electrodeposition in the presence of surfactants

Zinc electrodeposition onto a steel substrate in the presence of surfactants with different charged head groups, namely, anionic sodium dodecylsulfate (SDS), cationic dodecyltrimethylammonium bromide (CTAB), and nonionic octylphenolpoly(ethyleneglycolether) n , n = 10 (Triton X-100), was studied by both chronoamperometric and atomic force microscopy (AFM) techniques. AFM analysis shows that Zn electrodeposition, in the absence of surfactants, begins in the underpotential deposited (UPD) region with the formation of different-sized circular aggregates with a random distribution, which indicates that progressive nucleation takes place. In the presence of SDS, CTAB, and Triton X-100 no change of the electrodeposit morphology is observed as a function of the surfactant presence. The Zn UPD deposition is confirmed by chronoamperometric measurements performed in this potential region. In what concerns the bulk deposition region, where the hydrogen evolution that goes together with the zinc deposition, the analysis of the current-time transients indicates that the Zn electrodeposition occurs by instantaneous nucleation and three-dimensional growth controlled by diffusion in the surfactant-free solution and in the presence of SDS. When the CTAB and Triton X-100 were added to the bath, change from instantaneous to progressive nucleation arises as a consequence of the simultaneous adsorption of the surfactant that inhibits the nucleation sites. The kinetic parameters values obtained from the Heerman and Tarallo model are in accordance with the dimensionless analysis of the transients. AFM confirms the effect of surfactants on the zinc bulk deposition.

Self-assembled monolayers of Vitamin B12 disulphide derivatives on gold

Using the self-assembly technique, novel monolayers on gold have been prepared from new cobyrinate dialkyl disulphide derivatives. The successful formation is proved by cyclic voltammetry and by in situ ellipsometry. The electrochemical characterisation by reductive desorption allows to estimate the surface coverage and reveals that the presence of two cobyrinates introduces some disorganisation in the monolayer. More packed and organised monolayers have been observed in systems containing only one terminal redox centre. From ellipsometric measurements a possible orientation of the cobyrinate centre in the adsorbed monolayer is modelled. The modified electrodes display electrocatalytic activity for the reduction of dissolved oxygen.

A Biomimetic Platform to Study the Interactions of Bioelectroactive Molecules with Lipid Nanodomains

In this work, we developed a biomimetic platform where the study of membrane associated redox processes and high-resolution imaging of lipid nanodomains can be both performed, based on a new functional gold modification, l-cysteine self-assembled monolayer. This monolayer proved to be ideal for the preparation of defect-free planar supported lipid bilayers (SLBs) where nanodomains with height difference of ∼1.5 nm are clearly resolved by atomic force microscopy. Single and multicomponent lipid compositions were used, leading to the formation of different phases and domains mimicking the lateral organization of cellular membranes, and in all cases stable and continuous bilayers were obtained. These platforms were tested toward the interaction with bioelectroactive molecules, the antioxidant quercetin, and the hormone epinephrine. Despite the weak interaction detected between epinephrine and lipid bilayers, our biomimetic interface was able to sense the redox process of membrane-bound epinephrine, obtain its surface concentration (9.36 × 10(-11) mol/cm(2) for a fluid bilayer), and estimate a mole fraction membrane/water partition coefficient (Kp) from cyclic voltammetric measurements (1.13 × 10(4) for a fluid phase membrane). This Kp could be used to quantitatively describe the minute changes observed in the photophysical properties of epinephrine intrinsic fluorescence upon its interaction with liposome suspensions. Moreover, we showed that the lipid membrane stabilizes epinephrine structure, preventing its oxidation, which occurs in neutral aqueous solution, and that epinephrine partition and mobility in membranes depends on lipid phase, expanding our knowledge on hormone membrane interactions.