Electrochemical Fine-Tuning of the Chemoresponsiveness of Langmuir-Blodgett Graphene Oxide Films.

Graphene oxide has been widely deployed in electrical sensors for monitoring physical, chemical, and biological processes. The presence of abundant oxygen functional groups makes it an ideal substrate for integrating biological functional units to assemblies. However, the introduction of this type of defects on the surface of graphene has a deleterious effect on its electrical properties. Therefore, adjusting the surface chemistry of graphene oxide is of utmost relevance for addressing the immobilization of biomolecules, while preserving its electrochemical integrity. Herein, we describe the direct immobilization of glucose oxidase onto graphene oxide-based electrodes prepared by Langmuir-Blodgett assembly. Electrochemical reduction of graphene oxide allowed to control its surface chemistry and, by this, regulate the nature and density of binding sites for the enzyme and the overall responsiveness of the Langmuir-Blodgett biofilm. X-ray photoelectron spectroscopy, surface plasmon resonance, and electrochemical measurements were used to characterize the compositional and functional features of these biointerfaces. Covalent binding between amine groups on glucose oxidase and epoxy and carbonyl groups on the surface of graphene oxide was successfully used to build up stable and active enzymatic assemblies. This approach constitutes a simple, quick, and efficient route to locally address functional proteins at interfaces without the need for additives or complex modifiers to direct the adsorption process.

Interaction of cationic surfactants with DPPC membranes: effect of a novel Nα-benzoylated arginine-based compound.

Cationic amino acid-based surfactants are known to interact with the lipid bilayer of microorganism resulting in cell death through a disruption of the membrane topology. To elucidate the interaction of a cationic surfactant synthesized in our lab, investigations involving Nα-benzoyl-arginine decyl amide (Bz-Arg-NHC10), and model membranes composed by 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were done. Bz-Arg-NHC10was able to penetrate into DPPC monolayers up to a critical pressure of 59.6 mN m-1. Differential scanning calorimetry revealed that as the concentration of Bz-Arg-NHC10 increased, the main transition temperature of DPPC slightly decreased. Atomic force microscopy (AFM) in situ experiments performed on supported DPPC bilayers on mica allowed monitoring the changes induced by Bz-Arg-NHC10. DPPC bilayer patches were partially removed, mainly in borders and bilayer defects for 50 µM Bz-Arg-NHC10 solution. Increasing the concentration to 100 µM resulted in a complete depletion of the supported bilayers. Surface plasmon resonance (SPR) experiments, carried out with fully DPPC bilayers covered chips, showed a net increase of the SPR signal, which can be explained by Bz-Arg-NHC10 adsorption. When patchy DPPC bilayers were formed on the substrate, a SPR signal net decrease was obtained, which is consistent with the phospholipids' removal observed in the AFM images. The results obtained suggest that the presence of the benzoyl group attached to the polar head of our compound would be the responsible of the increased antimicrobial activity against gram-negative bacteria when compared with other arginine-based surfactants.

Measurement of relative lung perfusion with electrical impedance and positron emission tomography: an experimental comparative study in pigs.

BACKGROUND: Electrical impedance tomography (EIT) with indicator dilution may be clinically useful to measure relative lung perfusion, but there is limited information on the performance of this technique. METHODS: Thirteen pigs (50-66 kg) were anaesthetised and mechanically ventilated. Sequential changes in ventilation were made: (i) right-lung ventilation with left-lung collapse, (ii) two-lung ventilation with optimised PEEP, (iii) two-lung ventilation with zero PEEP after saline lung lavage, (iv) two-lung ventilation with maximum PEEP (20/25 cm H2O to achieve peak airway pressure 45 cm H2O), and (v) two-lung ventilation under unilateral pulmonary artery occlusion. Relative lung perfusion was assessed with EIT and central venous injection of saline 3%, 5%, and 10% (10 ml) during breath holds. Relative perfusion was determined by positron emission tomography (PET) using 68Gallium-labelled microspheres. EIT and PET were compared in eight regions of equal ventro-dorsal height (right, left, ventral, mid-ventral, mid-dorsal, and dorsal), and directional changes in regional perfusion were determined. RESULTS: Differences between methods were relatively small (95% of values differed by less than 8.7%, 8.9%, and 9.5% for saline 10%, 5%, and 3%, respectively). Compared with PET, EIT underestimated relative perfusion in dependent, and overestimated it in non-dependent, regions. EIT and PET detected the same direction of change in relative lung perfusion in 68.9-95.9% of measurements. CONCLUSIONS: The agreement between EIT and PET for measuring and tracking changes of relative lung perfusion was satisfactory for clinical purposes. Indicator-based EIT may prove useful for measuring pulmonary perfusion at bedside.

Impact of sphingomyelin acyl chain (16:0 vs 24:1) on the interfacial properties of Langmuir monolayers: A PM-IRRAS study.

Membrane structure is a key factor for the cell`s physiology, pathology, and therapy. Evaluating the importance of lipid species such as N-nervonoyl sphingomyelin (24:1-SM) -able to prevent phase separation- to membrane structuring remains a formidable challenge. This is the first report in which polarization-modulated infrared reflection-absorption spectroscopy (PM-IRRAS) is applied to investigate the lipid-lipid interactions in 16:0 vs 24:1-SM monolayers and their mixtures with 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol (Chol) (DOPC/SM/Chol 2:1:1). From the results we inferred that the cis double bond (Δ15) in 24:1-SM molecule diminishes intermolecular H-bonding and chain packing density compared to that of 16:0-SM. In ternary mixtures containing 16:0-SM, the relative intensity of the two components of the Amide I band reflected changes in the H-bonding network due to SM-Chol interactions. In contrast, the contribution of the main components of the Amide I band in DOPC/24:1-SM/Chol remained as in 24:1-SM monolayers, with a larger contribution of the non-H-bonded component. The most interesting feature in these ternary films is that the CO stretching mode of DOPC appeared with an intensity similar to that of SM Amide I band in DOPC/16:0-SM/Chol monolayers (a two-phase [Lo/Le] system), whereas an extremely low intensity of the CO band was detected in DOPC/24:1-SM/Chol monolayers (single Le phase). This is evidence that the unsaturation in 24:1-SM affected not only the conformational properties of acyl chains but also the orientation of the chemical groups at the air/water interface. The physical properties and overall H-bonding ability conferred by 24:1-SM may have implications in cell signaling and binding of biomolecules.

Phase-segregated Membrane Model assessed by a combined SPR-AFM Approach.

Model biomembranes can provide valuable insights into the properties of complex biological membranes. Among several techniques, Surface Plasmon Resonance (SPR) provides a label-free analysis of the interactions of bioactive molecules with biomembranes with an experimental setup that allows mimicking biological environments. Nevertheless, protocols that enable the preparation of stable supported membrane systems with reproducible structural and functional properties on the biosensor chip are still needed. In this work, we present a simple protocol to modify SPR substrates that allows the formation of a phase-segregated supported lipid bilayer (SLB). SLBs are formed by fusion of lipid vesicles of pure phospholipids (DMPC, DPPC and DOPC) and of a ternary mixture (DOPC/16:0 SM/Cho in 2:1:1 molar ratio) on a SPR gold sensor chip covered with a dithiothreitol monolayer. The formation of a SLB on the SPR sensing surface in a reproducible way was assessed by the combined use of the SPR technique with AFM. The interaction of a cholesterol-extracting drug with SLBs was studied as a model of membrane-lipophilic biomolecule interaction. The proposed strategy allowed us to obtain a membrane model where phase coexistence is present and where Cho depletion from ternary mixtures was comparable to the extraction results reported for human erythrocytes.

Volume expansion of erythrocytes is not the only mechanism responsible for the protection by arginine-based surfactants against hypotonic hemolysis.

A novel arginine-based cationic surfactant Nα-benzoyl-arginine dodecylamide (Bz-Arg-NHC12) was synthesized in our laboratory. In this paper we study the interaction of Bz-Arg-NHC12 with sheep and human red blood cells (SRBC and HRBC respectively) due to their different membrane physicochemical/biophysical properties. SRBC demonstrated to be slightly more resistant than HRBC to the hemolytic effect of the surfactant, being the micellar structure responsible for the hemolytic effect in both cases. Moreover, besides the hemolytic effect, a dual behavior was observed for the surfactant studied: Bz-Arg-NHC12 was also able to protect red blood cells against hypotonic lysis for HRBC in a wide range of surfactant concentrations. However, the degree of protection showed for SRBC was about 50% lower than for HBRC. In this regard, a remarkable volume expansion was evidenced only for SRBC treated with Bz-Arg-NHC12, although no correlation with the antihemolytic potency (pAH) was found. On the contrary, our surfactant showed a greater pAH when human erythrocytes were submitted to hypotonic stress, with a low volume expansion, showing a higher amount of solubilized phospholipids in the supernatant when compared with SRBC behavior. Surface plasmon resonance measurements show the molecular interaction of the surfactant with lipid bilayers from HRBC and SRBC lipids, demonstrating that in the latter neither microvesicle release or lipid extraction occurred. Our results demonstrate that the volume expansion of erythrocytes is not the only mechanism responsible for the protection by surfactants against hypotonic hemolysis: volume expansion could be compensated via microvesicle release or by the extraction of membrane components upon collisions between red blood cells and surfactant aggregates depending on the membrane composition.

Alimentos funcionales: análisis de la recomendación en la práctica diaria / Functional foods: analysis of the recommendation in the daily practice

Introducción: La investigación científica, la innovación tecnológica y la tendencia hacia lo saludable revolucionaron la industria alimenticia con el nuevo concepto de “alimentos funcionales”. Surgen en Japón en los años 80 y actualmente el mercado internacional se encuentra en pleno auge. Los Licenciados en Nutrición, como nexo entre ciencia, industria y consumidor, constituyen una pieza clave y cumplen un rol fundamental en educación y asesoramiento. El objetivo del estudio fue establecer el nivel de conocimiento que poseen los Licenciados en Nutrición en éste área sobre el concepto de “alimento funcional”, fuentes de información y capacitación utilizadas, recomendación y uso adecuado.Material y métodos: se realizó un estudio cualitativo, descriptivo y transversal. Se entrevistaron 57 Licenciados de Ciudad de Buenos Aires y Conurbano bonaerense.Resultados: La mitad de los entrevistados tenía información previa sobre los alimentos funcionales y, aunque el 63% desconocía su definición, los identificaban correctamente. Los más nombrados fueron los diseñados. Las fuentes bibliográficas y medios de información más nombrados fueron: “Internet”, “libros”, “congresos”. El 91% consideró importante conocerlos y/o usarlos por el beneficio a la salud y utilidad. El resto no los consideró importantes por ausencia de necesidad, falta de información y desconfianza. El 72% recomendaría alimentos funcionales entre los que destacan lácteos (con probióticos, fitoesteroles y omega 3), cereales, frutas y hortalizas (semillas de lino, chía, salvado de avena). Entre las causas de no recomendación destacan precio y rótulos poco claros. Existe un marcado interés sobre capacitación y uso adecuado. Conclusiones: Los conocimientos que poseen los expertos en nutrición sobre alimentos funcionales son claves ya que deben interpretar y “traducir” la información científica y de la industria a la comunidad en general, como fuentes confiables e idóneas.

Phospholipid bilayers supported on thiolate-covered nanostructured gold: in situ Raman spectroscopy and electrochemistry of redox species.

Thiol-covered nanostructured gold has been tested as a platform for the preparation of high-area phospholipid bilayer systems suitable for optical and electrochemical sensing. In situ and ex situ Raman spectroscopy and electrochemical measurements are made to study methylene blue (MB) and flavin-adenine dinucleotide (FAD) incorporation into dimyristoylphosphatidylcholine (DMPC) bilayers prepared by vesicle fusion on dithiothreitol (DTT)-covered nanostructured gold. Results show that lipophilic positively charged MB molecules are incorporated in the bilayer reaching the DTT-gold interface. On the other hand, the negatively charged FAD molecules are immobilized at the outer part of the phospholipid bilayer and cannot be electrochemically detected. Our results demonstrate that DTT-covered nanostructured gold provides a suitable high-area platform for phospholipid membranes that are able to separate and sense different kinds of molecules and biomolecules.

Self-assembled dithiothreitol on Au surfaces for biological applications: phospholipid bilayer formation.

Self-assembly of dithiothreitol (DTT) on Au(111) from solution deposition has been studied by X-ray photoelectron spectroscopy and electrochemical data. DTT molecules self-assemble on Au(111) in a lying-down configuration irrespective of the concentration and temperature. XPS and electrochemical data indicate a DTT surface coverage of theta approximately 0.16 with two S-head-Au covalent bonds per DTT molecule. The DTT monolayer turns the Au surface hydrophilic enough to allow the formation of fluid dimyristoylphosphatidylcholine (DMPC) bilayer domains by vesicle fusion as revealed by in situ atomic force imaging. Methylene blue (MB) and flavin adenine dinucleotide (FAD) have been used as probes to study molecule transport across the bilayer.

Immobilization of methylene blue on self-assembled iodine monolayers on gold.

The immobilization of methylene blue (MB) on iodine-covered Au(111) is studied by electrochemical techniques, scanning tunneling microscopy (STM), Auger Electron Spectroscopy (AES), and Raman spectroscopy. Results show that MB species are efficiently adsorbed on the square root of 3 x square root of 3 R30 degrees I lattice on Au(111). The electrochemical behavior of the adsorbed MB molecules is reversible, indicating a relatively fast electron transfer from the Au(111) surface to the immobilized MB species through the iodine layer. STM images with molecular resolution are consistent with adsorption of MB dimers on a square root of 3 x square root of 3 R30 degrees I lattice placed atop of the Au(111) substrate. Results are compared to those obtained for MB immobilized on Au(111) covered by S(n) (n = 3-8) surface structures.