SENSING IMMOBILIZED MOLECULES OF STREPTAVIDIN ON A SILICON SURFACE BY MALDI-TOF MASS SPECTROMETRY AND FLUORESCENCE MICROSCOPY

Indexación: Web of Science; Scielo.A hydrogen-terminated Si (111) surface was modified to form an aminoterminated monolayer for immobilization of streptavidin. Cleavage of an N-(ω-undecylenyl)-phthalimide covered surface using hidrazine yields an amino group-modified surface, which serves as a substrate for the attachment of biotin and subsequently streptavidin. We used surface analytical techniques to characterize the surface and to control the course of functionalization before the immobilization of streptavidin. To confirm the presence of the streptavidin Texas red on the surface two powerful techniques available in a standard biochemical laboratory are used, Fluorescence Microscopy and MALDI-TOF that allow us to detect and determine the immobilized streptavidin. This work provides an avenue for the development of devices in which the exquisite binding specificity of biomolecular recognition is directly coupled to a biosensor. In addition, we have demonstrated that MALDI-TOF and fluorescence microscopy are useful techniques for the characterization of silicon functionalized surfaces.http://ref.scielo.org/gm87c

Nanomechanics of Langmuir-Blodgett films

La caracterització topogràfica i nanomecànica de pel·lícules moleculars és important degut al creixent interès que aquestes desperten, tant des del punt de vista científic com tecnològic. Com a cas particular tenim les pel·lícules Langmuir i Langmuir- Blodgett (LB), les quals permeten el control de l'àrea per molècula i per tant de la nanoestructura de la mostra. Les pel·lícules Langmuir poden caracteritzar-se en la interfase aire-aigua mitjançant les isotermes de pressió superficial-àrea i amb el Microscopi d'Angle de Brewster (BAM). Les mesures de pressió superficial proporcionen informació global de la pel·lícula mentre que el BAM proporciona imatges òptiques d'àrees de dimensions mil·limètriques amb resolució lateral a escala micromètrica. Les pel·lícules Langmuir poden ser transferides a un substrat pla (pel·lícules LB) i poden estudiar-se a escala nanomètrica mitjançant les Microscopies de Sonda Local (SPMs). Entre elles, la Microscòpia de Forces Atòmiques proporciona imatges topogràfiques, mentre que la Microscòpia de Forces Laterals i l'Espectroscòpia de Forces proporcionen informació sobre les propietats nanotribològiques i nanomecàniques de les pel·lícules. En aplicar aquestes tècniques a l'estudi de pel· lícules mixtes, proporcionen informació sobre miscibilitat, separació de fases, estructura de dominis i propietats mecàniques. Degut a la possibilitat de controlar la pressió superficial de la pel·lícula, es pot correlacionar aquest valor amb l'estructura i el comportament nanomecànic de les capes.The topographical and nanomechanical characterization of molecular films is an important issue due to the increasing interest in this kind of 2-dimensional structure, both from a scientific and technological point of view. In particular, Langmuir and Langmuir-Blodgett (LB) films have been widely studied as it is possible to control the area per molecule in the layer, with the consequent control over the sample nanostructure. Langmuir films can be characterized at the air-water interphase by using surface pressure-area isotherms and Brewster Angle Microscopy (BAM). Surface pressure measurements provide information on the films as a whole and BAM provides optical images of areas in the millimetric range with lateral resolution on the micrometric scale. Langmuir films can be transferred onto an atomically flat substrate and the transferred films (LB films) can be studied in the nanometric range using Scanning Probe Microscopies (SPMs). Of these, Atomic Force Microscopy provides topographical information, while Lateral Force Microscopy and Force Spectroscopy provide information about the nanotribological and nanomechanical properties of the films. These techniques, when applied to the study of mixed films, provide information about miscibility, phase separation, domain structure and mechanical properties. In this respect, SPMs can provide information at a nanometric level that it is not available using BAM. Thanks to the possibility of controlling the film surface pressure, correlation between sample nanostructure and nanomechanics can be established

Nanomechanics: A new approach for studying the mechanical properties of materials

Mitjançant l'espectroscòpia de forces atòmiques s'ha estudiat la resposta nanomecànica a la nanoindentació de la superfície més estable d'un material trencadís FCC, com és ara el MgO (100). L'expulsió del material en forma de capes demostra que la fallida trencadissa implica, de fet, l'inici de la deformació plàstica o estrès crític, i que la deformació plàstica posterior consisteix en una sèrie d'esdeveniments discrets. Es pot determinar amb precisió el mòdul de Young, E, a partir de la regió de deformació elàstica mitjançant una mecànica senzilla, atesa l'absència de dislocacions induïdes per la nanoindentació. Amb aquesta finalitat s'ha desenvolupat un nou model fisicomatemàtic, que té en compte les interaccions laterals. El valor de l'estrès crític de fricció també s'ha calculat i comentat. Com a conseqüència d'aquesta expulsió en capes, també s'ha estudiat la resposta nanomecànica de superfícies de capes primes (gruix & 1 µm) de molècules orgàniques altament orientades, ja que es tracta de materials en capes amb interaccions de tipus Van der Waals. També en aquests materials la superfície es deforma plàsticament i presenta discontinuïtats discretes en les corbes d'indentació, associades ara a les capes moleculars expulsades per l'indentador. En el cas del metall quasiunidimensional tetratiofulvalè tetracianoquinodimetà (TTFTCNQ), el valor del mòdul de Young, E & 20 GPa, coincideix amb l'obtingut per altres mètodes. En el cas de la fase ! del radical p-nitrofenil nitronil nitròxid (p-NPNN) no es disposa d'informació per a monocristalls, i el valor obtingut per a les capes primes és de E & 2 GPa.Atomic force spectroscopy was used to study the nanomechanical response to nanoindentations on the most stable face (100) of FCC brittle materials such as MgO and alkali halides. The layered expulsion of material demonstrates that brittle failure results from the critical stress brought on by plastic deformation and that plastic deformation consists of a series of discrete events. Due to the absence of indentation- induced dislocations, Young?s modulus E can be correctly estimated from the elastic deformation region using simple mechanics. A new model is developed taking into account lateral interactions. Critical shear stress is also evaluated and discussed. As a result of the layered expulsion we also studied the nanomechanical response of surfaces of highly-oriented molecular organic thin films (ca. 1 µm thickness) because these are Van der Waals layered materials. The surfaces were again found to deform plastically and there were discrete discontinuities in the indentation curves, representing the molecular layers being expelled by the penetrating tip. Here, the Hertz model is quite good at revealing the role of lateral interactions in the indentation process. For the quasi-one-dimensional metal tetrathiafulvalene tetracyanoquinodimethane (TTF-TCNQ) the value of Young?s modulus, E & 20 GPa, coincides with that obtained by other bulk methods. For the !-phase of the p-nitrophenyl nitronyl nitroxide (p-NPNN) radical, no information is available for single crystals and the estimated value obtained for the film is E & 2 GPa

Electrochemistry of LB films of mixed MGDG:UQ on ITO

Peer ReviewedPostprint (author’s final draft

Biomimetic monolayer films of digalactosyldiacylglycerol incorporating plastoquinone

The photosynthesis is the process used by plants and bacteria cells to convert inorganic matter in organic thanks to the light energy. This process consist on several steps, being one of them the electronic transport from the photosystem II to the cytochrome thanks to plastoquinone-9 (PQ). Here we prepare membranes that mimic the characteristics and composition of natural photosynthetic cell membranes and we characterize them in order to obtain the PQ molecules position in the membrane and their electrochemical behaviour. The selected galactolipid is digalactosyldiacylglycerol (DGDG) that represents the 30% of the thylakoid membrane lipid content. The results obtained are worthful for several science fields due to the relevance of galactolipids as anti-algal, anti-viral, anti-tumor and anti-inflammatory agents and the antioxidant and free radical scavenger properties of prenylquinones.; Both pure components (DGDG and PQ) and the DGDG:PQ mixtures have been studied using surface pressure-area isotherms. These isotherms give information about the film stability and indicate the thermodynamic behaviour of the mixture and their physical state. The Langmuir-Blodgett (LB) film has been transferred forming a monolayer that mimics the bottom layer of the biological membranes. This monolayer on mica has been topo-graphically characterized using AFM and both the height and the physical state that they present have been obtained. Moreover, these monolayers have been transferred onto ITO that is a hydrophilic substrate with good optical and electrical features, so that, it is suitable for studying the electrochemical behaviour of these systems and it is a good candidate for energy producing devices. (C) 2015 Elsevier B.V. All rights reserved.Peer ReviewedPostprint (author’s final draft

Electrochemical behaviour of mixed LB films of ubiquinone - DPPC

The structure and the electrochemical behaviour of Langmuir and Langmuir-Blodgett (LB) films of the biological ubiquinone-10 (UQ) and a mixture of dipalmytoilphosphatidylcholine (DPPC) and UQ at the molar ratios DPPC:UQ 5:1 and 10:1 have been investigated. The surface pressure-area isotherms of the Langmuir films and the AFM images of the LB films show the formation of a monolayer in the DPPC:UQ mixture till a certain surface pressure is attained, and then at higher surface pressures the UQ is progressively expelled. The cyclic voltammograms of DPPC:UQ LB films formed on indium tin oxide, ITO, at different surface pressures show one reduction and one oxidation peak at low surface pressures, but two or even more reduction and oxidations peaks at medium and high surface pressures. The electrochemical behaviour is correlated with the film structure

Structure and Nanomechanics of Model Membranes by Atomic Force Microscopy and Spectroscopy: Insights into the Role of Cholesterol and Sphingolipids

Biological membranes mediate several biological processes that are directly associated with their physical properties but sometimes difficult to evaluate. Supported lipid bilayers (SLBs) are model systems widely used to characterize the structure of biological membranes. Cholesterol (Chol) plays an essential role in the modulation of membrane physical properties. It directly influences the order and mechanical stability of the lipid bilayers, and it is known to laterally segregate in rafts in the outer leaflet of the membrane together with sphingolipids (SLs). Atomic force microscope (AFM) is a powerful tool as it is capable to sense and apply forces with high accuracy, with distance and force resolution at the nanoscale, and in a controlled environment. AFM-based force spectroscopy (AFM-FS) has become a crucial technique to study the nanomechanical stability of SLBs by controlling the liquid media and the temperature variations. In this contribution, we review recent AFM and AFM-FS studies on the effect of Chol on the morphology and mechanical properties of model SLBs, including complex bilayers containing SLs. We also introduce a promising combination of AFM and X-ray (XR) techniques that allows for in situ characterization of dynamic processes, providing structural, morphological, and nanomechanical information

Direct Measurement of the Nanomechanical Stability of a Redox Protein Active Site and Its Dependence upon Metal Binding

The structural basis of the low reorganization energy of cupredoxins has long been debated. These proteins reconcile a conformationally heterogeneous and exposed metal-chelating site with the highly rigid copper center required for efficient electron transfer. Here we combine single-molecule mechanical unfolding experiments with statistical analysis and computer simulations to show that the metal-binding region of apo-azurin is mechanically flexible and that high mechanical stability is imparted by copper binding. The unfolding pathway of the metal site depends on the pulling residue and suggests that partial unfolding of the metal binding site could be facilitated by the physical interaction with certain regions of the redox protein.We are grateful to A. Donaire and I. Díez-Pérez for discussions, and to the Catalan government (grant 2014SGR-1251), the Spanish government (grant CTQ2013-43892R) and the European Research Council (PELE ERC-2009-Adg 25027) for financial support.Peer ReviewedPostprint (author's final draft