RESUMO
We study the interaction between one aptamer and its analyte (the MnSOD protein) by the combination of surface-enhanced Raman scattering and multivariate statistical analysis. We observe the aptamer structure and its evolution during the interaction under different experimental conditions (in air or in buffer). Through the spectral treatment by principal component analysis of a large set of SERS data, we were able to probe the aptamer conformations and orientations relative to the surface assuming that the in-plane nucleoside modes are selectively enhanced. We demonstrate that the aptamer orientation and thus its flexibility rely strongly on the presence of a spacer of 15 thymines and on the experimental conditions with the aptamer lying on the surface in air and standing in the buffer. We reveal for the first time that the interaction with MnSOD induces a large loss of flexibility and freezes the aptamer structure in a single conformation.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Análise Espectral RamanRESUMO
In this contribution, we report that conformational changes of molecules that are often buried in a wide-distributed Gaussian distribution can be discerned by analyzing the dynamics of specific Raman lines. We investigate the pertinence of the auto- and cross-correlation functions applied to the dynamics of three Raman lines of an amino acid, the tryptophan. The cross-correlation between intensity and the Raman band is an indicator of the charge transfer during the diffusion limited reaction of tryptophan and the gold surface. The Péclet number Pe can provide a valuable indicator of the convective and/or diffusive features of each Raman band. Adsorption induced conformation changes can be identified using the autocorrelation of the multiples states within the Raman band centered at 1550 cm-1.
Assuntos
Triptofano/química , Ouro/química , Nanopartículas Metálicas/química , Conformação Molecular , Análise Espectral Raman , Eletricidade EstáticaRESUMO
In this contribution, we provide new insights on the temporal fluctuations of surface enhanced Raman spectra (SERS) of large single molecules such as proteins. Because they can only fit partly into small active volume, SERS analysis is referred to spectral pointillism where only protein subdomains are shined and the whole protein landscape is built from the dynamics of successive individual spectra. By applying our approach on bovine serum albumin, we show that single protein subdomains are mostly comprised of three distinct amino acids. Surface amino acids such as lysine are preferentially detected in the open form of the protein. The investigation of the tryptophan Fermi doublet in the single protein regime is highly instructive on the protein conformation. We finally demonstrate that spectral pointillism enables to correlate individual amino acids with structural information.
Assuntos
Aminoácidos/química , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismo , Triptofano/química , Triptofano/metabolismo , Animais , Bovinos , Conformação Proteica , Análise Espectral RamanRESUMO
Surface enhanced Raman scattering (SERS) spectroscopy becomes increasingly used in biosensors for its capacity to detect and identify single molecules. In practice, a large number of SERS spectra are acquired and reliable ranking methods are thus essential for analysing all these data. Supervised classification strategies, which are the most effective methods, are usually applied but they require pre-determined models or classes. In this work, we propose to sort SERS spectra in unknown groups with an alternative strategy called Fourier polar representation. This non-fitting method based on simple Fourier sine and cosine transforms produces a fast and graphical representation for sorting SERS spectra with quantitative information. The reliability of this method was first investigated theoretically and numerically. Then, its performances were tested on two concrete biological examples: first with single amino-acid molecule (cysteine) and then with a mixture of three distinct odorous molecules. The benefits of this Fourier polar representation were highlighted and compared to the well-established statistical principal component analysis method.
Assuntos
Análise Espectral Raman , Algoritmos , Técnicas Biossensoriais , Cisteína/química , Modelos Teóricos , Método de Monte Carlo , Odorantes/análise , Análise Espectral Raman/métodosRESUMO
Pulmonary surfactant is a complex mixture of phospholipids and proteins and forms a thin film at the lung alveolar interface separating air from liquid environment. The film reduces the work of breathing during repeatable compressions of the alveoli which form a characteristic multilayer upon compression. In this work, we investigated the structure of bovine lipid extract surfactant (BLES). We analysed the BLES films by atomic force microscopy (AFM) and spectroscopic ellipsometry (SE) in order to provide combined characterization of both morphology and thickness of surfactant films. We show how the spectroscopic ellipsometry can be used to supplement the data obtained by AFM. We demonstrate that indium tin oxide (ITO) substrate used for spectroscopic ellipsometry is preferable over glass substrate to enhance the optical contrast. An optical model was proposed to account for non-uniform film morphology. We obtained good correlations between the multilayer surface coverage, determined by both AFM and SE. SE measures the thickness of the first uniform monolayer as 2.6 nm that cannot be achieved by AFM imaging alone.
Assuntos
Lipídeos/análise , Proteínas/análise , Tensoativos/análise , Compostos de Estanho/química , Animais , Bovinos , Microscopia de Força Atômica , Análise Espectral , Propriedades de SuperfícieRESUMO
In the present work, the combination of chemical immobilization with electron beam lithography enables the production of sensitive and reproducible SERS-active areas composed of stochastic arrangements of gold nanoparticles. The number of nanoparticles was varied from 2 to 500. Thereby a systematic analysis of these SERS-active areas allows us to study SERS efficiency as a function of the number of nanoparticles. We found that the experimental parameters are critical, in particular the size of the SERS-active area must be comparable to the effective area of excitation to obtained reproducible SERS measurements. The sensitivity has also been studied by deducing the number of NPs that generate the enhancement. With this approach we demonstrates that the maximum enhancement, the best sensitivity, is obtained with the smallest number of nanoparticles that is resonant at a given excitation wavelength.
Assuntos
Cristalização/métodos , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Análise Espectral Raman/métodos , Luz , Substâncias Macromoleculares/química , Teste de Materiais , Conformação Molecular , Tamanho da Partícula , Espalhamento de Radiação , Propriedades de SuperfícieRESUMO
We report the changes in the electrical properties of the lipid-protein film of pulmonary surfactant produced by excess cholesterol. Pulmonary surfactant (PS) is a complex lipid-protein mixture that forms a molecular film at the interface of the lung's epithelia. The defined molecular arrangement of the lipids and proteins of the surfactant film gives rise to the locally highly variable electrical surface potential of the interface, which becomes considerably altered in the presence of cholesterol. With frequency modulation Kelvin probe force microscopy (FM-KPFM) and force measurements, complemented by theoretical analysis, we showed that excess cholesterol significantly changes the electric field around a PS film because of the presence of nanometer-sized electrostatic domains and affects the electrostatic interaction of an AFM probe with a PS film. These changes in the local electrical field would greatly alter the interaction of the surfactant film with charged species and would immediately impact the manner in which inhaled (often charged) airborne nanoparticles and fibers might interact with the lung interface.
Assuntos
Colesterol/química , Lipídeos/química , Membranas Artificiais , Proteínas/química , Surfactantes Pulmonares/química , Eletricidade Estática , Animais , Bovinos , Colesterol/metabolismo , Colesterol/farmacologia , Metabolismo dos Lipídeos , Microscopia de Força Atômica , Proteínas/metabolismoRESUMO
Optical antennas are elementary units used to direct optical radiation to the nanoscale. Here we demonstrate an active control over individual antenna performances by an external electrical trigger. We find that by an in-plane command of an anisotropic load medium, the electromagnetic interaction between individual elements constituting an optical antenna can be controlled, resulting in a strong polarization and tuning response. An active command of the antenna is a prerequisite for directing light wave through the utilization of such a device.
RESUMO
Microcantilevers were first introduced as imaging probes in Atomic Force Microscopy (AFM) due to their extremely high sensitivity in measuring surface forces. The versatility of these probes, however, allows the sensing and measurement of a host of mechanical properties of various materials. Sensor parameters such as resonance frequency, quality factor, amplitude of vibration and bending due to a differential stress can all be simultaneously determined for a cantilever. When measuring the mechanical properties of materials, identifying and discerning the most influential parameters responsible for the observed changes in the cantilever response are important. We will, therefore, discuss the effects of various force fields such as those induced by mass loading, residual stress, internal friction of the material, and other changes in the mechanical properties of the microcantilevers. Methods to measure variations in temperature, pressure, or molecular adsorption of water molecules are also discussed. Often these effects occur simultaneously, increasing the number of parameters that need to be concurrently measured to ensure the reliability of the sensors. We therefore systematically investigate the geometric and environmental effects on cantilever measurements including the chemical nature of the underlying interactions. To address the geometric effects we have considered cantilevers with a rectangular or circular cross section. The chemical nature is addressed by using cantilevers fabricated with metals and/or dielectrics. Selective chemical etching, swelling or changes in Young's modulus of the surface were investigated by means of polymeric and inorganic coatings. Finally to address the effect of the environment in which the cantilever operates, the Knudsen number was determined to characterize the molecule-cantilever collisions. Also bimaterial cantilevers with high thermal sensitivity were used to discern the effect of temperature variations. When appropriate, we use continuum mechanics, which is justified according to the ratio between the cantilever thickness and the grain size of the materials. We will also address other potential applications such as the ageing process of nuclear materials, building materials, and optical fibers, which can be investigated by monitoring their mechanical changes with time. In summary, by virtue of the dynamic response of a miniaturized cantilever shaped material, we present useful measurements of the associated elastic properties.
RESUMO
Atomic force microscopy (AFM) can be used to reveal intimate details about the effect of anesthetics on phospholipid bilayers. In AFM, surfaces are probed using a tip revealing lateral structural features at 10-20-nm resolution and height features at 0.5-nm resolution. Additionally, information on the viscoelasticity of the surface can be gained by examining the forces of tip-surface interactions. This is also known as force spectroscopy. In this chapter, the use of AFM to observe and quantify anesthetic-induced changes in phospholipid bilayers is detailed. The procedures developed to create supported phospholipid bilayers are described and the techniques developed to generate the best AFM images and force spectroscopy results have been revealed.
Assuntos
Anestésicos/química , Bicamadas Lipídicas/química , Microscopia de Força Atômica , Fosfolipídeos/química , Avaliação Pré-Clínica de Medicamentos , Elasticidade , Propriedades de SuperfícieRESUMO
Measuring the thickness of biological films remains a difficult task when using differential measurements by atomic force microscopy (AFM). The use of microstructured substrates combined with a selective adsorption constitutes an alternative to tribological measurements. The statistical thickness analysis of biological layers, especially via the dispersion measurements, can provide a way to quantify the molecular orientation. AFM thicknesses were then compared with those obtained optically by spectroscopic ellipsometry (SE) and surface plasmon resonance enhanced ellipsometry (SPREE). The biolayers could then be modeled using a vertical gradient of optical index, which reflects height dispersions. Thiol-modified DNA strands of various lengths account for a good biological model for the study of the strand motion in air and in liquid.
Assuntos
DNA/química , DNA/ultraestrutura , Membranas Artificiais , Microscopia de Força Atômica/métodos , Ressonância de Plasmônio de Superfície/métodosRESUMO
The toxicity of inhaled nanoparticles entering the body through the lung is thought to be initially defined by the electrostatic and adhesive interaction of the particles with lung's wall. Here, we investigated the first step of the interaction of nanoparticles with lung epithelial cells using atomic force microscope (AFM) as a force apparatus. Nanoparticles were modeled by the apex of the AFM tip and the forces of interaction between the tip and the cell analyzed over time. The adhesive force and work of adhesion strongly increased for the first 100s of contact and then leveled out. During this time, the tip was penetrating deeply into the cell. It first crossed a stiff region of the cell and then entered a much more compliant cell region. The work of adhesion and its progression over time were not dependent on the load with which the tip was brought into contact with the cell. We conclude that the initial thermodynamic aspects and the time course of the uptake of nanoparticles by lung epithelial cells can be studied using our experimental approach. It is discussed how the potential health threat posed by nanoparticles of different size and surface characteristics can be evaluated using the method presented.
Assuntos
Pulmão/citologia , Microscopia de Força Atômica , Adesividade , Animais , Células Epiteliais/fisiologia , Masculino , Nanopartículas , Ratos , Ratos Sprague-Dawley , TermodinâmicaRESUMO
Atomic force measurements were performed on supported pulmonary surfactant (PS) films to address the effect of cholesterol on the physical properties of lung surfactant films. We recently found that cholesterol in excess of a physiological proportion abolishes surfactant function, and is the reason that surfactant fails to lower the surface tension upon compression. In this study, we investigated how the loss of mechanical stability observed earlier is related to the local mechanical properties of the film by local force measurements. The presence of 20% of cholesterol in bovine lipid extract surfactant (BLES) resulted in a decrease of the observed adhesive interaction, and an increase in rigidity of the film. We discuss the implication the increased rigidity might have on the functional failure of PS.
Assuntos
Colesterol/farmacologia , Bicamadas Lipídicas/farmacologia , Surfactantes Pulmonares/química , 1,2-Dipalmitoilfosfatidilcolina/farmacologia , Animais , Bovinos , Bicamadas Lipídicas/química , Microscopia de Força Atômica , Modelos Moleculares , Fosfatidilcolinas/farmacologia , Surfactantes Pulmonares/análise , Propriedades de Superfície/efeitos dos fármacos , Tensão Superficial/efeitos dos fármacosRESUMO
The measurement of the thickness of DNA films on microarray as a function of the medium (liquid, air) is gaining importance for understanding the signal response of biosensors. Thiol group has been used to attach DNA strands to gold micropads deposited on silicon surface. Atomic force microscopy (AFM) was employed in its height mode to measure the change in the pad thickness and in its force mode to measure the indentation depth of the nanofilm. A good coherence between the height and force modes is observed for the film thickness in air. The adhesion force was found to be an alternative way to measure the surface coverage of the biolayer at nanoscopic scale. However the force analysis (compression, steric and electrostatic) provides baseline information necessary to interpret the AFM height image in liquid. Analysis of the film thickness distribution shows that the height of the DNA strands depends on both the DNA strand length (15-35 base pairs) and the environment (air, liquid). In air, longer strands lay down onto gold surface whereas the charge reversal of gold in liquid causes a repulsion of longer strands, which stand up.
Assuntos
Materiais Revestidos Biocompatíveis/química , DNA/química , DNA/ultraestrutura , Membranas Artificiais , Microscopia de Força Atômica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Adsorção , Ar , Materiais Revestidos Biocompatíveis/análise , DNA/análise , Teste de Materiais , Conformação de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Tamanho da Partícula , Soluções , Propriedades de Superfície , Aderências TeciduaisRESUMO
The quantification of microstructural strains at the surface of materials is of major importance for understanding the reactivity of solids. The present paper aims at demonstrating the potentialities of the atomic force microscopy (AFM) for mapping the three-dimensional surface strain field on patterned tensile specimens. Electron beam (e-beam) lithography has been used to deposit 16 x 16 arrays of gold-squared pads. Monitoring the evolution of such a pattern under applied strain allows to quantify the triaxial strains both at the micro-(plastic) domain and nanoscale (elastic) domain vs. applied strain. The proposed method was applied to stainless steels after 4.5% plastic strain.
RESUMO
We report on the near-field imaging of microstructured polymer layers deposited on an homogeneous metal thin film on which a surface plasmon mode is excited. The microstructures in the polymer layers are designed by electron beam lithography, and the near-field imaging is performed with a photon scanning tunneling microscope (PSTM). We show that, despite their very small height, the microstructures can be conveniently imaged with a PSTM thanks to the field enhancement at the surface of the metal thin film supporting the surface plasmon. The influence of the illumination conditions on the contrast of the PSTM images is discussed. In particular, we show that both the field enhancement and the near-field intensity distribution around the microstructures depend dramatically upon the illumination conditions, leading to the conclusion that the PSTM is well suited for spatially resolved near-field surface plasmon sensing purposes.
RESUMO
The development of biosensors based on DNA hybridization requires a more precise knowledge of the thermodynamics of the hybridization at a solid interface. In particular, the selectivity of hybridization can be affected by a lot of parameters such as the single-strand (ss)DNA density, the pH, the ionic strength or the temperature. The melting temperature, T(m), is in part a function of the ionic strength and of the temperature and therefore provides a useful variable in the control of the selectivity and sensitivity of a DNA chip. The electrochemical technique has been used to determine the T(m) values when the probe is tethered by a DNA self-assembled monolayer (SAM). We have built a special thin layer cell, which allows the recording of the cyclic voltammogram under controlled temperature conditions. T(m) has been determined by recording the thermogram (current versus temperature) of a redox indicator on a double-stranded hybrid (dsDNA) modified electrode and comparison with the corresponding ssDNA response. T(m) of supported DNA varies linearly with the ionic strength. The stability of the SAMs has been considered and comparison between T(m) in solution and on a solid support has been discussed.
Assuntos
DNA/química , DNA/metabolismo , Eletroquímica/métodos , DNA/genética , Eletrodos , Desnaturação de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Concentração Osmolar , Oxirredução , Sensibilidade e Especificidade , Temperatura , TermodinâmicaRESUMO
The variation in resonance response of microcantilevers was investigated as a function of pressure (10(-2)-10(6)Pa) and temperature (290-390K) in atmospheres of helium (He) and dry nitrogen (N(2)). Our results for a silicon cantilever under vacuum show that the frequency varies in direct proportion to the temperature. The linear response is explained by the decrease in Young's modulus with increasing the temperature. However, when the cantilever is bimaterial, the response is nonlinear due to differential thermal expansion. Resonance response as a function of pressure shows three different regions, which correspond to molecular flow regime, transition regime, and viscous regime. The deflection in flow transition regime resulting from thermal variation has minimal effect on frequency. The frequency variation of the cantilever is caused mainly by changes in the mean free path of gas molecules.
RESUMO
The reactivity of the palladium shaped as a microcantilever is investigated as a function of the hydrogen stoichiometry. A small cell holding the microcantilever is designed to monitor the deflection and the flexural resonance response from high vacuum to a hydrogen gas pressure of several bars. The measurements show that the Young's modulus is accurate if the cantilever is thick enough to be described by a continuum mechanics approach. The orientation distribution function of the palladium grains determined by X-ray diffraction enables to correlate Young's modulus measured using microcantilevers with the elastic constant tensor issued from the literature. The surface stress induced by the dissociation of H(2) in palladium surface depends mainly on the cantilever cross-section. Cantilever response was found to be extremely sensitive to both the palladium lattice expansion induced by the insertion of hydrogen atoms into octahedral sites of palladium and the electronic affinity between palladium and hydrogen.
RESUMO
An electrochemical methodology for bio-molecule sensing using an array of well-defined nanostructures is presented. We describe the fabrication by e-beam lithography of nanoelectrodes consisting of a 100 micro m x 50 micro m area containing interdigitated electrodes of 100 nm in width and interelectrode distance of 200 nm. Sensitivity and response time of the nanoelectrodes are compared to the responses of macro- and microelectrodes. The specificity of the sensor is studied by modifying the gold electrodes with DNA. The technique enables to characterize both single and double-stranded DNA of 15 nucleotides. A special electrochemical cell is adapted to control the temperature and measure the DNA concentration by UV analysis. The electrochemical method requires no label on the DNA, only redox mediators were used.