A method is presented for the covalent attachment of oligonucleotides to silicon (100) surfaces patterned with micron-scale features. UV light exposure of hydrogen-terminated silicon (100) coated with alkenes functionalized with N-hydroxysuccinimide ester groups results in Si-C bonded monolayers. The N-hydroxysuccinimide ester surfaces act as a template for the subsequent covalent attachment of DNA oligonucleotides. In order to create patterns of surface attached DNA oligonucleotides with high density, the surface attachment chemistry has been investigated and optimised. Micron-scale patterning of surfaces was achieved by exposure with UV laser light via a mask. DNA oligonucleotide patterns, with feature sizes of several microns, were reliably produced over large areas. The patterned surfaces were characterised with scanning electron microscopy, epifluorescence microscopy and ellipsometry. Hybridisation with fluorescent label- and gold nanoparticle-conjugates of the complementary oligonucleotide is achieved. The methods offer reliable approaches for the creation of micron-scale motifs of DNA on surfaces.
The present work takes advantage of the intrinsic localisation of two-photon fluorescence excitation to develop two-photon fluorescence recovery after photobleaching -TP-FRAP - as a method to assess fluorophore dynamics with microscopic resolution. Numerical simulations are proposed to improve data interpretation beyond the usual frame of FRAP data analysis. This work was developed for measuring the dynamics of cytoskeleton proteins. The apical face of epithelial cells is covered with a dense set of microvilli, the main components of which have been identified and localized at the ultrastructural level, but their dynamic organisation remains largely unknown. To understand the apical morphogenesis and to assess the dynamics of cytoskeleton proteins that might underlie the steady-state morphology, GFP-fusion proteins were expressed. Using TP-FRAP, fluorophore dynamics could be resolved between the plasma membrane and the cytosol, and interpreted in terms of diffusive mobility or exchange rates within and between these two compartments. This is applied in particular to ezrin, a membrane-actin linker protein localized in the cytosol and at the plasma membrane, which plays a key role in coupling signal transduction to cortical morphogenesis. TP-FRAP experiments in conjunction with ezrin mutaganesis and numerical modelling strongly suggest a fast cyclic renewal dynamics with three sequential membrane binding states with distinct mobilities and biochemical reactivities. This paper presents a detailed account of the instrumental design and the numerical method developed to interpret recovery data. This approach should be more generally useful to locally assess the dynamics of protein turnover in submicroscopic structures, and resolve its molecular basis.
Phosphorylation and dephosphorylation, which are the most remarkable posttranslational modifications, are considered to be important chemical reactions that control the activation of proteins. We examine the phosphorylation analysis method by measuring the infrared absorption peak of phosphate group that observed at about 1070cm-1 (9.4μm) with Fourier Transform Infrared Spectrometer (FT-IR). This study indicates that it is possible to identify a phosphorylation by measuring the infrared absorption peak of phosphate group observed at about 1070 cm-1 with FT-IR method. As long as target peptides have the same amino acid sequence, it is possible to identify the phosphorylated sites (threonine, serine and tyrosine).
For the measurement of biomolecular interactions such as immunoreactions it is often necessary to prepare reporter molecules to detect small biomolecules. In many cases fluorescence markers are used to detect the binding between molecules. These markers, however, can influence the examined reaction. A label-free optical detection method based on the principle of a Young interferometer offers an alternative solution. This technology allows real-time, kinetic analysis of antigene-antibody reactions or the detection of a specific analyte without elaborate sample preparation. Especially reactions where it is inconvenient or impossible to use markers can be detected with this method. In this paper, an interferometric device based on a planar waveguide as sensing element is presented. The system yields a high resolution with respect to surface mass coverage and a low sensitivity towards undesired external influences. Interferometric sensors theoretically have the highest detection limits among label-free bionsensors.
A microinterface for ElectroSpray Mass Spectrometry (ESI-MS), acting as an electrochemical cell, was developed for the on-chip electrochemical tagging of cysteines in peptides and proteins during electrospray ionisation which occurs via a 1,4-Michael addition. Benzoquinones are electrogenerated from the corresponding hydroquinones to react specifically with cysteine residues and the production of the resulting adducts is followed by MS. The electrotagging efficiency was tested on L-cysteine using several hydroquinones bearing either electrodonating or withdrawing groups. The reaction kinetics was determined by electrochemical techniques such as Cyclic Voltammetry (CV) and was checked by MS experiments. In both experiments, carboxymethylhydroquinone was found to be the best tagging reagent. The electrochemical tagging was successfully applied to the identification of target proteins through the labelling of peptides coming from their proteolytic digestion. The information on the number of cysteine residues in proteolytic peptides was found to enhance the protein identification confidence.
Protein and polysaccharide nano-patterning is of prime interest for biological applications but also for applications in the field of diffractive optics. In this work, we used a photo-nano-patterning process based on light interferences through a photo-sensitive material for patterning polysaccharides and polypeptides pure and mixed gels of gelatin, hyaluronan, and chitosan. Chromium ions were incorporated in the gels to render them photo-sensitive. Polyelectrolyte multilayer thin films of poly(L-lysine)/hyaluronan were also investigated either by incorporating chromium ions or by adsorbing a photo-sensitive hyaluronan. Depending on the weights ratios of the polymers, respectively gelatin/chitosan and gelatin/hyaluronan, the gel surfaces exhibit different fringe patterns, as can be visualized by atomic force microscopy. The diffracted intensity characterizing the holographic grating was also depending on gel type. Pure gelatin gels was taken as the reference material. The best results in terms of surface patterns and diffracted intensities were obtained for the gelatin/chitosan gels prepared at acidic pH and exposed at energies ranging from 100 to 400 mJ/cm2. Our results show that surface patterns of various depths and structures can be created by the photo-patterning technique on biological polymers. These results open new perspectives for the surface control of biological materials but also for making use of the optical properties of these biocompatible biopolymers.
Molecular motors are multicomponent molecular structures that consume energy to induce motion and to generate forces. Their dynamics covers various time and length scales and critically depends on chemical-mechanical coupling, external forces and molecular properties such as diffusion, particle distribution and density. The complex behavior of these systems consequently offers a formidable challenge for theoretical descriptions and numerical approaches that aim to provide a computational laboratory for a fundamental analysis of the underlying interaction mechanisms as well as interpretations or to study control of the system's behavior. Coupling a linear molecular motor system to an energy supply can induce movement of the motor molecules along a filamentous structure. The complex dynamics of bound (i.e. attached to a filament) and free (i.e. diffusing in the surrounding medium) molecular motors thereby may depend on the diffusive properties of the molecules and on the excitation process driving the motor system. Our theory is therefore based on spatially dependent Fokker-Planck equations for the dynamics of bound and free motors. The model considers spatially inhomogeneous transition rates coupling the energetic sublebels of the molecules as well as spatial fluctuations and diffusion. Computational modelling of the spatio-temporal dynamics of molecular motors shows that both, molecular diffusion and bandwidth of the transition rate set an upper limit to the efficiency of the motor progression. A sufficiently small molecular diffusion as well as a thorough adjustment of transition rates lead to a regular forward propagation while for high diffusion and improperly chosen rates spatio-temporally diverging particle distributions may evolve. Suitable excitation conditions for efficient movement-control are discussed.
In this paper we are going to introduce optical methods for blood cell characterization performed by standard hematology analyzers. As a consequence, we are going to focus on methods that are, or will be, compatible with an average cost, commercial, blood cell counter.
Photopolymers are widely used to elaborate diffractive components by light wavefront engineering. Photopolymer material is light structured through wavefront engineering causing interferences that will be printed in photopolymer layer to constitute the diffractive structure. We show that good diffractive components results can be obtained with photoprotein produced by metal doping of selected proteins coming form biology or biotechnology. We validate this for one application concerning the diffractive storage.
Surface plasmon resonance imaging is an optical method that allows the real time detection of small changes in the physical properties of a dielectric medium near a metallic surface. Using proper surface functionalization and structuration, this technique can be applied to the realization of optical biochips where multiple unlabeled interactions can be monitored. More precisely, thanks to the use of an adequate optical set-up built around a gold surface realized by self assembled monolayers or electrocopolymerization, we studied DNA:DNA interactions with potential application to genetic diagnostic and DNA:protein interactions to demonstrate the ability of the system to determine simultaneously different affinity constants.
Direct methods (spatial differentiation), fractal analysis and spectral analysis (“spectrum enhancement”) have been applied to extract morphological descriptors from images of cytoskeletal microtubules. Images had been obtained from experiments on cultured cells (rat hepatocytes). Principal components analysis has been applied to morphological descriptors. An image classifier has thus been trained to tell normal (control) microtubule structures from those treated by a given concentration of a fungicide for a given time. Validation has been performed on sets of new images of the same two classes. Then the classifier has been used to rank the morphology of microtubules treated at lower doses and to quantify structural recovery after exposure. The paper is the first account of extensive morphological classification of microtubules and paves the way to a dose-response relationship based on quantitative morphology.