The persistence length of adsorbed dendronized polymers

The persistence length of cationic dendronized polymers adsorbed onto oppositely charged substrates was studied by atomic force microscopy (AFM) and quantitative image analysis. One can find that a decrease in the ionic strength leads to an increase of the persistence length, but the nature of the substrate and of the generation of the side dendrons influence the persistence length substantially. The strongest effects as the ionic strength is being changed are observed for the fourth generation polymer adsorbed on mica, which is a hydrophilic and highly charged substrate. However, the observed dependence on the ionic strength is much weaker than the one predicted by the Odijk, Skolnik, and Fixman (OSF) theory for semi-flexible chains. Low-generation polymers show a variation with the ionic strength that resembles the one observed for simple and flexible polyelectrolytes in solution. For high-generation polymers, this dependence is weaker. Similar dependencies are found for silica and gold substrates. The observed behavior is probably caused by different extents of screening of the charged groups, which is modified by the polymer generation, and to a lesser extent, the nature of the substrate. For highly ordered pyrolytic graphite (HOPG), which is a hydrophobic and weakly charged substrate, the electrostatic contribution to the persistence length is much smaller. In the latter case, we suspect that specific interactions between the polymer and the substrate also play an important role.

Scaling properties of DNA knots studied by atomic force microscopy

Erika Ercolini

The main subject of the present thesis is the experimental study of the scaling properties of DNA knots of different complexities by tapping mode atomic force microscopy (AFM) in air. Homo- or heterogeneous mixture of DNA knot types were deposited onto mica in regimes of (i) strong adsorption, which induces a kinetic trapping of the molecules, and of (ii) weak adsorption, which permits relaxation on the surface. The contour of each knotted molecule was analyzed by a box counting algorithm, giving the number of boxes containing a part of the molecule, N(L), as a function of the box size, L, allowing to recover the relation N(L) ≈ L-df, where df is the fractal dimension and ν = 1/df is the scaling exponent. This relationship is complicated by the presence of a persistence length of DNA (about 50 nm) which introduces a crossover from a rigid rod behavior to a self-avoiding walk behavior. In (i) the radius of gyration of the adsorbed DNA knot scales with the 3D Flory exponent ν ≈ 0.58 within error. In (ii), the value ν ≈ 0.66, intermediate between the 3D and 2D (ν = 3/4) exponents, was found, indicating an incomplete 2D relaxation or a different polymer universality class. A different analysis, where the fractal dimension was determined by a customized box counting algorithm giving the knot mass as a function of the box size, yielded compatible results. In the case of weak binding conditions, AFM images show evidence of the localization of knot crossings, which is an effect theoretically predicted for knotted polymers confined in two dimensions (flat knots). Part of this thesis is dedicated to a fascinating application of AFM: the imaging of biomolecules in an aqueous buffer. In particular, images of plasmids adsorbed to mica strong enough to be visualized and loosely enough to see them moving in consecutive scans will be shown. The possibility of imaging under buffer molecules loosely anchored to the substrate opens the way to the investigation of biological processes near physiological conditions. The first step of the study of homologous recombination will be presented. We will also show images concerning our study of the activity of topoisomerase I on supercoiled plasmids. Clusters of DNA molecules and proteins were found when imaging in presence of magnesium, in agreement with recent findings by AFM in air. The research on topoisomerases and their interactions with inhibitors or poisons is a hot topic, being these enzymes targets of anti-cancer drugs. Our study paves the way to the investigation of the activity of topoisomerase II, the enzyme which removes knots from DNA. Finally, the principles of static and dynamic light scattering, and the results of dynamic light scattering on latex beads of known size will be presented. Particular emphasis will be given to the discussion of the contribution of light scattering techniques for future experiments on the study of the static and dynamic properties of DNA knotted molecules in solution. These experiments would contribute to shed light on the possible dependence of the gyration radius of knotted polymers on the knot type, and would allow testing theoretical predictions about their relaxation time.

EPFL2008

Stimuli-responsive nanostructured surfaces

Ana Maria Popa

The coupling of stimuli-responsive macromolecules to nanostructured surfaces opens the perspective for the fabrication and integration of "smart" micro- and nano-electro-mechanical systems (MEMS&NEMS) in which the smallest motile unit is the polymeric chain. The goal of this work was to develop thermoresponsive, durable nanoporous surfaces as a first step toward the fabrication of functional nanocontainers and nanovalves. The proposed strategy involves in a first step the design and implementation of a clean-room compatible, reproducible and up-scalable method for defining organized nanoporous arrays in MEMS compatible surfaces, such as silicon, silicon oxide and silicon nitride. The main objective of the second step was the covalent immobilization of responsive macromolecules on the obtained structures, as to provide the later with a "smart" behavior. The nanostructuring step was realized by block-copolymer assisted lithography. This newly developed technique enables the definition of nanoscale features on semiconductor substrates over large surface areas and in a cost effective manner. A novel method for patterning thin metal films by a lift-off approach, using spin coated block-copolymer micelles monolayers as a template, was investigated. The obtained nanoporous mask was used as resist for the structuring of hard substrates in plasma-assisted processes and structures with aspect ratios as high as 1:10 were be obtained. By using different block-copolymer micelles and spin-coating conditions, the proposed approach enables the variation of the nanopores' diameter from 40 to 80 nm and a surface coverage between 11.7% and 25.5 % in an independent manner. This technique was successfully integrated in a standard microfabrication process for the batch production of thin nanoporous silicon nitride membranes. The 100 nm thick membranes span over thousands of square micrometers and present porosities higher than 109 pores/cm2. They are thus competitive in terms of porosity with the commercially available polymeric membranes. In addition they are two orders of magnitude thinner than polymeric membranes, this making them extremely interesting for applications where permeation rate or response time is important. The rapid flow of water soluble small molecular species through the nanopores was demonstrated in a qualitative manner in this work. Another series of investigations targeted the functionalization of the available nanoporous surfaces with responsive molecules. Poly(N-isopropyl acrylamide) (PNIPAAM) was chosen as a model system. Its coupling to silicon surfaces by a "grafting-to" approach from melt, using an epoxy-functionalized silane as anchoring layer was studied. The analysis of the yielded grafting density showed results superior to those obtained by classic grafting-to approaches. The relation between the polymer's weight and its responsiveness was investigated in liquid media by atomic force microscopy and by dynamic force spectroscopy. Polymers with more than 500 monomers in the back bone were shown to exhibit enhanced responsiveness. Additionally, two bioengineered polymers from the class of elastin-like polymers (ELPs) were investigated as possible candidates for the fabrication of stimuli responsive nanovalves. The molecules were synthesized in collaboration with the Bioforge group (Universidad de Valladolid, Spain) and details of the bioproduction process are presented. A method for the covalent photoattachment of macromolecules using a benzophenone-functionalized silane, was tested in this work. First the capacity of the benzophenone silane to graft synthetic thermo-responsive polymers such as PNIPAAM was demonstrated. The temperature-induced morphology change of the photografted PNIPAAM was not affected by this anchoring strategy and was monitored by tapping AFM in liquid media. The investigations were then extended to the photo-immobilization of ELPs on nanoporous silicon surfaces and the XPS measurements revealed the success of this step. However no conformation modification of the proteins could be evidenced by tapping-mode AFM in liquid media under different ionic strengths.

EPFL2009

Scaling properties of DNA knots studied by atomic force microscopy

Erika Ercolini

EPFL2008

Stimuli-responsive nanostructured surfaces

Ana Maria Popa

EPFL2009