Biofunctionalization of PET/SiO2 surfaces for single molecule experiments and medical applications

PET/SiO2 layers were chemically modified to maintain immobilization of functional single molecules. GFP molecules provide an ideal system due to their stability and intrinsic fluorescence. GFP in vivo biotinylated within its NH2-terminal region and attached on the substrate via the biotinstreptavidin bond was further investigated with confocal microscopy, atomic force microscopy (AFM) and spectroscopic ellipsometry (SE). AFM revealed monolayered donut-like structures representing assemblies of biotinstreptavidinbiotinGFP immobilized onto PET/SiO2 surfaces via mPEG. In particular, regions with an approximate height of 12 nm, which approaches the molecular dimensions of the above complex given by molecular modeling, could be detected. The dimensions of the donut-like structures suggest a close-to-each-other positioning of the GFP molecules - which, however, retain their functionality, as evidenced by confocal microscopy. © 2011 World Scientific Publishing Company.

Electroosmotic flow analysis of a branched U-turn nanofluidic device.

In this paper, we present the analysis of electroosmotic flow in a branched -turn nanofluidic device, which we developed for detection and sorting of single molecules. The device, where the channel depth is only 150 nm, is designed to optically detect fluorescence from a volume as small as 270 attolitres (al) with a common wide-field fluorescent setup. We use distilled water as the liquid, in which we dilute 110 nm fluorescent beads employed as tracer-particles. Quantitative imaging is used to characterize the pathlines and velocity distribution of the electroosmotic flow in the device. Due to the device's complex geometry, the electroosmotic flow cannot be solved analytically. Therefore we use numerical flow simulation to model our device. Our results show that the deviation between measured and simulated data can be explained by the measured Brownian motion of the tracer-particles, which was not incorporated in the simulation.

Fibers with integrated mechanochemical switches: minimalistic design principles derived from fibronectin.

Inspired by molecular mechanisms that cells exploit to sense mechanical forces and convert them into biochemical signals, chemists dream of designing mechanochemical switches integrated into materials. Using the adhesion protein fibronectin, whose multiple repeats essentially display distinct molecular recognition motifs, we derived a computational model to explain how minimalistic designs of repeats translate into the mechanical characteristics of their fibrillar assemblies. The hierarchy of repeat-unfolding within fibrils is controlled not only by their relative mechanical stabilities, as found for single molecules, but also by the strength of cryptic interactions between adjacent molecules that become activated by stretching. The force-induced exposure of cryptic sites furthermore regulates the nonlinearity of stress-strain curves, the strain at which such fibers break, and the refolding kinetics and fraction of misfolded repeats. Gaining such computational insights at the mesoscale is important because translating protein-based concepts into novel polymer designs has proven difficult.

A Bayesian approach to tracking in single molecule fluorescence microscopy

Using fluorescence microscopy with single molecule sensitivity it is now possible to follow the movement of individual fluorophore tagged molecules such as proteins and lipids in the cell membrane with nanometer precision. These experiments are important as they allow many key biological processes on the cell membrane and in the cell, such as transcription, translation and DNA replication, to be studied at new levels of detail. Computerized microscopes generate sequences of images (in the order of tens to hundreds) of the molecules diffusing and one of the challenges is to track these molecules to obtain reliable statistics such as speed distributions, diffusion patterns, intracellular positioning, etc. The data set is challenging because the molecules are tagged with a single or small number of fluorophores, which makes it difficult to distinguish them from the background, the fluorophore bleaches irreversibly over time, the number of tagged molecules are unknown and there is occasional loss of signal from the tagged molecules. All these factors make accurate tracking over long trajectories difficult. Also the experiments are technically difficulty to conduct and thus there is a pressing need to develop better algorithms to extract the maximum information from the data. For this purpose we propose a Bayesian approach and apply our technique to synthetic and a real experimental data set.

Effects of KI encapsulation in single-walled carbon nanotubes by Raman and optical absorption spectroscopy.

The effect of KI encapsulation in narrow (HiPCO) single-walled carbon nanotubes is studied via Raman spectroscopy and optical absorption. The analysis of the data explores the interplay between strain and structural modifications, bond-length changes, charge transfer, and electronic density of states. KI encapsulation appears to be consistent with both charge transfer and strain that shrink both the C-C bonds and the overall nanotube along the axial direction. The charge transfer in larger semiconducting nanotubes is low and comparable with some cases of electrochemical doping, while optical transitions between pairs of singularities of the density of states are quenched for narrow metallic nanotubes. Stronger changes in the density of states occur in some energy ranges and are attributed to polarization van der Waals interactions caused by the ionic encapsulate. Unlike doping with other species, such as atoms and small molecules, encapsulation of inorganic compounds via the molten-phase route provides stable effects due to maximal occupation of the nanotube inner space.

Single event molecular signalling for estimation and control

Cell biology is characterised by low molecule numbers and coupled stochastic chemical reactions with intrinsic noise permeating and dominating the interactions between molecules. Recent work [9] has shown that in such environments there are hard limits on the accuracy with which molecular populations can be controlled and estimated. These limits are predicated on a continuous diffusion approximation of the target molecule (although the remainder of the system is non-linear and discrete). The principal result of [9] assumes that the birth rate of the signalling species is linearly dependent on the target molecule population size. In this paper, we investigate the situation when the entire system is kept discrete, and arbitrary non-linear coupling is allowed between the target molecule and downstream signalling molecules. In this case it is possible, by relying solely on the event triggered nature of control and signalling reactions, to define non-linear reaction rate modulation schemes that achieve improved performance in certain parameter regimes. These schemes would not appear to be biologically relevant, raising the question of what are an appropriate set of assumptions for obtaining biologically meaningful results. © 2013 EUCA.