The mechanical properties of Rhodococcus RC291 were measured using force spectroscopy equipped with a bacterial cell probe. Rhodococcal cells in the late growth stage of development were found to have greater adhesion to a silicon oxide surface than those in the early growth stage. This is because there are more extracellular polymeric substances (EPS) that contain nonspecific binding sites available on the cells of late growth stage. It is found that EPS in the late exponential phase are less densely bound but consist of chains able to extend further into their local environment, while the denser EPS at the late stationary phase act more to sheath the cell. Contraction and extension of the EPS could change the density of the binding sites, and therefore affect the magnitude of the adhesion force between the EPS and the silicon oxide surface. By treating rhodococcal EPS as a surface-grafted polyelectrolyte layer and using scaling theory, the interaction between EPS and a solid substrate was modelled for the cell approaching the surface which revealed that EPS possess a large capacity to store charge. Changing the pH of the surrounding medium acts to change the conformation of EPS chains.

ÐÐÐÐÐÐÐÐÐWe consider the behaviour of single molecules on surfaces and, more generally, in confined environments. These are loosely split into three sections: single molecules in biology, the physics of single molecules on surfaces, and controlled (directed) diffusion. With recent advances in single molecule detection techniques, the importance and mechanisms of single molecule processes such as localised enzyme production and intracellular diffusion across membranes has been highlighted, emphasising the extra information that cannot be obtained with techniques, which present average behaviour. Progress has also been made in producing artificial systems that can control the rate and direction of diffusion, and because these are still in their infancy (especially in comparison to complex biological systems), we discuss the new physics revealed by these phenomena. Introduction: single molecule diffusionThe motion of the very small has been studied for a long time, [1,2] beginning with the initial microscopic observations of pollen on water in 1827 and continuing in the present day with a vast array of molecular diffusion studies. Initially such studies concentrated on the average -2 -motion of an ensemble of molecules as techniques such as fluorescence recovery after photobleaching [3] (FRAP) were not sensitive enough to observe an individual particle. Despite this, averaging techniques have been, and continue to be, very successful in probing protein dynamics [4][5][6] and protein-protein interactions, [7,8] as well as determining average diffusion coefficients of molecules within a small region. [9,10] Recent advances in experimental techniques have allowed the motion and interactions of single molecules to be studied with improved accuracy. Some of these techniques, such as atomic force microscopy, [11,12] total internal reflection fluorescence (TIRF) imaging, [13,14] and super-resolution imaging, [15][16][17] have allowed direct images to be produced which provide insight into the orientation, [18] clustering, or changes to a molecule within a system. Time-stop imaging techniques have also been used to determine the kinetic properties of a system. [19] However these are somewhat limited by the equipment in terms of exposure time, acquisition rate, and size of detection region. In general, imaging is useful as it provides visual confirmation of the region under study. We show in Figure 1 data exemplifying why single molecule imaging reveals more information than ensemble averaging techniques; here molecular motion is smeared out of the signal when the data are averaged (box 4 in Figure 1), but time-stop imaging reveals a complex molecular trajectory (box 3 in Figure 1).Techniques designed to examine the diffusive properties of molecules within a sample, such as neutron spin echo, [20][21][22] dynamic light scattering, [23,24] fluorescence correlation spectroscopy (FCS), [25][26][27][28] and single mode optical fibre detectors, [29] generally do not involve imaging in real space, but require spectroscopic determ...

We describe a fluorescence correlation spectroscopy investigation into the diffusion of fluoresceintagged dextran (FDEX) in a poly(methacrylic acid) (PMAA) hydrogel. The temperature dependence of FDEX diffusion is shown to follow Zimm behaviour in pure water, and the decrease in the diffusion coefficient when in the PMAA hydrogel has been modelled. The addition of acid and alkali (HCl and NaOH respectively) not only control the swelling and collapse of the hydrogel but also reveal a strong pH dependence of the dextran diffusion coefficient, which shows a (non-monatonic) increase with pH. The addition of NaCl and CaCl 2 salts similarly showed evidence of network swelling, most notably at low salt concentration, but also that the diffusion coefficient within the gel at these low concentrations is larger that in the equivalent solution without the hydrogel, indicating that the combination of hydrogel and salt works to increase the diffusion coefficient above that in pure water.

The diffusion of a polyzwitterion, poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC), in aqueous solution containing different alkali halides was studied by fluorescence correlation spectroscopy at single molecule level. It was found that the halide anion has a greater effect on the radius of zwitterionic PMPC molecules than alkali cations, which is due to the mechanism by which PMPC molecules interact with the surrounding hydrogen bond network of water molecules and adsorbed ions. With the addition of salt, the size of PMPC remains constant while its diffusion coefficient is reduced slightly, although larger cations (e.g. K +) result in slightly increased diffusion coefficient for 1 M potassium chloride-based solutions. This enhanced diffusion coefficient is attributed to the decrease in the viscosity of the aqueous solution on the addition of salt. When the counter-ion was varied in potassium-based salts, different effects were observed for different anions, resulting a reduction in the diffusion coefficient as a function of salt concentration. This reduction was modest for KBr, but significant for KI. Overall, no discernible changes were observed as the size of the PMPC coil was varied, except in case of KI for which a significant increase was observed at higher ionic strength. Divalent cations (Ca 2+ and Mg 2+), produced similar effects to those found for monovalent cations. These effects are explained by the interaction of PMPC with the hydrogen bond network of water molecules and with the adsorbed ions.

The diffusion of rhodamine-labeled poly(ethylene glycol) (r-PEG) within surface-grafted poly(ethylene glycol) (s-PEG) layers in aqueous solution at 18 °C was measured by fluorescence correlation spectroscopy. The diffusion coefficient of r-PEG within s-PEG was controlled by the grafting density, σ, and scaled as σ–1.42±0.09. It is proposed that a characteristic blob size associated with the grafted (brush) layer defines the region through which the r-PEG diffusion occurs. The diffusion coefficients for r-PEG in semidilute solution were found to be similar to those in the brushes.