Single Molecule Tracking Studies of Lower Critical Solution Temperature Transition Behavior in Poly(N-isopropylacrylamide)

Abstract

In this thesis project, single molecule tracking, SMT, experiments in poly(nisopropylacrylamide, pNIPAAm, were carried out using probe fluorophores that had partitioned into the polymer. When the pNIPAAm switches from expanded to collapsed at elevated temperatures, the free volume accessible to the diffusing molecules decreases and trajectories become more confined.The work presented here can be best understood when organized into two categories-SMT trajectory analysis techniques and SMT results in pNIPAAm. In the first category, four techniques for analyzing SMT data -confinement level analysis, time series analysis and statistical analysis of lateral diffusion, multistate kinetics, and a newly developed, radius of gyration evolution analysis -were compared using a set of sample fluorophore trajectories obtained from diffuse probe in surface-tethered pNIPAAm. The five SMT trajectories, ranging in length from 41 to 273 steps, were all successfully analyzed by all four techniques, provided two important criteria were met: enough steps to define the motion were acquired in the trajectory, generally on the order of 50 steps, and the fast and slow diffusion coefficients differed by at least a factor of 5. Beyond that, the four trajectory analysis methods studied provide partially confirmatory and partially complementary information. SMT data resulting from more complex physical behavior may well benefit from using these techniques in succession to identify and sort populations.In the second category of research, spatial and temporal heterogeneities in expanded and collapsed surface bound pNIPAAm films were studied by SMT experiments. Tracking data were analyzed using two methods involving radius of gyration (R g ) evolution and confinement iii level calculations. These two analysis techniques were used to elucidate the range of behaviors displayed by hundreds of single molecules, which exhibited complex diffusion. The main conclusions that were drawn from this work were: 1) small molecule probe behavior in pNIPAAm is dictated by the free volume within surface tethered chains, and the distribution of probe behavior can be used to gain nanometer-scale information about the state of the polymer brush at high and low temperatures, 2) confinement level calculations and radius of gyration evolution results show a larger degree of confinement at higher temperatures, measureable in higher percentage of confined steps in a trajectory, longer periods of confined events, and smaller area of confined zones.

Direct Nanoscopic Measurement of Laminar Slip Flow Penetration of Deformable Polymer Brush Surfaces: Synergistic Effect of Grafting Density and Solvent Quality

“…As shown in Table S1, for the two extreme conditions in this study, in going from 192 to 2952 μm/s, D app values increase from 2.7 × 10 –12 to 1.5 × 10 –11 cm 2 /s for the low-density film under good solvent conditions (100% MeOH), whereas for the high-density film in a poor solvent (31 mol % MeOH/H 2 O), the corresponding D app values only increase from 9.5 × 10 –13 to 1.5 × 10 –12 cm 2 /s. These values clearly reflect distinctive changes in D app due to external flow as well as solvent quality, yet still are reasonable as small molecule diffusion coefficients in dense polymer brushes. ,, …”

Molecule motion at polymer brush interfaces from single‐molecule experimental perspectives

“…SMT has already been applied to study various complex material systems such as membrane‐incorporated molecule diffusion in cells, molecular transport in sol‐gel films, and intracellular delivery of nanomedicines . The example in Figure (b) exhibits the trajectories of individual fluorescence probes inside a polymer brush layer obtained by combining SMT with TIRF …”

“…Although the combination of FCS and single‐molecule polarization is useful in examining the diffusive and orientational dynamics of fluorescence probes in polymer brushes, it lacks spatial resolution and is incapable of detecting spatial heterogeneity in translational diffusions. Using SMT, Elliott et al investigated the diffusion of small fluorescent probes inside PNIPAM brushes of an estimated grafting density of ∼0.1 chains/nm 2 and a swollen brush thickness of 115 nm in deionized water, which is comparable to the excitation depth in a TIRF microscopic setup . Figure shows representative results.…”

Imaging Fluorescence-Correlation Spectroscopy for Measuring Fast Surface Diffusion at Liquid/Solid Interfaces

“…This concept was demonstrated for diffusion of fluorescently labeled alkanoic acids at a fusedsilica−hydrocarbon 22 and methylated-silica−water interfaces 23 and of amphiphilic dyes in stimulus-responsive thin films of poly(N-isopropylacrylamide) below and above their critical transition temperature. 24,25 Single-molecule imaging and tracking have also been adapted to porous silica films and particles, to investigate the influence of pore structure and surface interactions on molecular transport within both thin porous sol−gel films 26−29 and reversed-phase chromatographic silica particles. 30 These measurements reveal spatial heterogeneities in silica structure that lead to localized variations in molecular diffusivities, where molecular motions depend on the chemistry of the fluorescent probe molecule, the ordering of the film during deposition, and treatment of the pore surface.…”