We have evaluated the effect of varying three key parameters for Fluorescence Correlation Spectroscopy analysis, first in the context of a one species/one environment system, and then in a complex system composed of two species, or conversely, two environments. We establish experimentally appropriate settings for the (1) minimum lag time, (2) maximum lag time, and (3) averaging times over which an autocorrelation is carried out, as a function of expected diffusion decay time for a particular solute, and show that use of appropriate settings plays a critical role in recovering accurate and reliable decay times and resulting diffusion constants. Both experimental and simulated data were used to show that for a complex binary system, to extract accurate diffusion constants for both species, decay times must be bounded by adequate minimum and maximum lag times as dictated by the fast and slow diffusing species, respectively. We also demonstrate that even when constraints on experimental conditions do not permit achieving the necessary lag time limits for both of the species in a binary system, the accuracy of the recovered diffusion constant for the one species whose autocorrelation function is fully time-resolved is unaffected by uncertainty in fitting introduced by the presence of the second species.

Diffusive transport within complex environments is a critical piece of the chemistry occurring in such diverse membrane systems as proton exchange and bilayer lipid membranes. In the present study, fluorescence correlation spectroscopy was used to evaluate diffusive charge transport within a strong polyelectrolyte polymer brush. The fluorescent cation rhodamine-6G was used as a counterion probe molecule, and the strong polyelectrolyte poly(styrene sulfonate) was the polymer brush. Such strong polyelectrolyte brushes show promise for charge storage applications, and thus it is important to understand and tune their transport efficiencies. The polymer brush demonstrated preferential solvation of the probe counterion as compared to solvation by the aqueous solvent phase. Additionally, diffusion within the polymer brush was strongly inhibited, as evidenced by a decrease in diffusion constant of 4 orders of magnitude. It also proved possible to tune the transport characteristics by controlling the solvent pH, and thus the ionic strength of the solvent. The diffusion characteristics within the charged brush system depend on the brush density as well as the effective interaction potential between the probe ions and the brush. In response to changes in ionic strength of the solution, it was found that these two properties act in opposition to each other within this strong polyelectrolyte polymer brush environment. A stochastic random walk model was developed to simulate interaction of a diffusing charged particle with a periodic potential, to show the response of characteristic diffusion times to electrostatic field strengths. The combined results of the experiments and simulations demonstrate that responsive diffusion characteristics in this brush system are dominated by changes in Coulombic interactions rather than changes in brush density. More generally, these results support the use of FCS to evaluate local charge transport properties within polyelectrolyte brush systems, and demonstrate that the technique shows promise in the development of novel polyelectrolyte films for charge storage/transport materials.

Single molecule polarization and fluorescence correlation spectroscopy were used to evaluate heterogeneous transport mechanisms of molecular ions within supported polyelectrolyte brushes. Modes of diffusive transport include periods of significantly restricted rotational motion, often maintained over tens of milliseconds; periods of fast molecular rotation; and occasional adsorption of fluorescent probe molecules in the brush. The studies reveal rapid switching between orientational states during each observed mode of motion. Through quantitative analysis of state occupation times, the rate constants for transitions from weakly associated to strongly associated states were extracted. Additionally, the pH dependence of the ion transport rates in the brush exhibits an abrupt, rather than continuous, trend. These single molecule studies demonstrate the presence of dynamic anisotropic interactions between the charged molecular probe and the polymer brush and provide experimental evidence of stimuli responsive switchable transport functionality in the polyelectrolyte brush.

Fluorescence correlation spectroscopy and single molecule burst analysis were used to measure the effects of glass surface interactions on the diffusion of two common fluorescent dyes, one cationic and one anionic. The effects of dye–surface interactions on measured diffusion rates as a function of distance from the surface were investigated. Use of a three-axis piezo stage, combined with reference calibration measurements, enabled the accurate acquisition of surface-distance-dependent transport data. This analysis reveals attractive interactions between the cationic dye and the surface, which significantly alter the extracted diffusion values and persist in the measurements up to 1.0 μm from the surface. The Coulomb attraction between the cationic dye and the surface also results in rare, long-lived association events that lead to irreproducibility in extracted diffusion values. In addition to an assignment of the association lifetime for these events, this paper demonstrates that, if experiments must be performed with cationic probes near a glass surface, the use of solution electrolytes can eliminate deleterious dye–surface interactions, as the dyes were tested in a variety of environments. Finally, our data demonstrate that a better dye choice is an anionic probe, which exhibits no depth dependence of diffusion characteristics above a glass surface.

Ion-exchange chromatography (IEX) relies on electrostatic interactions between the adsorbent and the adsorbate, and is used extensively in protein purification. Conventional IEX utilizes ligands that are singly charged and randomly dispersed over the adsorbent, creating a heterogeneous distribution of potential adsorption sites. Clustered-charge ion exchangers exhibit higher affinity, capacity, and selectivity than their dispersed-charge counterparts of the same total charge density. In the present work, we monitored the transport behavior of an anionic protein near clustered-charge adsorbent surfaces using Fluorescence Correlation Spectroscopy. We can resolve protein free diffusion, hindered diffusion and association with bare glass, agarose-coated, and agarose-clustered peptide surfaces, demonstrating that this method can be used to understand and ultimately optimize clustered charge adsorbent and other surface interactions at the molecular scale.

The present work reports on in situ observations of the interaction of organic dye probe molecules and dye-labeled protein with different poly(ethylene glycol) (PEG) architectures (linear, dendron, and bottle brush). Fluorescence correlation spectroscopy (FCS) and single molecule event analysis were used to examine the nature and extent of probe–PEG interactions. The data support a sieve-like model in which size-exclusion principles determine the extent of probe–PEG interactions. Small probes are trapped by more dense PEG architectures and large probes interact more with less dense PEG surfaces. These results, and the tunable pore structure of the PEG dendrons employed in this work, suggest the viability of electrochemically-active materials for tunable surfaces.

Single-molecule fluorescence spectroscopy is employed to reveal 3-dimensional details of the mechanisms underpinning ion transport in a polyelectrolyte thin film possessing polymer-brush nanoscale order. The ability to resolve fluorescence emission over three discrete polarization angles reveals that these ordered materials impart 3-dimensional orientation to charged, diffusing molecules. The experiments, supported by simulations, report global orientation parameters for molecular transport, track dipole angle progressions over time, and identify a unique transport mechanism: translational diffusion with restricted rotation. Generally, realization of this experimental method for translational diffusion in systems exhibiting basic orientation should lend itself to evaluation of transport in a variety of important, ordered, functional materials.

Conspectus Functional polymers have a wide variety of applications ranging from energy storage to drug delivery. For energy storage applications, desirable material properties include low cost, high charge storage and/or mobility, and low rates of degradation. Isotropic thin films have been used for many of these types of applications, but research suggests that different structures such as polymer brushes can improve charge transport by an order of magnitude. Supported polymer brush structures produced by ‘grafting-from’ polymerization methods offer a framework for a controlled study of these materials on the molecular-scale. Using these materials, researchers can study the basis of hindered diffusion because they contain a relatively homogeneous polyelectrolyte membrane. In addition, researchers can use fluorescent molecular probes with different charges to examine steric and Coulombic contributions to transport near and within polymer brushes. In this Account, we discuss recent progress in using fluorescence correlation spectroscopy, single-molecule polarization-resolved spectroscopy, and a novel 3-dimensional orientational technique to understand the transport of charged dye probes interacting with the strong polyanionic brush, poly(styrene sulfonate). Our preliminary experiments demonstrate that a cationic dye, Rhodamine 6G, probes the brush as a counterion, and diffusion is therefore dominated by Coulombic forces, which results in a ten-thousand-fold decrease in the diffusion coefficient in comparison with free diffusion. We also support our experimental results with molecular dynamics simulations. Further experiments show that up to 50 percent of the time, Rhodamine 6G translates within the brush without significant rotational diffusion, which indicates a strong deviation from Fickian transport mechanisms (In which translational and rotational diffusion are related directly through parameters such as chemical potential, size, solution viscosity, and thermal properties). To understand this oriented transport, we discuss the development of an experimental technique that allows us to quantify the 3-dimensional orientation on the time scale of intra-brush transport. This method allowed us to identify a unique orientational transport direction for Rhodamine 6G within the poly(styrene sulfonate) brush and to report preliminary evidence for orientational dye ‘hopping.’