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The proposal's goal is to understand the relationship between charge/mass transport within a supported polyelectrolyte membrane, and the associated local polymer structure, with the overall objective of tuning this response.  The specific aims are 1) to investigate and understand diffusive ion transport within supported polyelectrolyte brushes 2)  to investigate and understand probe ion orientation within supported polyelectrolyte brushes.  Progress made on this project in the past year has involved understanding how charged interfaces influence dye transport.  Additionally, we have developed advanced signal processing algorithms for noisy single molecule data.  Both of these projects were necessary steps towards our overall goal of understanding transport in supported polyelectrolyte brushes. 

Project 1: Dye Diffusion at Glass Interfaces

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 glass surfaces used in the work were plasma cleaned, resulting in high purity –OH terminated silica surfaces. 

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. Repulsive interactions for the anionic dye were also observed by using single event blip frequency analysis. 

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. By using additional single-molecule techniques such as bin dwell analysis, it was possible to extract association lifetimes for the long-lived surface interactions.    

The overall impact of this work lies in that it provides general guidelines for performing dynamics experiments near glass surfaces.  We showed 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. Our data demonstrate that a better dye choice is an anionic probe, which exhibits no evidence of depth-dependent diffusion characteristics above a glass surface.  The results and data discussed above were presented in a publication in Langmuir, as noted in the publications list.

Project 2: Advanced Data Analysis Techniques

The resolution of heterogeneous interactions probed in single-molecule experiments is dominated by shot-noise.  Among the several techniques for processing single-molecule time series data, wavelet shrinkage is proven to reduce noise.  Standard wavelet algorithms can also introduce unwanted artifacts when acting on discontinuous signals, because they are not customized for the discrete nature of single-molecule data.  In this project, modifications to the basic method that are specific to smFRET were developed and tested on simulated systems.  It was found that, despite its simplicity compared to its more modern cousins, the Haar wavelet basis produces the most optimally denoised estimates.  Other efforts were spent assessing various thresholding methods, developing a time-localized noise estimator, and implementing a translation-invariant wavelet transformation to reduce artifacts associated with discontinuities and inadequate distinction of noise.  The time-local estimator enhances noise reduction by 5-20 %, and translation-invariant transformation nearly eliminates the aforementioned artifacts.  Kinetic parameters extracted from denoised estimates are accurate to within 5 % of the simulated values.  Overall, the improved resolution results in the complete and accurate characterization of both simple and complex smFRET systems.  Additional results of these efforts include compiled analysis algorithms that are made publicly available on our web site for others’ implementation and testing.  The methods developed and the interpretations resulted in a paper under revision at the Journal of Physical Chemistry B, as noted in the publications list.