Single-Molecule Spectroscopy and Fluorescence Correlation Spectroscopy of the Lateral Transport of the T3 Promoter Primer at a Chemical Interface

Abstract

Fluorescence correlation spectroscopy reveals that an oligonucleotide, the T3 promoter primer, undergoes only lateral diffusion when adsorbed to the interface of water and silica chemically modified with a hydrocarbon. The autocorrelation decay fits well to the model of simple diffusion, reporting a diffusion coefficient of 1.8 × 10−6 cm2/s. Single-molecule resolution of bursts for the T3 promoter primer reveals that rare, strong adsorption punctuates the lateral diffusion. Removal of the strong adsorption events from the data set, followed by autocorrelation, shows the actual diffusion coefficient to be 2.8 × 10−6 cm2/s, which is comparable to other oligonucleotides of the same size at the same interface. The single-molecule measurements show that average duration of strong adsorption is 0.2 s, and the average fraction of strongly adsorbed molecules is 10% of the molecules at the interface. While single-molecule spectroscopy reveals a process not evident in fluorescence correlation spectroscopy, the precision of the parameters describing strong adsorption is limited by the statistics of small numbers. Fluorescence correlation spectroscopy is suited to observing a much larger number of events, which is needed for high precision. The two methods are complementary: single-molecule spectroscopy gives estimates of the chemical parameters needed for design of the fluorescence correlation spectroscopy, achieving precise measurements with an accurate interpretation.

Dye Diffusion at Surfaces: Charge Matters

“…The present analysis reveals that there is a prolonged interaction of the R6G with the surface, consistent with chemisorption events described earlier . In conjunction with the frequency analysis data presented in Figure , and the large standard deviations shown in Figure , the dwell time data support the presence of rare, long-lived association events that are consistent with adsorption/desorption processes . As shown in Figure b, and consistent with the FCS data, the Alexa dye conforms to a model of simple diffusion, even close to the coverslip.…”

Heterogeneous Translational Dynamics of Rhodamine B in Polyelectrolyte Multilayer Thin Films Observed by Single Molecule Microscopy

“…This rectangular appearance can be caused by the rapid movement of a dye molecule into the confocal observation zone, followed by reversible immobilization. Single-molecule events with a similar appearance have been reported by other investigators in systems where adsorption is known to take place. , Similar dye adsorption has also been reported in PEM thin films and polyelectrolye solutions on the macroscopic level.…”

“…Based on the evaluation of single events, the mathematical model employed for fitting and interpreting the autocorrelation curves includes terms for both diffusive motion and adsorption (or nanoconfined diffusion) processes that occur over a large range of time scales. Because each translational term in the autocorrelation function is independent, a sum of kinetic parameters for adsorption and diffusion can be utilized to model situations exhibiting more than one process . All the autocorrelated data was fit to the following equation, which also accounts for detector after-pulsing (a common phenomenon in FCS) and photobleaching: -0.82pc G false( normalτ false) = true( A normalp .25em normale − normalτ / normalτ AP − A normalp + 1 true) [ a 1 + 4 D normalτ / normalω 2 + ∑ i italicn b i e − k i τ ] newline10pc true( P .25em normale − k PB normalτ − P + 1 true) …”

On the pH-Responsive, Charge-Selective, Polymer-Brush-Mediated Transport Probed by Traditional and Scanning Fluorescence Correlation Spectroscopy

“…There are two additional explanations for this slow component. The first is that this component is due to the adsorption/desorption equilibrium between the anionic Alexa dye and local cationic traps within the PDMAEMA, as has been reported for faster processes using other methods . An alternate explanation involves trapping of dyes in brush surface irregularities, which also would not depend on brush thickness.…”

“…The first is that this component is due to the adsorption/desorption equilibrium between the anionic Alexa dye and local cationic traps within the PDMAEMA, as has been reported for faster processes using other methods. 38 An alternate explanation involves trapping of dyes in brush surface irregularities, which also would not depend on brush thickness. To distinguish the likely mechanism at the current time would require the development and optimization of both complex simulations and alternate polymer synthesis techniques, both of which are outside of the scope of the current work, which focuses both on verifying the tunable internal chemistry of weak polyelectrolyte brushes and the identification of a new diffusion time scale on the order of seconds for Alexa interacting with PDMAEMA in acidic conditions.…”