Fluorescence Correlation Spectroscopy, a Tool to Investigate Supramolecular Dynamics:  Inclusion Complexes of Pyronines with Cyclodextrin

Abstract

The control of supramolecular systems requires a thorough understanding of their dynamics on a molecular level. We present fluorescence correlation spectroscopy (FCS) as a powerful spectroscopic tool to study supramolecular dynamics with single molecule sensitivity. The formation of a supramolecular complex between beta-cyclodextrin (beta-CD) as host and pyronines Y (PY) and B (PB) as guests is studied by FCS. Global target analysis of full correlation curves with a newly derived theoretical model yields in a single experiment the fluorescence lifetimes and the diffusion coefficients of free and complexed guests and the rate constants describing the complexation dynamics. These data give insight into the recently published surprising fact that the association equilibrium constant of beta-CD with PY is much lower than that with the much bulkier guest PB. FCS shows that the stability of the complexes is dictated by the dissociation and not by the association process. The association rate constants are very similar for both guests and among the highest reported for this type of systems, although much lower than the diffusion-controlled collision rate constant. A two-step model including the formation of an encounter complex allows one to identify the unimolecular inclusion reaction as the rate-limiting step. Simulations indicate that this step may be controlled by geometrical and orientational requirements. These depend on critical molecular dimensions which are only weakly affected by the different alkyl substituents of PY and PB. Diffusion coefficients of PY and PB, of their complexes, and of rhodamine 110 are given and compared to those of similar molecules.

Dynamics of Supramolecular Association Monitored by Fluorescence Correlation Spectroscopy

“…[13,16,[22][23][24][25][26] Here we need a solution which describes two interconverting fluorescent species with different diffusion coefficients observed under Gaussian illumination. Under the further assumption that the relaxation time of the reaction is much shorter than the typical diffusion times of guest and complex (fast exchange), the correlation function shown by Equation (4) is obtained for diffusion and reaction: [27] …”

“…[27] Briefly, the set-up comprises a continuous-wave argon ion laser for excitation at 515 nm (PY and PB) and 496.5 nm (R123), a confocal inverted microscope with high aperture objective (Olympus 60 , NA 1.20, water immersion), a pinhole of 100 mm diameter, single-photon avalanche photodiodes (SPCM-AQR-14, Perkin-Elmer Vaudreuil, Quebec, Canada), and TCSPCmodules (SPC 132, Becker & Hickl GmbH, Berlin, Germany). Correlation curves were calculated with a fast home-built LabVIEW routine (National Instruments).…”

“…They have the same xanthene skeleton, but methyl and ethyl substituents, respectively, at the amino side groups (Figure 4 b), so that the role of steric effects of the guest molecule can be analyzed. The dynamic studies with b-CD have already been published, [27] whereas those with g-CD represent new results. "Soft cages" were modeled by micelles formed by the aggregation of well-known non-ionic surfactants such as Triton X-100 (TX100) [ Figure 4 c].…”

“…In most cases the value of K obtained in fluorescence and absorption titrations is fixed in the fits of the correlation curves, in order to improve the accuracy of other fit parameters as t B and k À . [27,33] For the system PY:g-CD it was not possible to determine the association and dissociation rate constants since the reaction term appears at very short times, where the quality of the data is worse and the anti-bunching term interferes.…”

Single‐Molecule Monitoring of the Structural Switching Dynamics of Nucleic Acids through Controlling Fluorescence Blinking

“…À was obtained through autocorrelation analysis using the following Equation (1) [43][44][45] ( Figure 2c). …”