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.

The systematic description of the complex photophysical behaviour of pyrene in surfactant solutions in combination with a quantitative model for the surfactant concentrations reproduces with high accuracy the steady-state and the time resolved fluorescence intensity of pyrene in surfactant solutions near the cmc, both in the monomer and in the excimer emission bands. We present concise model equations that can be used for the analysis of the pyrene fluorescence intensity in order to estimate fundamental parameters of the pyrene-surfactant system, such as the binding equilibrium constant K of pyrene to a given surfactant micelle, the rate constant of excimer formation in micelles, and the equilibrium constant of pyrene-surfactant quenching. The values of the binding equilibrium constant K(TX100)=3300·10³ M⁻¹ and K(SDS)=190·10³ M⁻¹ for Triton X-100 (TX100) and SDS micelles, respectively, show that the partition of pyrene between bulk water and micelles cannot be ignored, even at relatively high surfactant concentrations above the cmc. We apply the model to the determination of the cmc from the pyrene fluorescence intensity, especially from the intensity ratio at two vibronic bands in the monomer emission or from the ratio of excimer to monomer emission intensity. We relate the finite width of the transition region below and above the cmc with the observed changes in the pyrene fluorescence in this region.

The oligomers formed during the early steps of amyloid aggregation are thought to be responsible for the neurotoxic damage associated with Alzheimer's disease. It is therefore of great interest to characterize this early aggregation process and the aggregates formed, especially for the most significant peptide in amyloid fibrils, Amyloid-β(1-42) (Aβ42). For this purpose, we directly monitored the changes in size and concentration of initially monomeric Aβ42 samples, using Fluorescence Correlation Spectroscopy. We found that Aβ42 undergoes aggregation only when the amount of amyloid monomers exceeds the critical aggregation concentration (cac) of about 90 nM. This spontaneous, cooperative process resembles surfactants self-assembly and yields stable micellelike oligomers whose size (≈50 monomers, R h ≈ 7-11 nm) and elongated shape are independent of incubation time and peptide concentration. These findings reveal essential features of in vitro amyloid aggregation, which may illuminate the complex in vivo process.Alzheimer's disease (AD) is a neurodegenerative disease characterized by the presence of Amyloid-β plaques in the brain. Although the causal relationship between these protein fibrillar aggregates and the neurodegenerative disease has not been established yet, the 'amyloid hypothesis' , that accumulation and aggregation of amyloid-β peptide initiates a cascade of neurodegenerative events, has been widely accepted [1][2][3] . Impairment of Amyloid-β clearance in AD patients seems to be the main cause for accumulation of the peptide 4,5 . It is thought that the neurotoxic species that trigger the amyloid cascade leading to neurodegeneration are early non-fibrillar aggregates, which may also be the precursors of the amyloid fibrils 2,6-8 . The dominant peptides in amyloid fibrils are Amyloid-β(1-42) (Aβ42) and Amyloid-β(1-40) (Aβ40), with Aβ42 being the more fibrillogenic of the two, with a much stronger tendency to aggregate [9][10][11] .There is ample literature on the mechanism underlying amyloid fibril formation 12,13 . Most kinetic studies agree on a complex nucleation-growth mechanism, where the differences in the microscopic rates and in the relevance of secondary nucleation processes determine the degree of aggregation and can account for the differences between Aβ40 and Aβ42 11,14 . For such nucleation-dependent processes, a critical aggregation concentration (cac) is predicted, above which aggregation takes place 10 . For Aβ40 the formation of micelle-like intermediates was reported, with a critical concentration in the micromolar range [15][16][17] , whereas recent studies have found nanomolar cac values for both Aβ40 and Aβ42 [18][19][20] . The latter values fit better with the reported physiological concentrations of Aβ in the picomolar to nanomolar range which may be locally higher due to accumulation or impairment of clearance 4,5,18,21,22 . A possible reason for the discrepancy in the cac values may be the strong adsorption of Amyloids Aβ40 and Aβ42 to interfaces 23 , which can lead to great ...

An empirical model for the concentrations of monomeric and micellized surfactants in solution is presented as a consistent approach for the quantitative analysis of data obtained with different experimental techniques from surfactant solutions. The concentration model provides an objective definition of the critical micelle concentration (cmc) and yields precise and well defined values of derived physical parameters. The use of a general concentration model eliminates subjective graphical procedures, reduces methodological differences, and thus allows one to compare directly the results of different techniques or to perform global fits. The application and validity of the model are demonstrated with electrical conductivity, surface tension, NMR chemical shift, and self-diffusion coefficient data for the surfactants SDS, CTAB, DTAB, and LAS. In all cases, the derived models yield excellent fits of the data. It is also shown that there is no need to assume the existence of different premicellar species in order to explain the chemical shifts and self-diffusion coefficients of SDS as claimed recently by some authors.

Thioflavin T is a highly sensitive fluorescent marker of amyloid fibrils that has been widely used for in vitro biomedical assays. However, neither its complex photophysical behavior nor its binding mode to amyloid fibrils are still well understood. We present a detailed analysis of the photophysical properties of Thioflavin T in various media, including solvents and solvent mixtures of different viscosities as well as fibrillar and globular proteins. We propose a model that explains the strong wavelength dependency of the Thioflavin T fluorescence and the large fluorescence enhancement in certain environments. We determine the binding affinities and the fluorescence properties of Thioflavin T bound to amyloid-β (1-42) fibrils and to bovine serum albumin and discuss the sensitivity and the specificity of this probe to amyloid aggregates. These results allow us to assess the suitability of Thioflavin T for quantitative determinations in biomedical studies.

The host-guest complexation between an Alexa 488 labelled adamantane derivative and β-cyclodextrin is studied by Fluorescence Correlation Spectroscopy (FCS). A 1:1 complex stoichiometry and a high association equilibrium constant of K = 5.2 × 104 M−1 are obtained in aqueous solution at 25 °C and pH = 6. The necessary experimental conditions are discussed. FCS proves to be an excellent method for the determination of stoichiometry and association equilibrium constant of this type of complexes, where both host and guest are nonfluorescent and which are therefore not easily amenable to standard fluorescence spectroscopic methods.

A novel fluorescent host−guest material, molecular sieves of AlPO4-5-type doped with 2,2‘-bipyridyl-3,3‘-diol, was prepared and spectroscopically characterized. The composite crystals show a pronounced optical anisotropy, indicating a high degree of alignment of the guest molecules within the zeolitic pore system. A mean tilting angle of 22° was found for the orientation of the individual dye molecules in the straight channels. The corrected fluorescence emission spectra were determined, and time-resolved fluorescence studies revealed that the dye molecules are preferentially found in three different types of microenvironment. By invoking pH-dependent studies of the dye in aqueous solution, we could trace these spectroscopic features back to two main influences, coadsorbed water within AlPO4-5 pores and guest−host interactions with a few relatively weak Brønsted acid (defect) sites of the inorganic host network.

Supramolecular binding is a key process in many biological systems and in newly developed supramolecular assemblies. Most of the scientific work on these systems is focused on their structural properties and on the thermodynamics of the association process. However, the underlying dynamics are usually much less known, in spite of the great importance they have during the binding process in these highly dynamic systems. Understanding supramolecular binding in biological systems and controlling the functionality of new synthetic supramolecular systems can only be achieved through knowledge of the structure-dynamics relationship. There is a strong need for suitable techniques which cover the typically wide time interval of the association dynamics and which do not need a perturbation of the system. We briefly review high-resolution fluorescence correlation spectroscopy (FCS) as a technique to monitor supramolecular dynamics and to give information on how structure determines the dynamics of host-guest association. The comparison of hosts and guests with different structures shows that geometrical and orientational requirements determine the association rate constant, whereas the dissociation is defined by the strength of specific interactions. As model hosts cyclodextrins and micelles are studied.