Four amphiphilic block copolymers polyisobutylene-block-poly(methacrylic acid) (IB m -MAA n ; m = 70−134, n = 52−228) were synthesized and transferred into aqueous medium at pH 10−12. Their structure in solution was characterized by fluorescence correlation spectroscopy (FCS), static and dynamic light scattering (SLS, DLS), analytical ultracentrifuge (AUC), and by transmission electron microscopy (TEM) with freeze-fracturing and staining techniques. DLS data, AUC sedimentation traces, and TEM images indicate at least two different kinds of particles. TEM shows spherical micelles; however, especially for polymers with larger hydrophobic blocks, additional particles are observed. FCS shows extremely low critical micelle concentrations (cmc < 0.3 mg/L). The main part of the particles consists of micelles with diameters from 15 to 50 nm, built by 130−200 block copolymer molecules. Aggregation numbers and diameters are consistent with a model recently proposed by Förster et al. (J. Chem. Phys. 1996, 104, 9956−9970). The packing densities are determined from the hydrodynamic diameters and the aggregation numbers; they vary between 6 and 32%. For large hydrophobic block lengths additional structures are found, in most cases with a narrow size distribution. The origin of these structures is discussed.

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Raman correlation spectroscopy-intensity fluctuation spectroscopy of Raman-scattered light from a small sample volume-has been demonstrated. A modified confocal Raman microscope is used to observe number fluctuations of colloidal particles caused by Brownian motion. Correlation analysis of the fluctuations yields mobility and number density of the particles. In mixed systems, particles are differentiated according to their chemistry by selecting characteristic Raman bands for detection. Cross correlation of the signals from different Raman bands has been demonstrated. This method extends the domain of optical fluctuation spectroscopies to Raman scattered light, combining the chemical identification obtained by Raman scattering with the structural and dynamical information obtained by correlation spectroscopy. ͓S1063-651X͑98͒50403-5͔

We report on the application of fluorescence correlation spectroscopy (FCS) to observe the interaction between thrombin and thrombin inhibitors. Two site-specific fluorescent labels were used to distinguish between inhibitors directed to the active site, the exosite, or both binding sites of thrombin. For several well-known inhibitors of thrombin, the binding sites observed by FCS correspond to previous studies. The interaction of the recently discovered thrombin inhibitor ornithodorin from the tick Ornithodorus moubata with thrombin was investigated. It was found that this inhibitor, like hirudin and rhodniin, binds to both the active site and exosite of thrombin simultaneously. This study shows the feasibility of FCS as a sensitive and selective method for observing protein-ligand interactions. As an additional technique, simultaneous labeling with both fluorescent labels was successfully demonstrated.

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