Fluorescence Correlation Spectroscopy Study of TAMRA Diffusion in Poly(vinyl-alcohol) and Ficoll70 Solutions
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0897-J02-06.1
Fluorescence Correlation Spectroscopy Study of TAMRA Diffusion in Poly(vinyl-alcohol) and Ficoll70 Solutions. Ariel Michelman-Ribeiro1,2, Ferenc Horkay1, Ralph Nossal1, Hacène Boukari1 1 Laboratory of Integrative & Medical Biophysics, National Institutes of Health, Bethesda, MD. 2 Physics Department, Boston University, Boston, MA. ABSTRACT We compare fluorescence correlation spectroscopy (FCS) measurements of the fluorescent nanoparticle, TAMRA, diffusing in non-fluorescent -hence invisible- poly(vinylalcohol) (PVA) or Ficoll70 solutions as a function of the polymer concentration, c. We determine changes of the translational diffusion coefficient of TAMRA and fit the data with the universal scaling law ( D ~ exp[−α (c / c * )ν ] ) to extract information about solvent quality. For PVA, we find ν = 0.74, suggesting that water in this case acts as a good solvent, whereas ν = 1.02 in Ficoll70 solutions, indicating theta-like behavior. INTRODUCTION A typical use of fluorescence correlation spectroscopy (FCS) is to determine the diffusion coefficient of nanoscopic particles moving in dilute solutions, yielding the same coefficient as obtained from dynamic light scattering (DLS). However, FCS has several advantages over DLS [1-5]: 1) detection specificity of fluorescence targets when the latter move in a complex, non-fluorescent, environment; 2) use of small (nanomolar) concentrations of the targeted particles and small volumes (~10-40 µl); 3) ability to study targets having a wide range of sizes, including particles as small as 1 nm; 4) applicability to both in-vivo and in-vitro conditions. Our objective is to exploit these properties to extend FCS to investigations of the behavior of macromolecules in concentrated solutions, gels, and cellular systems –a problem of interest to both basic research and applied engineering [6-11]. In this paper we describe results of analysis of FCS measurements of TAMRA (MW=430 Da), a small fluorescent probe (~ 1.6 nm), moving within non-fluorescent poly(vinyl alcohol) (PVA) polymer and Ficoll70 solutions. PVA is a neutral, water-soluble, linear polymer commonly used as a model for tissue engineering matrices [6,9-12]. Ficoll70 is a water-soluble, highly-branched sucrose-polymer that behaves as an ideal neutral sphere. It is used in perfusion experiments and research studies of the effects of crowding on, for example, the enzymatic activity or protein stability [7-8]. However, the solutions demonstrate different behaviors as the polymer concentrations are increased. Above a threshold concentration (c* ~ 2.5% W/V) PVA chains entangle to form a flexible network that can be cross-linked to create a gel [6,12]. According to scaling theory of polymers, the PVA solution is characterized by a mesh size, ξ , that varies as ξ ≈ rg (c / c * ) −ν , where rg , c * , and ν are, respectively, the radius of gyration of the polymer chain, the critical concentration, and the solvent quality (ν = 0.75 , good solvent; ν = 1 , theta condition) [13]. For Ficoll70 the particles appear unentangled
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