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Fluorescence Correlation Spectroscopy of Nanoparticles

The possibility of using nanoparticles as superior labels and sensors that do not photobleach in environmental and biological studies has sparked wide-spread interest. In order to apply nanoparticles in these areas, it is necessary to understand the diffusion of nanoparticles in a liquid environment. We are applying correlation techniques, mainly fluorescence correlation spectroscopy (FCS), to extract the diffusion constants of nanoparticles in solution. The diffusion constant is a direct measure of the total nanoparticle size, which includes the inorganic core and the organic capping material. FCS measures spontaneous intensity fluctuations caused by small deviations from equilibrium when molecules or nanoparticles enter and leave a detection area. The largest fluctuations are observed when only a few molecules/particles are present in a small detection volume with the ultimate limit being a single molecule/particle at a given time. Sufficient signal to noise for single molecule/particle FCS can be achieved through minimizing the detection volume by focusing a laser beam to a diffraction limited spot combined with high quantum yield photodetectors.

The figure above shows autocorrelation curves for 40 (red) and 100 nm (blue) dye beads in water (left graph). Fits (lines) to the data (symbols) give the time it takes the beads to diffuse through the confocal excitation volume. With the known dimensions of the excitation volume the diffusion constant is calculated, which is directly related to the size of the beads by the Stokes – Einstein equation. A fluorescence transient of 100 nm beads (inset, left graph) shows individual fluorescence bursts, which confirm that less than one molecule is present in the excitation volume. Additional information can be obtained by calculating an intensity histogram of single bursts from the fluorescent transients (right graph).

We are applying FCS to measure the diffusion of nanoparticles to understand their mobility under different conditions, e.g. for different core sizes and shapes, varying surface capping materials, or with an applied external field. In addition to determining the hydrodynamic radius of the nanoparticles, this approach allows us to follow binding and dissociation between the the nanoparticle surface and molecular targets or substrates as well as among nanoparticles including nanoparticle aggregation. In particular, we are interested in understanding heterogeneous systems, which contain mixtures of different nanoparticles. Our FCS studies are carried out in close interaction with the Landes Lab.


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