Reduction of Artifacts in Fluorescence Correlation Spectroscopy Due to Sample Adsorption on Optical Glass Surfaces

The preparation of glass cell surfaces that are chemically functionalized with poly(ethylene glycol) (PEG) chains to reduce sample adsorption and their use in fluorescence correlation spectroscopy (FCS) is described. Optical glass coverslips were acid etched and reacted with either 750 Mr PEG (PEG-750) or 5000 Mr PEG (PEG-5000) to produce adsorption-resistant optical surfaces. FCS data for Nile red-loaded Triton X-100 micelles (NR-TX-100) and Alexa Fluor 555-labeled proteins, bovine serum albumin (BSA-A555), lipidized BSA (lipid-BSA-A555), and three low molecular weight dyes deposited on PEGylated coverslips were evaluated. Measurement artifacts due to sample adsorption on the PEG-5000 functionalized coverslips were reduced significantly for the majority of test materials. Calculations of translational diffusion coefficients and Stokes radii confirmed the effectiveness of this approach. PEG-5000 functionalized coverslips were demonstrated as more effective in inhibiting adsorption than PEG-750 functionalized coverslips. Neither of the functionalized coverslips inhibited the adsorption of one test compound, rhodamine B, a dye that adsorbs strongly on glass surfaces. The use of longer PEG chains in conjunction with chemical cross-linking is proposed for producing a denser, less porous PEG layer for the prevention of strongly glass-adsorbing fluorophores that do not interact with the PEG layer.