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

Abstract

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.

Spatial Control of Epsin-induced Clathrin Assembly by Membrane Curvature

“…Glass coverslips were passivated by covalent attachment of PEG according to earlier studies ( 21 ). Briefly, glass coverslips were cleaned with 3 n NaOH for 5 min and rinsed with water.…”

Variable surface transport modalities on functionalized nylon films revealed with single molecule spectroscopy

“…[1][2][3][4] Functionalization of a membrane or surface changes the interfacial chemical properties and thus can tune the ability of a surface to interact with target molecules [5][6][7][8][9] without changing the other bulk properties of the membrane. 10,11 The performance of a functionalized surface for ltration or separation is difficult to determine a priori because heterogeneous surface dynamics cause deviations from predictive models, [12][13][14] especially when the target molecules are large and complex like proteins. 15,16 Consequently, most such surfaces are designed via iterative optimization, but understanding the mechanistic details of surface transport, adsorption and heterogeneity will allow for more efficient bottom-up design.…”

Characterization of Particle Size and Surface Adsorption for SiO2Abrasives Used in Chemical Mechanical Planarization via Fluorescence Correlation Spectroscopy

“…24 The instrument's 488 and 543 nm lasers were used as excitation sources for R110 and A546-containing samples, respectively. The excitation laser beam was passed though neutral density filters to reduce its focused power on the samples to approximately 10 μW.…”

“…FCS Measurements.-FCS measurements were performed using a custom-built system employing an inverted fluorescence microscope as described previously. 24 The instrument's 488 and 543 nm lasers were used as excitation sources for R110 and A546-containing samples, respectively. The excitation laser beam was passed though neutral density filters to reduce its focused power on the samples to approximately 10 μW.…”