TIRF microscopy as a screening method for non-specific binding on surfaces

We report a method for studying nanoparticle-biosensor surface interactions based on total internal reflection fluorescence (TIRF) microscopy. We demonstrate that this simple technique allows for high throughput screening of non-specific adsorption (NSA) of nanoparticles on surfaces of different chemical composition. Binding events between fluorescent nanoparticles and functionalized Zeonor® surfaces are observed in real-time, giving a measure of the attractive or repulsive properties of the surface and the kinetics of the interaction. Three types of coatings have been studied: one containing a polymerized aminosilane network with terminal –NH2 groups, a second film with a high density of –COOH surface groups and the third with sterically restraining branched poly(ethylene)glycol (PEG) functionality. TIRF microscopy revealed that the NSA of nanoparticles with negative surface charge on such modified coatings decreased in the following order –NH2 > -branched PEG > –COOH. The surface specificity of the technique also allows discrimination of the degree of NSA of the same surface at different pH.

Total internal reflection fluorescence microscopy is used for non-specific adsorption screening of nanoparticles on functionalized surfaces. Binding of fluorescent nanoparticles is observed in real-time, measuring attractive or repulsive surface properties.Figure optionsDownload high-quality image (97 K)Download as PowerPoint slideResearch highlights
► TIRF microscopy is useful for screening of non-specific surface–particle interactions.
► Optimal surface characteristics determined result in lowest non-specific binding of particles.
► Substrate-particle charge repulsion had the strongest effect in decreasing the background.
► Charge repulsions outperformed effects of coatings based on steric hindrance and water hydration.
► COOH surfaces provide enough repulsion to antibody coated particles for low non-specific binding.