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

Abstract

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 ÐNH 2 groups, a second film with a high density of Ð 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 ÐNH 2 > -branched PEG > -COOH. The surface specificity of the technique also allows discrimination of the degree of NSA of the same surface at different pH. KeywordsTotal Internal Reflection, Fluorescence, Microscopy, Non-Specific Binding, SurfaceParticle Interaction IntroductionCurrent research in biomedical diagnostics is directed at inexpensive, highly sensitive, miniaturized devices, in which usually disposable substrates or chips are used. Such devices are required to measure analytes in microliter volume samples, typically at picomolar level. In fluorescence-based assays, the signal can be greatly enhanced with the use of dye doped nanoparticles as fluorescent labels [1][2][3][4]. However, in a successful diagnostics device, signal-to-noise ratio is paramount to its performance and effectively controls the sensitivity. Controlling the noise and background contribution, dictated by non-specific binding of the non-analyte constituents of the sample remains very challenging. The chemical composition of the substrate surface and its interactions with the active surface area of the dye-doped nanoparticles, therefore, play a key role in the performance of diagnostic platform. In this work, we focus on surface functionalization of the substrate ZeonorÒ, a cyclo-olefin polymer (COP), which is characterized by ease of injection molding for biochip fabrication and relatively low autofluorescence, which is an important factor for fluorescence-based applications including TIRF microscopy.Total Internal Reflection Fluorescence (TIRF) microscopy [5] is presented here as a very useful and fast method to compare non-specific adsorption of detection molecules on different surfaces. This method allowed for rapid screening of surfaces to find their ability to prevent non-specific binding. Total internal reflection occurs an any interface between materials of different refractive index inside the material of higher refractive index provided the angle of incidence of the light is above the critical angle defined as:where n 1 is the refractive index of the high refractive index mat...

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