NSTI BioNano 2010

Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) as an Analytical Tool for in situ Measurement of Adsorption and Dissociation of Proteins to Polystyrene and Semiconductor Nanoparticles (invited presentation)

S. Corry, W.L. Downey
Molecular Probes / Life Technologies, US

Keywords: molecular probes, life technologies

Abstract:

As nanoparticles have extremely large relative surface areas, behavior of these particles tends to be governed by surface chemistry effects. In a complex biological system, strong nanoparticle interactions with proteins, carbohydrates, nucleic acids, lipids, and even small molecules such as vitamins modulate toxic effects and environmental impact. However, existing techniques for characterizing interfacial interactions between biomolecules and other materials require a separation step, high vacuum, and/or significant time and monetary expense. We have developed an in situ, real-time, high-throughput, experimental framework using time-resolved fluorescence resonance energy transfer (TR-FRET) to characterize interactions between lanthanide-labeled biomolecules and fluorescent nanoparticles. Fluorescent semiconductor nanocrystals known as quantum dots (QDs) have been used extensively in immunohistochemistry (IHC), immunocytochemistry (ICC), and flow cytometry. In all these applications, the particles are exposed to a specific biological target (generally a protein or hapten) amidst a background of biomolecules. Although serum protein abundance spans several orders of magnitude, albumin and immunoglobulins (IgGs) comprise over 60% of total protein, so interactions between particles and these proteins have particular importance in biomedical applications. Biomolecules labeled with a Terbium-containing fluorophore were used to probe specific and non-specific interactions with QDs containing different surface functionalities, including QD conjugates of biotin, streptavidin, and IgG. Adsorption to the particles was well fit with a modified Langmuir isotherm. We demonstrated that bovine serum albumin (BSA) binds to QDs with a greater strength than IgG does, and that PEGylation of QDs significantly reduces non-specific binding. Size exclusion chromatography (SEC) was used to generate independent adsorption isotherms, and ranking of binding interactions was consistent between TR-FRET and SEC analytical methods. Due to the ubiquity of polystyrene (PS) in biomedical devices such as microtiter plates, we extended our characterization techniques for QDs to fluorescent PS microspheres. Besides measuring non-specific adsorption of BSA and IgG, we utilized the TR-FRET technique for real-time, in situ measurement of surfactant-induced dissociation of the adsorbed proteins. Though adsorption to spherical particles is qualitatively different from adsorption to flat surfaces, this analytical framework enables rapid prototyping and testing of PS materials. Besides applicability to flat PS surfaces, the ability to monitor effects of surfactants has implications for designing new textile materials and laundry detergents.
 
Program | Symposia | Speaker Exhibitor | Press Venue Registration |
Short Courses | News | Subscribe | Contact | Site Map
© Copyright 2009 TechConnect World. All Rights Reserved.