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Fractal-Like Silver Aggregates Enhance the Brightness and Stability of Single-Molecule Fluorescence

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Abstract:

This paper describes a method to significantly enhance single-molecule fluorescence detection in confocal microscopy, demonstrating that fractal-like silver structures significantly improve dye stability and brightness. The experiments compared two immunoassay models based on the deposition of rabbit IgG on silver structures. The experiments were performed with the fluorophore-labeled protein at low picomolar concentrations. Well-separated bright spots were still easily distinguishable. Under standardized conditions we observed increased photostability and brightness enhancement for the dyes that were immobilized on the surface using a primary antibody. In contrast, when the unlabeled primary antibody was immobilized on the surface and the labeled secondary antibody was placed at a larger distance, we observed only a modest enhancement of fluorescence. Furthermore, based on backscattered reflected light images, it was proven that the observed fluorescence enhancements originate from the areas with deposited silver nanostructures. Fractal-like substrates are relatively easy to prepare. We believe that with their superior performance, they should find wide applications in single-molecule studies in which a longer observation time is required.

Keywords: FLIM; FLUORESCENCE LIFETIME IMAGING MICROSCOPY; FRACTAL-LIKE STRUCTURES; METAL-ENHANCED FLUORESCENCE; SINGLE-MOLECULE DETECTION

Document Type: Research Article

DOI: http://dx.doi.org/10.1366/10-06112

Affiliations: 1: Center for Commercialization of Fluorescence Technologies, Department of Molecular Biology and Immunology, UNTHSC, Fort Worth, Texas 76107, USA; Department of Biophysics, Institute of Physics, Marie Curie-Sklodowska University, 20-031 Lublin, Poland 2: University of Texas at Tyler, Tyler, Texas 75799, USA 3: Center for Commercialization of Fluorescence Technologies, Department of Molecular Biology and Immunology, UNTHSC, Fort Worth, Texas 76107, USA 4: Center for Commercialization of Fluorescence Technologies, Department of Molecular Biology and Immunology, UNTHSC, Fort Worth, Texas 76107, USA; Department of Microbiology, Institute of Biology, The Jan Kochanowski University in Kielce, 25-406 Kielce, Poland 5: Center for Commercialization of Fluorescence Technologies, Department of Molecular Biology and Immunology, UNTHSC, Fort Worth, Texas 76107, USA; Department of Cell Biology and Anatomy, UNTHSC, Fort Worth, Texas 76107, USA

Publication date: February 1, 2011

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