Analysis of metal enhanced fluorescence on near-IR fluorophores

Fluorescence is a highly sensitive, precise, and convenient detection technique that is widely used in chemistry, molecular biology and clinical laboratories. Fluorescence in the near-IR (700 - 900 nm) offers higher molar absorptivity and significantly lower background signals from scatter than those generated by visible wavelength excitation. The advantageous characteristics of near-IR fluorescence, primarily the reduced background signals, make this region of the spectrum ideal for enhancement by metal nanostructures. Though multiple groups have successfully demonstrated metal enhanced fluorescence, there remain several challenges in transferring this technology from the research stage to the commercial stage. Using a LI-COR Odyssey® Infrared Imaging System, we quantitatively analyzed the effects of silver particle geometries, including size, shape, and density of metal nanostructures, on the fluorescence enhancement of Near-IR fluorophores. Using silver island film coated glass slides, we were able to obtain an 18-fold enhancement of IRDye®700 and a 15-fold enhancement of IRDye®800 labeled DNA oligos over dye on plain glass. We further analyzed the silver-coated glass surfaces for enhancement reproducibility and linearity. We demonstrated that the metal enhanced emissions remained reproducible across a slide surface, and remained linear over several orders of magnitude. Finally, using a highly quenched labeled protein, we were able to show an enhancement and release of the quenched fluorescence, generating a 40-fold enhancement in the fluorescence emissions when spotted on a silver nanostructure coated glass slide. Generating silver nanostructure coated slides that enhance fluorescence while maintaining linearity and reproducibility will provide a class of new tools benefiting molecular biologists.