Sensitive Biosensing Using Plasmonic Enhancement of Fluorescence by Rapid Thermal Annealed Silver Nanostructures

Although immunofluorescence assays have been used for decades, they are not traditionally capable of quantifying biomarkers at ng/ml levels. Plasmonic enhancement of fluorescence using metallic surface nanostructures has exhibited potential in amplifying the fluorescence signal, which could reduce limits of detection and increase sensitivity. However, current methods for fabricating metallic nanostructures, such as e-beam nanolithography, colloidal lithography, and colloidal self-assembly, require complicated processes and have various drawbacks. In this work, we describe a nanostructure fabrication process we have developed based on the dewetting of thin silver films by rapid thermal annealing, which is suitable for large areas. The nanostructures were then coated with a thin silica film to protect them and also to control the distance between the metallic surface and fluorophores, an important parameter when tuning metal enhancement of fluorescence. Antibody-antigen immunoassays utilizing immunoglobulin G were applied to evaluate the fluorescence enhancement from these nanostructures. A nearly 19-fold increase in the fluorescence intensity was achieved with an optimized structure. Calibration of the resulting plasmonically enhanced immunofluorescence sensor showed that it is nearly 13 times more sensitive than the non-enhanced version and was capable of quantification of a biomarker at ~ 1 ng/ml levels. A model nanostructure was constructed for which finite difference time domain simulations were in good agreement with experimental results, allowing for optimization of process conditions for the further generation of nanostructures.