Near-Infrared Surface-Enhanced Fluorescence Using Silver Nanoparticles in Solution

Fluorescence spectroscopy is a widely used detection technology in many research and clinical assays. Further improvement to assay sensitivity may enable earlier diagnosis of disease, novel biomarker discovery, and ultimately, improved outcomes of clinical care along with reduction in costs. Near-infrared, surface-enhanced fluorescence (NIR-SEF) is a promising approach to improve assay sensitivity via simultaneous increase in signal with a reduction in background. This dissertation describes research conducted with the overall goal to determine the extent to which fluorescence in solution may be enhanced by altering specific variables involved in the formation of plasmonactive nanostructures of dye-labeled protein and silver nanoparticles in solution, with the intent of providing a simple solution that may be readily adopted by current fluorescence users in the life science research community. First, it is shown that inner-filtering, reabsorption of the emitted photons, can red-shift the optimal fluorophore spectrum away from the resonant frequency of the plasmon-active nanostructure. It is also shown that, under certain conditions, the quality factor may be a better indicator of SEF than the commonly accepted overlap of the fluorophore spectrum with the plasmon resonance of the nanostructure. Next, it is determined that streptavidin is the best choice for carrier protein, among the most commonly used dye-labeled detection antibodies, to enable the largest fluorescence enhancement when labeled with IRDye 800CW and used in combination with silver nanoparticles in solution. It is shown that the relatively small and symmetric geometry of streptavidin enables substantial electromagnetic-field confinement when bound to silver nanoparticles, leading to strong and reproducible enhancement. The role of silver nanoparticle aggregation is demonstrated in a dropletbased microfluidic chip and further optimized in a standard microtiter-plate format. A NIR-SEF technology based on aggregation with optimized salt concentration demonstrates a fluorescence signal enhancement up to 2530-fold while improving the limit-of-detection over 1000-fold. Finally, the NIR-SEF technology is applied to demonstrate 42-fold improvement in sensitivity of the clinically-relevant biomarker, alpha-fetoprotein, along with a 16-fold improvement in limit-of-detection. Advisor: Gregory R. Bashfor

A Near-Infrared, Surface-Enhanced, Fluorophore-Linked Immunosorbent Assay

The enzyme-linked immunosorbent assay is commonly used for research and clinical applications but typically suffers from a limited linear range and is difficult to multiplex. The fluorophore-linked immunosorbent assay is a closely related technique with good linear range and the ability to detect multiple antigens simultaneously but is typically less sensitive. Here, we demonstrate a near-infrared, surface-enhanced fluorophore-linked immunosorbent assay with sensitivity comparable to its enzyme-linked counterpart. A 59-fold enhancement to sensitivity (slope of linear fit) and an 8-fold improvement in LOD are demonstrated on a direct assay with rabbit immunoglobulin-G as a model system. The technique is also tested on a clinically relevant assay to detect alpha-fetoprotein, in which a 42-fold enhancement to sensitivity is demonstrated along with a 16-fold improvement in LOD. The technique enables these accomplishments while maintaining the entire traditional assay protocol and simply adding two steps at the end. This technique may prove superior to current protocols for biomarker research and clinical diagnoses, which require high sensitivity along with quantitation over an extended range