Understanding the complex biological signaling pathways within individual living cells and their inter-relationship with each other has long been a goal of systems biology. This requires the ability to simultaneously monitor a large number of biochemical species with both temporal and spatial resolution. Currently, techniques such as high throughput screening represent the state-of-the-art in such analyses, with the ability to monitor hundreds of different species in a single analysis. However, such techniques typically require the cells to be lysed and analyzed via time course measurements, providing limited temporal resolution and minimal to no spatial distribution information about the chemical species measured. This dissertation describes the development of a novel class of intracellular surface-enhanced Raman scattering (SERS) immuno-nanosensors with the potential for multiplexed analyses of as many as 50–100 different chemical species in a location specific fashion, within complex biological environments; thereby providing the first means of potentially monitoring multiple biochemical species in a non-destructive and real-time fashion in living cells. Development and characterization of these nanosensors was accomplished through a series of studies aimed at developing sensitive and selective SERS probes that could be fabricated through a generic process. The first portion of this work involved developing a novel SERS sensing platform, which resulted in: (a) discovery and characterization of unique multilayer SERS enhancements from silver SERS substrates, (b) evaluation of the effects of different size nanostructures on the enhancements from these multilayer substrates, (c) evaluation of the role dielectric spacer materials on these multilayer enhancements, and (d) expansion of this multilayer phenomenon to gold SERS substrates, providing biologically compatible sensor platforms that are stable for months. Detailed studies on the optical properties, SERS signal enhancing ability, reproducibility of signal (i.e., <5.2% RSD) as well as surface roughness were investigated.

Following optimization of the multilayer SERS substrate platforms, development of a generic antibody binding process was achieved, allowing fabrication of different immuno-nanosensors that are capable of the label-free detection of specific proteins in cell-like environments. Upon optimization, the nanosensors were evaluated by monitoring their response to various antigens in complex biological environments.