Surface-enhanced Raman scattering (SERS) is a powerful tool for intracellular analyses due its minimally invasive nature and molecular specificity. This research focuses on optimizing the sensitivity of SERS in order to widely apply it to the detection of ultra-trace biomolecules within individual living cells. Recent results have shown that large SERS enhancement factors (EF) can be achieved with multi-layer SERS substrates. To fabricate multi-layer SERS substrates, alternating layers of metal films and dielectric spacers are cast over a non-confluent monolayer of nanostructures. Individual particles of these substrates are then immobilized with antibodies to develop SERS-based immuno-nanosensors. The multi-layer SERS EFs can be increased by the use of appropriate dielectric spacer to fabricate the substrates. To further understand the effect of dielectric spacers on the multi-layer SERS EFs, this talk discusses the characterization of the SERS enhancements of multi-layer metal film on nanostructure SERS substrates fabricated with self-assembled monolayer (SAM) spacers. Monolayers with different chain lengths have been systematically capped with varying amount of metal films. It was found that the SERS EFs depend on the nature of the monolayer formed and the amount of metal film deposited on the monolayer. Using optimal SAMs and the appropriate amount of metal film overlayers, SAM multi-layer SERS substrate with optimized SERS intensity can be fabricated. This talk will also explore the functionalization of the SERS substrates with appropriate bio-recognition elements to develop SERS-based immuno-nanosensors.© (2010) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.