Dynamic intracellular analysis has important applications in areas like biomedical research, defense and security and many others. Although, there are several methods for intracellular analysis, surface enhanced Raman scattering (SERS) is becoming a preferred transduction method for such applications, due to its narrow spectral bandwidth, large SERS enhancement factors and high sensitivity. In our laboratory, SERS-based immuno-nanosensors are being developed and optimized for real-time, dynamic, and multiplexed analysis of molecular interaction within individual living cells. These nanosensors are fabricated by drop coating silica nanospheres onto a microscope slide. A film of SERS active metal is deposited on the nanospheres to form metal film over nanospheres (MFON), which are then removed from the slide by mechanical processes. The MFONs are functionalized with antibodies that target specific proteins under investigation. Radiation induced cell perturbation is minimized by the use of a HeNe laser for excitation at 632.8 nm. To improve SERS enhancement, different types of metal deposited substrates have been studied with multilayer- MFON (MULTI-FON) substrates demonstrating ideal enhancement. This paper evaluates the SERS enhancement of MULTI-FONs with self-assembled monolayers (SAMs) spacers sandwiched between layers of the metal film. Monolayers with carboxylic acid tail groups and different chain lengths are used as spacers in order to evaluate the effect of spacer length and chain functionalities on the SERS enhancement. The paper also discusses the effect of solvent used for the monolayer formation on the sensitivity of the SAM MULTI-FON SERS substrates.© (2009) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.