Surface-enhanced Raman scattering (SERS) has become an attractive analytical tool for intracellular analyses due to its
minimally invasive nature and molecular specificity. However, highly reproducible and optimized SERS substrates have
been seen as a key to developing SERS as a reliable analytical methodology. This research focuses on optimizing self
assembled monolayer (SAM)-based multilayer SERS substrates for a wide range of applications, including ultratrace
detection of biomolecules within individual living cells. Multilayer SERS substrates are comprised of alternating layers
of metal film and dielectric spacer cast on a monolayer of nanostructures. Using these substrates, varying degrees of
SERS enhancement factors (EF) have been achieved, some as large as 10-fold relative to optimized single film over
nanostructures substrates. To gain a mechanistic understanding of multilayered SERS enhancements, SAMs have been
used to systematically vary spacer thickness. The results revealed spacer-dependent SERS EFs. To further the
understanding of multilayer SERS enhancement, this work discusses the use of terminating functional groups in the
optimization of SAM multilayer SERS substrates. SAMs having various functional groups were used as dielectric
spacers to systematically vary the dielectric constant. To investigate the effect of the pH on the uniformity of the SAMs
and their multilayer SERS enhancement, SAMs were formed in alkylthiol solutions of different pH and the subsequent
SERS enhancement were evaluated. It was found that using alklythiol SAMs with appropriate terminating functional
groups the SAM multilayer can achieve SERS EFs ranging between 10<sup>8</sup> and10<sup>10</sup> and the substrates yielded highly
reproducible SERS signals. The effect of the pH on the SERS enhancement is selective on the type of the terminating
functional group of the alkylthiol used for SAM formation.
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.
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