Polymer nanosphere lithography (NSL) masks were etched in oxygen plasma prior to metal deposition, which tunes the localized surface plasmon resonance (LSPR) and the interparticle distance resulting in greater Raman amplification than conventional film over nanosphere (FON). Various nanosphere masks were investigated, using nanosphere sizes of 220, 360, 450, 520, and 650 nm etched from 0 (conventional FON) to 10 min. Thereby, the film over etched nanospheres (FOEN) amplifies by up to a factor of 4 the Raman response of 4-nitrobenzenethiol (4-NBT) on Ag as compared to unetched FON. The LSPR response of FOEN and SEM analysis of the gap distance revealed that the optimal amplification results from a combination of tuning the gap (a gap/diameter of less than 1 improves the Raman response), matching the laser excitation wavelength (633 and 785 nm both investigated), and an increasing roughness of FOEN. Metal multilayers of Ag and Au were also deposited to investigate the effect on the Raman and LSPR response. While the LSPR response remains relatively invariable, the Raman signal from 4-NBT decreased significantly by increasing the number of layers when Ag is used as the outer layer. However, a bimetallic film composed of Ag underlayer with Au as an outerlayer further increased the Raman response by a factor of 1.6. Overall, the most intense Raman response for FOEN was obtained with 360 nm nanospheres, etched for 1.5 min to create a gap of 66 nm with an overall bimetallic film thickness of 175 nm, which was composed of an Ag underlayer of 87.5 nm and an Au outerlayer of 87.5 nm. Thus, by performing these simple modifications to FON, the Raman response can be increased by nearly 1 order of magnitude.

Ionic liquid self-assembled monolayers (SAM) were designed and applied for binding streptavidin, promoting affinity biosensing and enzyme activity on gold surfaces of sensors. The synthesis of 1-((+)-biotin)pentanamido)propyl)-3-(12-mercaptododecyl)-imidazolium bromide, a biotinylated ionic liquid (IL-biotin), which self-assembles on gold film, afforded streptavidin sensing with surface plasmon resonance (SPR). The IL-biotin-SAM efficiently formed a full streptavidin monolayer. The synthesis of 1-(carboxymethyl)-3-(mercaptododecyl)-imidazoliumbromide, a carboxylated IL (IL-COOH), was used to immobilize anti-IgG to create an affinity biosensor. The IL-COOH demonstrated efficient detection of IgG in the nanomolar concentration range, similar to the alkylthiols SAM and PEG. In addition, the IL-COOH demonstrated low fouling in crude serum, to a level equivalent to PEG. The IL-COOH was further modified with N,N'-bis (carboxymethyl)-l-lysine hydrate to bind copper ions and then, chelate histidine-tagged biomolecules. Human dihydrofolate reductase (hDHFR) was chelated to the modified IL-COOH. By monitoring enzyme activity in situ on the SPR sensor, it was revealed that the IL-COOH SAM improved the activity of hDHFR by 24% in comparison to classical SAM. Thereby, IL-SAM has been synthesized and successfully applied to three important biosensing schemes, demonstrating the advantages of this new class of monolayers.