Technical Abstract: Single molecular detection of pathogens and toxins of interest to food safety is within grasp using technology such as Atomic Force Microscopy. Using antibodies or specific aptamers connected to the AFM tip make it possible to detect a pathogen molecule on a surface. However, it also becomes necessary to trap that pathogen on the surface for detection. This can be carried out using linker molecules with a high affinity for chemical reaction with the pathogen. FTIR spectroscopy, along with AFM, has been used to study the surface characterization of certain linker molecules on a gold surface. A self assembled monolayer (SAM) of DSP was formed on the surface and NTA was then added to the surface to form a DSP-NTA linker. A different SAM of DSP-NTA was formed where the DSP and NTA had already reacted prior to being added to the surface. Both FTIR and AFM confirm that the order of addition of the linker to the surface affect the orientation and coverage of the linker molecule on the gold surface. This will affect the ability of the linker to effectively capture proteins on the surface for analysis.