To obtain chemical information on the nanoscale, the photothermal AFM-IR technique combines atomic force microscopy (AFM) with infrared (IR) spectroscopy, allowing for nanospectroscopy with resolutions down to 25 nm. AFM-IR in resonance enhanced mode is monolayer sensitive and opens up great opportunities for the study of biomolecular systems Here, AFM-IR in combination with classical IR reflection absorption spectroscopy (IRRAS) and spectroscopic ellipsometry (SE) is used, to study the individual fabrication steps of a recently developed electrochemical C9t aptasensor for the detection of S protein of the SARS-CoV-2 Omicron variant. To that end, aptamer binding and PEG blocking as well as the sensing of the S protein is examined. Following the morphological changes that occur in the respective steps allows conclusions on the molecular changes that happen.