Understanding and controlling antibody stability is crucial in biopharmaceutical development, as protein misfolding, denaturation, and aggregation - caused by stress factors such as temperature changes, pH variations, mechanical stress, or light exposure – can compromise the antibody's biological functionality.(1,2) These structural changes may lead to a loss of therapeutic efficacy or even trigger immune responses in patients.(2) This study investigates the potential of atomic force microscopy coupled to infrared spectroscopy (AFM-IR)(3) for nanoscale characterization of immunoglobulin G (IgG) antibodies in both their native and denatured states, captured by a biosensor, thus providing high-resolution chemical and structural insights into this model protein system. The introduction of a biosensor offers distinct selectivity for analysing the targeted antibodies, but the AFM-IR system requires a homogeneous sensor surface with a thin layer of covalently anchored selective capture proteins for IgG that absorb minimally in the targeted infrared regions, all while functioning under ambient conditions. A novel biosensor design is introduced, utilizing direct surface functionalization of silicon substrates with covalently immobilized antibody-capturing proteins. These proteins exhibit high yet reversible selective affinity for the Fc region of IgG(4), allowing for straightforward biosensor regeneration through pH variation, which enhances the biosensor’s applicability. The system’s early-stage success is demonstrated by silicon substrate functionalization with (3 Aminopropyl)triethoxysilane (APTES) and glutaraldehyde (GA), verified through AFM scans and water contact angle analysis of the surfaces. Ongoing research is focused on model protein binding tests, with particular emphasis on optimizing antibody-capturing protein G immobilization strategies. By integrating a reversible biosensor design for AFM-IR analysis, this work introduces a novel approach for studying native and denatured antibodies at the nanoscale. This approach holds the potential for enhancing quality control in biopharmaceutical production by enabling the development of inline sensors for antibody stability testing during bioprocessing. (1) Hawe, A.; Wiggenhorn, M.; Van De Weert, M.; Garbe, J. H. O.; Mahler, H.; Jiskoot, W. Forced Degradation of Therapeutic Proteins. J. Pharm. Sci. 2012, 101 (3), 895–913. https://doi.org/10.1002/jps.22812. (2) Pease III, L. F.; Elliott, J. T.; Tsai, D.-H.; Zachariah, M. R.; Tarlov, M. J. Determination of Protein Aggregation with Differential Mobility Analysis: Application to IgG Antibody. Biotechnol. Bioeng. 2008, 101 (6), 1214–1222. https://doi.org/10.1002/bit.22017. (3) Dazzi, A.; Glotin, F.; Carminati, R. Theory of Infrared Nanospectroscopy by Photothermal Induced Resonance. J. Appl. Phys. 2010, 107 (12), 124519. https://doi.org/10.1063/1.3429214. (4) Budde, B.; Schartner, J.; Tönges, L.; Kötting, C.; Nabers, A.; Gerwert, K. Reversible Immuno-Infrared Sensor for the Detection of Alzheimer’s Disease Related Biomarkers. ACS Sens. 2019, 4 (7), 1851 1856. https://doi.org/10.1021/acssensors.9b00631.