Understanding and controlling antibody stability is crucial in biopharmaceutical development, as protein misfolding, denaturation, and aggregation caused by stress factors (e.g. temperature changes, pH level, mechanical stress or light exposure) compromise the antibody's biological functionality.(1,2) These structural changes can lead to a loss of therapeutic efficacy or even trigger immune responses in patients.(2) This work explores the potential of atomic force microscopy coupled to infrared spectroscopy (AFM-IR)(3) for nanoscale characterization of immunoglobulin G (IgG) antibodies in both native and denatured states, captured by a biosensor, providing high-resolution chemical and structural insights into this model protein system. A novel biosensor design is presented, utilizing direct surface functionalization of silicon substrates with covalently immobilized antibody-capturing proteins. These proteins exhibit high but reversible affinity for the Fcconstant region of IgG(4), enabling selective antibody binding and straightforward biosensor regeneration via pH variation enhancing the biosensors’ applicability. The developed system demonstrates successful silicon substrate functionalization using (3-Aminopropyl)triethoxysilane (APTES) and glutaraldehyde (GA), with ongoing research focusing on optimizing antibody-capturing protein (protein G) immobilization strategies and conducting fluorescent-labelled antibody binding tests. By integrating a reversible biosensor design for AFM-IR analysis, this work introduces a new approach for studying native and denatured antibodies at the nanoscale. This approach has the potential to enhance quality control in biopharmaceutical production by enabling the setup as an inline sensor for antibody stability testing during bioprocessing applications. (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.