β‐Sheets Orientation in Physisorbed Protein Layers

Abstract Physisorption of antibodies onto surfaces is a low‐cost, rapid, and effective approach for immobilizing bioreceptors in applications such as bioelectronic sensors. However, there is a prevailing notion that physisorbed protein layers lack structural order, thus potentially compromising their stability and sensitivity compared to antibody films that are covalently attached to the substrate surface. This study demonstrates the preferential orientation of β‐sheets within the secondary structure of protein layers, specifically anti‐immunoglobulin G (anti‐IgG) and bovine serum albumin (BSA), when physisorbed onto gold (Au) thin films. Using polarization modulation infrared reflection‐absorption spectroscopy (PM‐IRRAS) and infrared attenuated total reflection (IR‐ATR) spectroscopy, it has been confirmed that the β‐strands in these protein layers are tilted relative to the surface normal by average angles of 75.3° ± 0.4° for anti‐IgG and of 79.3 ± 0.2° for BSA. These results are obtained by analyzing the orientation of the transition dipole moments (TDMs) associated with the amide I molecular vibrations derived from a comparison between experimental and simulated mid‐infrared spectra assuming isotropically oriented TDMs. The simulations incorporate refractive and absorption index dispersions obtained from the IR‐ATR spectra. Thus obtained findings offer valuable molecular‐level insights facilitating the design and optimization of biofunctionalized interfaces in advanced biomedical and biosensing applications.