Thickness Determination and Control in Protein-Based Biomaterial Thin Films

Controlling the thickness and uniformity of biomaterial films is crucial for their application in various fields including sensing and bioelectronics. In this work, we investigated film assemblies of an engineered repeat protein─specifically, the consensus tetratricopeptide repeat (CTPR) protein ─a system with unique robustness and tunability. We propose the use of microreflectance spectroscopy and apparent color inspection for the quick assessment of the thickness and uniformity of protein-based biomaterial films deposited on oxidized silicon substrates. Initially, we characterized the thickness of large, uniform, spin-coated protein films and compared the values obtained from microreflectance spectroscopy with those obtained from other typical methods, such as ellipsometry and atomic force microscopy. The excellent agreement between the results obtained from the different techniques validates the effectiveness of microreflectance as a fast, noninvasive, and affordable technique for determining the thickness of biomaterial films. Subsequently, we applied microreflectance spectroscopy to determine the thickness of drop-casted CTPR-based films prepared from small protein solution volumes, which present a smaller surface area and are less uniform compared to spin-coated samples. Additionally, we demonstrate the utility of apparent color inspection as a tool for assessing film uniformity. Finally, based on these results, we provide a calibration of film thickness as a function of the protein length and concentration for both spin-coated and drop-casted films, serving as a guide for the preparation of CTPR films with a specific thickness. Our results demonstrate the remarkable reproducibility of the CTPR film assembly, enabling the simple preparation of biomaterial films with precise thickness.