Resonance-Enhanced Force-Volume AFM-IR Implementation and Interpretation For Protein-Based Materials: A Study of Regenerated Silk Fibroin Films

Abstract

The objective of this study is to explore the ability of AFM-IR to characterize regenerated silk fibroin films such that collected spectra can be compared to typical spectra collected from far-field techniques. Additionally, the impact of AFM-IR parameters and material optical and thermal properties on peak location and probe depth are examined empirically and compared to the literature to better clarify the signal transduction scheme and impacts of dispersion on signal non-linearity. To achieve this, ATR FTIR and AFM-IR spectra of regenerated silk fibroin films are collected as well as multiplex frequency-sweep images and spectra from a regenerated silk fibroin wedge. Frequency-sweep data are used to establish the effective probing depth of AFM-IR as a function of laser pulse frequency. Spectra from the regenerated silk fibroin samples and a wedge are subsequently compared to literature peak assignments to determine dominant protein secondary structures at various effective probe depths. Our data indicate that laser pulse frequency does not effectively modulate probe depth, and that in the case of regenerated silk fibroin, probe depth is at least 2.5 $\mu$m. Spectral analysis revealed a difference in dominant protein secondary structure at the surface of regenerated silk fibroin films compared to the bulk and indicates aging driven by adsorption of water molecules and subsequent chain alteration due to interaction with hydrophobic side chains. Our findings indicate regenerated silk fibroin films largely take Silk I structure at the near-surface, and also reveal formerly uncharacterized turn structures in the bulk film which are only accessible using AFM-IR, appearing at coupled bands at 1263 cm-1 and 1670 cm1. Our results confirm the impact of sample and substrate thermal properties on modulating signal intensity, which may introduce spectral artifacts. This work furthers the fundamental understanding of the physical factors driving AFM-IR signal intensity and peak shifting, guides future studies investigating the impact of anomalous dispersion on IR peak position, and reveals changes in protein structure in regenerated silk fibroin films at various depths driven by water adsorption.

Date of Award	May 2025
Original language	English
Awarding Institution	Pennsylvania State University
Supervisor	Viviana M. Posada (Director) & Max Wetherington (Director)