Characterization of Substrates and Surface-Enhancement in Atomic Force Microscopy Infrared Analysis of Amyloid Aggregates.

Atomic force microscopy infrared (AFM-IR) spectroscopy is an emerging analytical technique that can be used to probe the structural organization of specimens with nanometer spatial resolution. A growing body of evidence suggests that nanoscale structural analysis of very small (<10 nm) biological objects, such as viruses and amyloid aggregates, requires substrates that must fit strict criteria of low surface roughness and low IR background, simultaneously. In this study, we examine the suitability of a broad range of substrates commonly used in AFM and IR fields, and we determined that silicon, zinc sulfide, and calcium fluoride are the most ideal substrates for nanoscale imaging of amyloid oligomers, protein aggregates that are directly linked to the onset and progression of neurodegenerative diseases. Our data show that these substrates provide the lowest roughness and the lowest background in the 800-1800 cm-1 spectral window from all examined AFM and IR substrates. We also investigate a contribution of surface enhancement in AFM-IR by the direct comparison of signal intensities from oligomers located on silicon and gold-coated silicon surfaces. We found that metallization of such substrates provides a factor of ~7 enhancements to the IR signal and induces an equivalent enhancement of the sample background in the 950-1250 cm-1 spectral region.