Infrared Nanospectroscopy of Air-Sensitive Biological Substrates Protected by Thin Hydrogel Films

The air sensitivity of many substrates, and specifically biosurfaces, presents an experimental challenge for their analysis by vibrational spectroscopy and, in particular, infrared microscopy on a nanometer scale. The recent development of atomic-force-microscopy-based infrared spectroscopy (AFM-IR), which circumvents the Abbe diffraction limit, allows nanoscale chemical characterization of surfaces. Additionally, this technique has been shown to work for thin films under aqueous environments but is limited to substrates up to 10 nm thick, thus ruling out application to many biological surfaces. To circumvent this restriction, we have utilized hydrogels to cover such surfaces and maintain a more physiologically representative environment for biological substrates. We show that it is feasible to use AFM-IR to chemically characterize this type of substrate buried under a thin hydrogel film. Specifically, this work describes the AFM-IR spectra of red blood cells under polyvinyl alcohol hydrogels.