An essential step during IAV infection is the pHinduced fusion of viral and host membrane from the specifically GD1a-bound state. This process is, at the same time, one of the most complex steps during infection and we sought to examine if the combination of SEIRA and tBLMs can detect the associated structural changes. Mimicking this process, we changed the pH from 7.4 to 5.0 to record the SEIRA difference spectra starting from the final spectra in Figure (see scheme in Figure ). We are using difference spectroscopy herein as it is a frequently employed approach to highlight changes upon a perturbation. These changes are observed as negative or positive signals for species that have disappeared or were created, respectively. In SEIRA they can be further interpreted as molecules that have moved away or towards from the surfaceenhancing region of the first 10 nm to the Au film (negative and positive signals, respectively). After pH change, we observe positive amide I and II bands at 1661 and 1550 cm -1 in our SEIRA difference spectra (Figure top), strongly indicating α-helical protein fragments moving closer to the membrane (note that the presented spectra have been corrected for background pHinduced changes in absence of the IAV, see Figure ). At the same time, we observe a negative signal at 1736 cm -1 assigned to ester C=O stretches of lipid molecules (Figure top). While both viral and tBLM lipids can contribute to this signal, it is sensible to ascribe this feature to the stronger enhanced tBLM lipids as they move further away from the surface-enhancing region. This would be in line with the proposed mechanism of pH-induced HA function. For instance, structural studies have shown that extensive structural changes of HA lead to an anchoring of the α-helical HA fusion peptide with the host membrane (Figure top). If the HA fusion peptide engaged successfully, the host membrane is then "pulled" towards the virus. The positive HA amide and negative tBLM lipid signals are therefore well in line with successful first steps ). A: A pH drop from 7.4 to 5.0 was performed with IAV bound via specific interactions to GD1a in tBLM with POPC or deuterated POPC-d82 as lipids to make use of the vibrational isotope effect. B: "pH 5.0 -pH 7.4" SEIRA difference spectra (black line) with POPC (top) and POPC-d82 (bottom) as lipids used in the tBLM; bands highlighted in purple refer to lipids and protein of IAV, whereas bands in blue are assigned to POPC/POPC-d82 of the tBLM. Light purple or light blue spectra beneath the SEIRA spectra are reference spectra of protein and lipids of IAV or POPC/POPC-d82 lipids in the tBLM, respectively. Reference spectra are shown with positive or negative intensity according to the interpretation of the SEIRA spectra (i.e. molecules approaching or removed from the SEIRA Au film). C: Schematic representation of structural changes observed in SEIRA difference spectra in B; protein structures are from PDB ID 6Y5G and 6YK. The pH change initiates structural changes of HA, which leads to the anchoring of HA's fusion peptide in the tBLM pulling the bilayer to the virus (