Enhanced Raman spectroscopy of proteins on native cell membranes stretched on super-hydrophobic surface

There is an urgency to fully overcome the limitations of known techniques (X-ray diffraction, NMR, TEM imaging) to solve the structure of roughly 70% of the cell membrane proteins. The limiting factors are the low yield in... [ view full abstract ]

There is an urgency to fully overcome the limitations of known techniques (X-ray diffraction, NMR, TEM imaging) to solve the structure of roughly 70% of the cell membrane proteins. The limiting factors are the low yield in extraction and crystallization of these proteins and/or the limited resolution of the instrument, plus the need of thousands of replicas for the same protein. Enhanced spectroscopy techniques are widely used in biological applications, due to their sensitivity, versatility and often low interaction/toxicity with a delicate sample. In recent work we have demonstrated the potential of a micro-fabricated super-hydrophobic device combined with different types of analysis (Raman spectroscopy, electron microscopy), to characterize single/few biological molecules with utmost sensitivity. Here we are performing SERS analysis of a specific subunit (SCN1a) of the Voltage gated Sodium channel (Na_v) on extracted cell membranes stretched over the pillars.

Basically, extracted cell membranes are stretched on top of the SHS, immuno-gold labeled for the SCN1a subunit and analyzed at the Raman spectrometer. To access the plasmonic effect on the gold nanoparticles, laser wavelength at 633 nm is used.

In Fig. 1 the experimental results are shown. The Raman mapping shows several areas of interest, including the ones identifying the membranes (Fig. 1d) and the gold nanoparticles per se (Fig. 1e). Moreover SERS effect is noticed nearby some of the gold nanoparticles (Fig. 1f, g). Several sharp peaks appear on the two spectra. Most of them are related to single aromatic aminoacids, like the ones at 643 cm^-1 (Tyr), 958, 1348, 1551 cm^-1 (Trp), 1049 cm^-1 (Phe).

These preliminary results show the potential of the method to chemically characterize single proteins by using SERS effect on a native cell membrane surface in dry, background free conditions. The signal enhancement given by the gold nanoparticle can be complemented by the topography and the plasmonic enhancement accessible with an AFM probe tip. This advancement will likely show the vibrational modes of the secondary structures of the protein thus giving access to structural information at the single molecule level.