Despite its potential for biomolecule characterization, FTIR microscopy (FTIRM) of samples at sub-micromolar concentration is extremely challenging using conventional approaches. A promising method for increasing the technique sensitivity is to take advantage from the electric field enhancement associated with the collective excitation of plasmonic resonance of nanoantennas assemblies, a technique called Collective Enhanced IR Absorption (CEIRA) . In order to obtain informative spectra with good SNR, a brilliant and broadband source is needed. Infrared Synchrotron radiation (IR-SR) possess both the aforementioned characteristics and its exploitation in the field of MIR plasmonic have been demonstrated .
In our study, gold nanoantennas arrays were simulated, designed and directly fabricated on CaF2 by electron beam lithography (EBL) to enhance the absorption in the 1400-1800 cm-1, with the aim to detect conformational variations in biologically relevant proteins. In vitro protein expression through recombinant technology is a well-established approach, although the purification yield could be very low (<200-300 µg of protein) for the less stable species and the crystallization very hard. By SR-CEIRA approach we got protein conformational details at femtomolar concentration, collecting spectra at 10x10 µm2 lateral resolution on protein monolayers, obtained by exploiting surface-functionalization thiols chemistry and amide coupling immobilization protocols.
We first obtained data from monolayers of reference proteins, Bovine Serum Albumin (BSA) and Concanavalin-A (Con A) and, by comparing them to the reference spectra collected on bulk samples, we confirmed the possibility to discern the difference in conformation between the two proteins. Conformational details on Epidermal growth factor Receptor (EGFR), a protein involved in the progression of lung cancer and expressed at the protein Production facility at Elettra, have been also recently obtained, and we are now working on the realization of fluidic devices for moving from de-hydrated to hydrated measurement conditions.
 Adato, R. et al. In-situ ultra-sensitive infrared absorption spectroscopy of biomolecule interactions in real time with plasmonic nanoantennas. Nat. Commun 4, 2154 (2013).
 Huck, C. et al.Surface-Enhanced Infrared Spectroscopy Using Nanometer-Sized Gaps. ACS Nano. 8 (2014) 4908–4914