Introduction:
Collective Enhanced IR Absorption (CEIRA) microscopy is a well-known method for increasing FTIR sensitivity by taking advantage from the collective resonant excitation of nanoantennas' arrays, that provides a huge electromagnetic field enhancement on the nano-scale [1]. Infrared signals of molecules located in these fields are enhanced by orders of magnitude, enabling a spectroscopic detection with unprecedented sensitivity [2].
Here, we present a multipurpose device made by adjacent gold nanoantennas arrays which allows the investigation of biomolecules in the entire MIR range. As a first application, we present a study on two model proteins, Bovine Serum Albumin (BSA) and Concanavalin-A (ConA), and one of biomedical interest: the Epithelial Growth Factor Receptor (EGFR)
Methods:
CEIRA substrates were fabricated by electron beam lithography. The repeating units of the arrays were centro-symmetric crosses, a geometry which permits to couple the antennas with both polarization of the light. Finite-difference time-domain simulations were used to calculate far-field and near-field signal profiles of the arrays.
Proteins were immobilized onto the gold surfaces by taking advantage from the gold/thiols chemistry. CEIRA substrates were integrated in fluidic devices in order to work in buffer and preserve protein native conformation. FTIR resonance spectra were acquired in reflection.
Results:
We benchmarked the performances of our devices using monolayers of reference proteins: BSA and Con A (Figure 1): model proteins were bonded to the surface via ammine coupling. Results confirmed the capabilities of our devices to discern the different conformation of the two proteins .
Then, EGFR was measured, a protein of particular interest since most of its mutations leads to lung cancer. EGFR was modified by adding to the original construct a His-tag, which was used for induce its selective binding in solution, without chemically modify the protein. In this way, we had the possibility to follow in real-time the binding of the molecule (Figure 2).
We believe that a platform with this sensitivity, few hundreds of molecules, has really the potentialities to become an advance high-throughput tool.
References:
[1] R. Adato et al. Nat.Commun. 2013, 4, 2154.[2] F. Neuberech et al. Chem.Rev. 2017,117, 5110-5145.
