Azurin self-assembled monolayers characterized by coupling electrical impedance Spectroscopy and spectroscopic ellipsometry

We present a combined study of azurin monolayers deposited on both bare and hexadecanethiol-covered gold surfaces carried out by coupling two independent techniques, in situ electrical impedance spectroscopy (EIS) and ex situ spectroscopic ellipsometry (SE). The combination of the two techniques which probe different sample properties in different environmental conditions results in a cross-consistent characterization of the optical and electrical properties of the protein layers. Moreover, it provides a reliable estimate of the layer thickness. Azurin self-assembles on bare gold forming a monolayer with a thickness of 3.2 +/- 0.2 nm, a value very close to the crystallographic height of the cylindrically shaped protein. When azurin is deposited on hexadecanethiol-covered gold, a protein layer thickness of 2.7 +/- 0.2 nm is obtained. This indicates that azurin forms a less dense layer when it is deposited on thiol-covered gold compared to the adsorption on bare gold. Both EIS and SE provide a direct estimate of the dielectric constant of the protein layer in the hydrated and dry states, respectively. In the case of the composite azurin/hexadecanethiol system, the combined EIS and SE analysis proves to be a valuable tool for the correct interpretation of the EIS data and allows for an estimate of the azurin and hexadecanethiol conductivity ratio.