Immunosensor surface functionalization by simple photochemical immobilization technique (PIT): A spectroscopic demonstration

Surface functionalization is a key step in biosensing and antibodies (Abs) occupy a key role in bio-recognition thanks to their superior specificity. When gold is the interacting surface, the recently introduced Photochemical Immobilization Technique (PIT) has shown to be quick, easy-to-use and very effective in tethering Abs oriented upright. The molecular base of PIT relies on the selective photo-reduction of the disulphide bridges in cys-cys/trp triads in IgGs, which in turn leads to the production of reduced SH groups with strong affinity towards noble metals. The selectivity of PIT arises from the presence of only 12 triads not all of them effective in yielding thiols. We demonstrate that by irradiating Abs in solution for only 30 s with UV light from an amalgam type lamp, the Abs are "activated" and approximately 7 thiols are produced. Once opened, SHs keep their properties for at least 200 s, a time long enough to convey Abs onto a gold surface on which they bind upright. The position of SH has been determined by mass spectrometry, which also confirms that only 8 thiols per Ab are reduced by UV and 4 of them (i.e. 2 disulphide bridges) are not exposed to the solvent lowering to two the number of bridges available for PIT. Moreover, SERS highlights that the signal from UV-treated Abs is much stronger than that coming from Abs physisorbed, thereby demonstrating higher proximity of Abs with the surface when they are UV-activated.