Optimization of the immunorecognition layer towards Brucella sp. on gold surface for SPR platform

A successful immunosensor is characterized by a proper antibody immobilization and orientation in order to enhance the antigen recognition. In this work, a thorough characterization of the antibody functionalized gold surface is performed to set up the best conditions to implement in an optical platform for the detection of Brucella sp. Two different strategies are evaluated, based on a random immobilization and on an oriented one: a direct antibody immobilization on carboxylic mixed polyethylene (PEG) self-assembled monolayer (SAM) or only carboxylic PEG SAM interface is compared to an oriented immobilization on a layer of protein G on the same PEG SAM interfaces. X-ray Photoelectron Spectroscopy (XPS), Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS) and contact angle (CA) are used to chemically characterize the gold functionalized surface and ToF-SIMS is also used to confirm the right antibody orientation. Optical characterization is applied to monitor the functionalization steps and fluorescence measurements are used to set up the proper experimental conditions and also to detect Brucella bacteria on the surface. Best results are obtained with a 10 ng/μl incubation solution of antibody immobilized, in an oriented way, on a mixed PEG SAM interface.