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.

Polymer Doping as a Novel Approach to Improve the Performance of Plasmonic Plastic Optical Fibers Sensors.

In this work, Fe2O3 was investigated as a doping agent for poly(methyl methacrylate) (PMMA) in order to enhance the plasmonic effect in sensors based on D-shaped plastic optical fibers (POFs). The doping procedure consists of immerging a premanufactured POF sensor chip in an iron (III) solution, avoiding repolymerization and its related disadvantages. After treatment, a sputtering process was used to deposit a gold nanofilm on the doped PMMA in order to obtain the surface plasmon resonance (SPR). More specifically, the doping procedure increases the refractive index of the POF's PMMA in contact with the gold nanofilm, improving the SPR phenomena. The doping of the PMMA was characterized by different analyses in order to determine the effectiveness of the doping procedure. Moreover, experimental results obtained by exploiting different water-glycerin solutions have been used to test the different SPR responses. The achieved bulk sensitivities confirmed the improvement of the plasmonic phenomenon with respect to a similar sensor configuration based on a not-doped PMMA SPR-POF chip. Finally, doped and non-doped SPR-POF platforms were functionalized with a molecularly imprinted polymer (MIP), specific for the bovine serum albumin (BSA) detection, to obtain dose-response curves. These experimental results confirmed an increase in binding sensitivity for the doped PMMA sensor. Therefore, a lower limit of detection (LOD), equal to 0.04 µM, has been obtained in the case of the doped PMMA sensor when compared to the one calculated for the not-doped sensor configuration equal to about 0.09 µM.