A 2D crystalline nanoarray of organothiols has been used for enhancing the detection sensitivity of Surface Plasmon Resonance assays. The nanoarrays have been fabricated by combining colloidal lithography and organothiol self assembly. In particular, immunoreaction produced by nano-spots with carboxylic function in a non-adhesive matrix of Polyethylene oxide (PEO) has been compared to uniform carboxylic surfaces. For the same concentration of analyte, the SPRi signal is much higher by using the 2D crystalline nanoarrayed surfaces.