Microcantilever-based sensing arrays for evaluation of biomolecular interactions

The controlled immobilization on a surface of biomolecules used as recognition elements is of fundamental importance in order to realize highly specific and sensible biosensors. Microcantilevers (MC) are nanomechanical sensors, which can be used as label free micro-sized mechanical transducers. MC resonant frequency is sensitively modified upon molecules adsorption, demonstrating an impressive mass resolution. A widely used approach for the immobilization of biorecognition elements on silicon substrates consists in the deposition of 3-aminopropyl-triethoxysilane (APTES) followed by the incubation with glutaraldehyde (GA) as a crosslinking agent. However, these derivatization processes produce a variable chemical functionalization because of the spontaneous polymerization of GA in aqueous solutions. With the aim of producing a more reliable chemical functionalization for protein immobilization, the deposition of a thin film of APTES by self-assembly followed by the modification of its amino groups into carboxyl groups by incubating in succinic anhydride (SA) is proposed. Moreover, the activation of these terminal carboxyl groups were performed by using the EDC/s-NHS protocol in order to enhance their reactivity toward primary amine groups present on biomolecules surface. This method was characterized from a physico-chemical point of view by means of compositional and morphological surface analysis. Moreover, data acquired after the application of this functionalization to a MC-based system showed a highly reproducible deposition of APTES/SA when compared to APTES/GA deposition process. APTES/SA derivatized MC arrays were then incubated with biomolecules for the study of its protein binding capability: the quantification of the grafted biomolecules was performed from the gravimetric data and compared with a theoretical surface density calculated through a molecular modeling tool, providing information about the orientation of the proteins tethered to the surface. In order to avoid or reduce non-specific protein interactions, Bovine Serum Albumin and ethanolamine were considered for their blocking capability. Finally, the detection of the envelope glycoprotein domain III of the Dengue virus type 1 based on immune-specific recognition through the DV32.6 antibody was performed, providing a stoichiometry ratio for the DIII-DV1/DV32.6 interaction. Currently, no cure or vaccine are available; thus, a better understanding of the interactions between the viruses and specific antibodies is expected to provide fundamental information for the development of a vaccine.