A MEMS (Micro Electro Mechanical Systems)approach to highly sensitive multiplexed biosensors

In the field of Micro Electro Mechanical Systems (MEMS), micro and nanomechanical resonators are playing a growing role in biosensing. Thanks to their extreme sensitivity, fast response and low cost, they can be competitive with current diagnostic systems. Nonetheless, the real challenge for new biosensing techniques consists in multiplexing functionality, the ability to detect more than one ligand simultaneously. In this direction, a promising approach is offered by micropillars technology, and in this project, the advantages coming from their geometry are exploited to develop a new highly sensitive and multiplexed biosensor. Micropillars are vertical oriented micromechanical resonators, used as mass sensors, in which the biomolecular adsorption is confined on their micron sized top area. This allows an easy quantification of the deposited mass and a reduction of the response time due to a faster diffusion mechanism. Moreover, pillars can be easily arranged in dense arrays of several thousand sensors in a square millimeter, offering a promising platform for multiplexing. In order to obtain a further improvement in sensibility, specificity and speed of analysis, an amplification of the signal, coming from the deposited mass, has been investigated. A sandwich assay approach based on functionalized gold nanoparticles has been applied and the signal amplification has been demonstrated using a biotin-streptavidin system. A multiple functionalization and a parallel read-out detection method have been also developed in order to drive the sensor towards a multiplexing system. The first target has been reached treating the pillar surface with a photocleavable thiol on which, after the UV laser exposure, -NH2 groups are available and can be used to introduce other functionalities, such as antibodies, through the reaction with –COOH groups. Afterwards, the best condition for the chain cleavage and the amine coupling have been investigated, using -COOH fully coated Quantum Dots as binding elements. Moreover, thanks to a further optimization of the pillar geometry, that lead to structures with a larger oscillation amplitude, the development of an innovative strategy for a parallel read-out based on CCD imaging and software image analysis has been carried out. Advantages from this method are demonstrated also for a single marker detection: acquiring simultaneously the frequency shift of tens pillars and applying a proper statistical analysis, it’s possible to overcome the variability of the single measures improving the device sensitivity. As proof of principle this method has been applied for the detection of PSMA (Prostate Specific Membrane Antigen) at diagnostically relevant concentrations (nM level) both from physiological solution and serum.