New micropatterning techniques for the spatial addressable immobilization of proteins

Bio-microdevices are miniaturised devices based on biologically derived components (e.g., DNA, proteins, and cells) combined or integrated with microfabricated substrates. These devices are of interest for numerous applications, ranging from drug discovery, to environmental monitoring, to tissue engineering. Before a bio-microdevice can be fully developed, specific fabrication issues need to be addressed. One of the most important is the spatial immobilization of selected biomolecules in specific micro-areas of the device. Among the biomolecules of interest, the controlled immobilization of proteins to surfaces is particularly challenging due to the complexity of these macromolecules and their tendency to lose bioactivity during the immobilization step. The present Thesis reports on three novel micropatterning techniques for the spatial immobilization of proteins with bioactivity retention and improved read-out of the resulting micropatterns. The technologies developed are based on three different micropatterning approaches, namely 1) direct-writing UV laser microablation (proLAB), 2) a novel microcontact printing method (μCPTA) and 3) a replica molding method combined with bead selfassembly (BeadMicroArray). The first two technologies, proLAB and μCPTA, are an implementation of existing techniques (laser ablation and μCP, respectively), whereas the third, i.e., the BeadMicroArray, is a totally new technique and type of patterning platform. ‘ProLAB’ is a technology that uses a micro-dissection tool equipped with a UV laser (the LaserScissors®) for ablating a substrate made of a layer of ablatable material, gold, deposited over a thin polymer layer. The latter layer is transparent to the laser but favours protein adsorption. In the present work microchannels were chosen as the structure of interest with the aim of arranging them in ‘bar-codes’, so to create an ‘information-addressable’ microarray. This platform was fabricated and its application to specific antigen binding demonstrated. The second technique that was developed is a microstamping method which exploits the instability of a high-aspect ratio rubber stamp fabricated via soft-lithography. The technique is denominated microcontact printing trapping air (μCPTA) since the collapsing of a rubber stamp made of an array of micro-pillars over a plane glass surface resulted in the formation of a large air gap around the entire array. The method can be successfully employed for printing micro-arrays of proteins, maintaining biological activity. The technique was compared with robotic spotting and found that microarrays obtained with the μCPTA method were more homogeneous and had a higher signal-tonoise ratio. The third technique developed, the BeadMicroArray, introduces a totally new platform for the spatial addressable immobilization of proteins. It combines replica molding with microbead self-assembling, resulting in a platform where diagnostic beads are entrapped at the tip of micropillars arranged in a microarray format. The fabrication of the BeadMicroArray involves depositing functional microbeads in an array of V-shaped wells using spin coating. The deposition is totally random, and conditions were optimised to fill about half the array during spin coating. After replica molding, the resulting polymer mold contains pyramid-shaped posts with beads entrapped at the very tip of the post. Thanks to the fabrication mode involved, every BeadMicroArray fabricated contains a unique geometric code, therefore assigning a specific code to each microarray. In the present work it was demonstrated that the functionality of the beads after replica molding remains intact, and that proteins can be selectively immobilized on the beads, for instance via biorecognition. The platform showed a remarkable level of selectively which, together with an efficient blocking towards protein non-specific adsorption, lead to a read-out characterized by a very good signal-to-noise. Also, after recognition, a code was clearly visible, therefore showing the encoding capacity of this unique microarray.