Luisa Filipponi

Protein patterning by microcontact printing using pyramidal PDMS stamps

Micro-contact printing, μCP, is a well-established soft-lithography technique for printing biomolecules. μCP uses stamps made of Poly(dimethylsiloxane), PDMS, made by replicating a microstructured silicon master fabricated by semiconductor manufacturing processes. One of the problems of the μCP is the difficult control of the printing process, which, because of the high compressibility of PDMS, is very sensitive to minute changes in the applied pressure. This over-sensitive response leads to frequent and/or uncontrollable collapse of the stamps with high aspect ratios, thus decreasing the printing accuracy and reproducibility. Here we present a straightforward methodology of designing and fabricating PDMS structures with an architecture which uses the collapse of the stamp to reduce, rather than enlarge the variability of the printing. The PDMS stamp, organized as an array of pyramidal micro-posts, whose ceiling collapses when pressed on a flat surface, replicates the structure of the silicon master fabricated by anisotropic wet etching. Upon application of pressure, depending on the size of, and the pitch between, the PDMS pyramids, an air gap is formed surrounding either the entire array, or individual posts. The printing technology, which also exhibits a remarkably low background noise for fluorescence detection, may find applications when the clear demarcation of the shapes of protein patterns and the distance between them are critical, such as microarrays and studies of cell patterning.

Microbeads on microposts: an inverted architecture for bead microarrays.

The rapid development of genomics and proteomics requires accelerated improvement of the microarrays density, multiplexing, readout capabilities and cost-effectiveness. The bead arrays are increasingly attractive because of their self-assembly-based fabrication, which alleviates many problems of top-down microfabrication. Here we present a simple, reliable, robust and modular technique for the fabrication of bead microarrays, which combines the directed assembling of beads in microstructures and PDMS-based replica molding. The beads are first self-assembled in pyramidal microwells fabricated by anisotropic etching of silicon substrates, then transferred on the apex of PDMS pyramids that replicate the silicon microstructures. The arrays are chemically and biochemically robust; they are spatially addressable and have the potential for being informationally addressable; and they appear to offer better readout capabilities than the classical microarrays.

Protein immobilisation on micro/nanostructures fabricated by laser microablation.

The performance of biomedical microdevices requires the accurate control of the biomolecule concentration on the surface, as well as the preservation of their bioactivity. This desideratum is even more critical for proteins, which present a significant propensity for surface-induced denaturation, and for microarrays, which require high multiplexing. We have previously proposed a method for protein immobilisation on micro/nanostructures fabricated via laser ablation of a thin metal layer deposited on a transparent polymer. This study investigates the relationship between the properties of the micro/nanostructured surface, i.e., topography and physico-chemistry, and protein immobilisation, for five, molecularly different proteins, i.e., lysozyme, myoglobin, α-chymotrypsin, human serum albumin, and human immunoglobulin. Protein immobilisation on microstructures has been characterised using quantitative fluorescence measurements and atomic force microscopy. It has been found that the sub-micrometer-level, combinatorial nature of the microstructure translates in a 3-10-fold amplification of protein adsorption, as compared to flat, chemically homogenous polymeric surfaces. This amplification is more pronounced for smaller proteins, as they can capitalize better on the newly created surface and variability of the nano-environments.

Negotiation of obstacles by fungi in micro-fabricated structures: to turn or not to turn?

Polymer microstructures were used to examine the manner in which fungal filaments negotiate obstacles in confined environments. When faced with an obstacle requiring a right-angle turn, two different responses were observed. In 21% of cases, hyphae turned around the corner and continued growth, while in the remaining 79% of cases, filaments continued apical growth into the corner, resulting in bending of the distal portions of the filament. The different reactions could not be linked to physical constraints (e.g., filament flexibility) since the filament deflection required to negotiate the obstacle was the same in all cases. Instead, the response appeared to be related to the original direction of growth at the time of filament formation (branching), with filaments turning only if the resultant growth vector was no more than 90/spl deg/ from their original branching vector. The results suggest that filaments are somehow able to retain a memory of their original branching direction, consistent with an overall survival strategy based on continued growth away from the colony center and into the surrounding environment.

Fungal growth in confined microfabricated networks

The understanding and control of cell growth in confined microenvironments has application to a variety of fields including cell biosensor development, medical device fabrication, and pathogen control. While the majority of work in these areas has focused on mammalian and bacterial cell growth, this study reports on the growth behavior of fungal cells in three-dimensionally confined PDMS microenvironments of a scale similar to that of individual hyphae. The general responses of hyphae to physical confinement included continued apical extension against barriers, resultant filament bending and increased rates of subapical branching with apparent directionality towards structure openings. Overall, these responses promoted continued extension of hyphae through the confined areas and away from the distal regions of the fungal colony. The induction of branching by apical obstruction provides a means of controlling the growth and branching of fungal hyphae through purposefully designed microstructures.

Immobilization of multiple proteins in polymer microstructures fabricated via laser ablation

Investigation of protein-polymeric surface interaction requires reliable practical techniques for evaluation of the efficiency of protein immobilization. In this study the efficiency of protein immobilization was evaluated using three different techniques: (1) protein-binding assay with fluorescent detection and (2) quantification, and (3) atomic force microscopy. This approach enables us to rapidly analyse the adsorption properties of different proteins. The comparative physico-chemical adsorption of α-chymotrypsin, human serum albumin, human immunoglobulin, lysozyme, and myoglobin in the micro-wells fabricated via a localized laser ablation of a protein-blocked thin gold layer (50 nm) deposited on a Poly(methyl ethacrylate) film has been studied. Correlations were observed between the quantitative and qualitative differences depending on both protein and polymeric surface hydrophobicity.

Protein interaction with combinatorial structures

Diazonaphthoquinone/novolak (DNQ) photoresist have the property of changing physical-chemical properties during exposure to UV light, which reflects in a change of the polymer hydrophobicity. A combinatorial surface having different exposed area was fabricated, in order to study the influence of hydrophobicity over protein adsorption and EDC-mediated covalent attachment. The results indicate two different behaviours, reflecting a substantial different mechanism of interaction. While protein adsorption decreased following the hydrophobicity decrease, covalent attachment increased, thus reflecting the effectiveness of the covalent mediator, which cross-links the protein to the carboxylic groups that form during exposure. Based on the results of the present work, a combinatorial microarray will be fabricated, to be used in the biosensor field.

Microcontact printing trapping air: A versatile tool for protein microarray fabrication

The present work introduces a new method for the fabrication of protein micro-patterns, microcontact printing trapping air. The method is based on microcontact printing, a well-established soft-lithographic technique for printing bioactive protein patterns. Usually, the stamp used is made of poly(dimethylsiloxane) obtained by replicating a lithographically microfabricated silicon master. In microcontact printing, the dimensions of the features in the stamp are critical, since the high compressibility of poly(dimethylsiloxane) causes high aspect ratio features to collapse, leading to the printing of undesired areas. In most cases, this is an unwanted effect, which interferes with the printing quality. In this work we used a poly(dimethylsiloxane)stamp bearing an array of micro-posts which, when placed over a flat surface, collapses with consequent formation of an air gap around the entire array. This effect is linked to the distance between the posts that form the array and can be exploited for the fabrication of protein microarrays having a remarkably low background noise for fluorescence detection.

Polymer microstructures for cellular growth studies

The understanding and control of cell growth in confined microenvironments has application to a variety of fields including cell biosensor development, medical device fabrication, and pathogen control. While the majority of work in these areas has focused on mammalian and bacterial cell growth, this study reports on the growth behavior of fungal cells in three-dimensionally PDMS microenvironments of a scale similar to that of individual hyphae. Confinement was found to affect filament branching rate and angle. Overall, fungal hyphae demonstrate much more coordinated behavior during confinement than observed during growth on simple planar unconfined substrates. The remarkable difference of fungal growth behaviour observed in the PDMS microenvironments compared to open, unrestricted environments suggests that three-dimensional microstructures could be used to control and alter fungal motility.