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.

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.

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.

Fungi use efficient algorithms for the exploration of microfluidic networks.

Fungi, in particular, basidiomycetous fungi, are very successful in colonizing microconfined mazelike networks (for example, soil, wood, leaf litter, plant and animal tissues), a fact suggesting that they may be efficient solving agents of geometrical problems. We therefore evaluated the growth behavior and optimality of fungal space-searching algorithms in microfluidic mazes and networks. First, we found that fungal growth behavior was indeed strongly modulated by the geometry of microconfinement. Second, the fungus used a complex growth and space-searching strategy comprising two algorithmic subsets: 1) long-range directional memory of individual hyphae and 2) inducement of branching by physical obstruction. Third, stochastic simulations using experimentally measured parameters showed that this strategy maximizes both survival and biomass homogeneity in microconfined networks and produces optimal results only when both algorithms are synergistically used. This study suggests that even simple microorganisms have developed adequate strategies to solve nontrivial geometrical problems.