Fluorescence Interference Contrast-enabled structures improve the microarrays performance.

Continuous improvements of the fluorescence-based sensitivity and specificity, required for high throughput screening, diagnostics, and molecular biology studies, are usually addressed by better readout systems, or better reporting elements. However, while Fluorescence Interference Contrast (FLIC), which modulates the fluorescence by materials-based parameters, has been used for decades to measure biomolecular interactions at nanometer-precision, e.g., for the study of molecular motors and membrane processes, it has been seldom used for high throughput or diagnostic microdevices. Moreover, the amplification of both the fluorescence signal, modulated by vertically-nano-calibrated structures, and the signal/background, modulated by laterally-micro-calibrated structures, has not been explored. To address this synergy, structures comprising optically transparent silicon oxide, tens of micrometers-wide and with thicknesses in the low hundreds of nanometers, which are able to promote the formation of standing waves if patterned on a reflective material, have been designed, fabricated and tested, for the use in DNA- and protein arrays. The light emitted by a fluorophore placed on top of the structures and reflected by a bottom mirror surface, e.g., silicon, platinum, is physically constrained to a region defined lithographically, both vertically and laterally, i.e., micro-pillars and -wells, resulting in an accurate identification and quantification of fluorescence. The signal/noise ratio on micro-/nano-structured substrates is comparable to that measured on planar substrates, but the physical confinement of the microarray spots results in a considerable increase of the intra-feature uniformity.

The threshold at which substrate nanogroove dimensions may influence fibroblast alignment and adhesion.

The differences in morphological behaviour between fibroblasts cultured on smooth and nanogrooved substrata (groove depth: 5-350 nm, width: 20-1000 nm) have been evaluated in vitro. The aim of the study was to clarify to what extent cell guidance occurs on increasingly smaller topographies. Pattern templates were made using electron beam lithography, and were subsequently replicated in polystyrene cell culture material using solvent casting. The replicates were investigated with atomic force microscopy (AFM). After seeding with fibroblasts, morphological characteristics were investigated using scanning electron microscopy (SEM) and light microscopy, in order to obtain qualitative and quantitative information on cell alignment. AFM revealed that the nanogroove/ridge widths were replicated perfectly, although at deeper levels the grooves became more concave. The smooth substrata had no distinguishable pattern other than a roughness amplitude of 1 nm. Interestingly, microscopy and image analysis showed that fibroblast after 4 h had adjusted their shape according to nanotopographical features down to cut-off values of 100 nm width and 75 nm depth. After 24 h culturing time, fibroblasts would even align themselves on groove depths as shallow as 35 nm. It appears depth is the most essential parameter in cellular alignment on groove patterns with a pitch ratio of 1:1. On the smooth substrata, cells always spread out in a random fashion. Analysis of variance (ANOVA) demonstrated that both main parameters, topography and culturing time, were significant. We conclude that fibroblast cells cultured on nanotopography experience a threshold feature size of 35 nm, below this value contact guidance does no longer exist.