Fluorescence Interference Contrast-enabled structures improve the microarrays performance

Abstract

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.