Generation of transducers for fluorescence-based microarrays with enhanced sensitivity and their application for gene expression profiling.

Abstract

The present paper describes a novel generation of microchips suitable for fluorescence-based assays, such as cDNA, oligonucleotide, or protein microarrays. The new transducers consist of a fully corrugated surface coated with a thin layer of Ta2O5 as a high refractive index material. Tuning of the incident excitation light beam to abnormal reflection geometry results in a confinement of the energy within the thin metal oxide layer. Consequently, strong evanescent fields are generated at the surface of these microchips and fluorophores located within the fields showed up to a 2 order of magnitude increase in fluorescence intensities relative to the epifluorescence signals. We have attributed this phenomenon as evanescent resonance (ER). Due to the surface architecture, propagation distances of the incident energy and fluorescence photons are in the micrometer range, thus preventing cross talk between adjacent regions. ER microchips offer a significant increase in fluorescence intensities in both "snapshot" fluorescence setups and commercial fluorescence scanners. The underlying principle of the novel chips is explained, and quantitative data on the fluorescence enhancement are provided. To demonstrate their potential, the novel chips are used to investigate the dependence of expression levels from metabolic genes in rat liver on drug treatment. In contrast to competitive hybridization, labeled samples were hybridized to individual ER microchips, and changes were observed by comparing with normalized data from different chips. Results obtained in gene expression profiling experiments with phenobarbital-treated rats are shown.