Up-converting phosphor reporters for nucleic acid microarrays

An important application of robotically spotted DNA microarrays is the moni toring of RNA expression levels(1,2). A clear limitation of this technology is the relatively large amount of RNA that is required per hybridization a s a result of low hybridization efficiency and limiting detection sensitivi ty provided by conventional fluorescent reporters(3,4). We have used a rece ntly introduced luminescent reporter technology, called UPT (up-converting phosphor technology(5,6)). Down-converting phosphors have been applied befo re to detect nucleic acids on filters using time-resolved fluorometry(7,8). The unique feature of the phosphor particles (size 0.4 mum) used here is t hat they emit visible light when illuminated with infrared (IR) light (980 nm) as a result of a phenomenon called up-conversiong(9,10). Because neithe r support material of microarrays nor biomolecules possess up-conversion pr operties, an enhanced image contrast is expected when these nonfading phosp hor particles are applied to detect nucleic acid hybrids on microarrays. Co mparison of the UPT reporter to cyanin 5 (Cy5) in a low-complexity model sy stem showed a two order of maginitude linear relationship between phosphor luminescence and target concentration and resulted in an excellent correlat ion between the two reporter systems for variable target concentrations (R- 2 = 0.95). However, UPT proved to be superior in sensitivity, even though a wide-field microscope equipped with a xenon lamp was used. This higher sen sitivity was demonstrated by complementary DNA (cDNA) microarray hybridizat ions using cDNAs for housekeeping genes as probes and complex cDNA as targe t. These results suggest that a UPT reporter technology in combination with a dedicated IR laser array-scanner holds significant promise for various m icroarray applications.