Detection of DNA using upconverting phosphor reporter probes

We have developed a nucleic acid detection assay using an upconverting phosphor DNA reagent as a reporter probe for the detection of target single-stranded DNA molecules. Upconverting phosphors allow the detection of DNA against zero background signal because low energy IR light is absorbed by a two-photon process and remitted as a visible photon, eliminating the presence of autofluorescence. The spectral properties of upconverting phosphors, which emit in the blue, red, or green region of the spectrum, are not sensitive to solvent conditions or to temperature and can therefore be used in a variety of buffers for the qualitative and quantitative detection of biological molecules. Short DNA oligonucleotides were covalently attached to the surface of submicron size phosphor particles and used to detect M13mp18(+) DNA targets captured by magnetic beads labeled with a capture probe specific to the target. This system allowed the detection on the order of femtomoles of M13mp18(+) strand DNA. Buffer conditions were optimized for the use of the phosphor reporters. The phosphor-labeled probes are stable in aqueous solution for 2 weeks and have a lyophilized shelf life of at least one month.