title: Quantitative detection of well-based DNA array using switchable lanthanide luminescence.
abstract: In this report a novel wash-free method for multiplexed DNA detection is demonstrated employing target specific probe pairs and switchable lanthanide luminescence technology on a solid-phase array. Four oligonucleotide capture probes, conjugated at 3' to non-luminescent lanthanide ion carrier chelate, were immobilized as a small array on the bottom of a microtiter plate well onto which a mix of corresponding detection probes, conjugated at 5' to a light absorbing antenna ligand, were added. In the presence of complementary target nucleic acid both the spotted capture probe and the liquid-phase detection probe hybridize adjacently on the target. Consequently the two non-luminescent label molecules self-assemble and form a luminescent mixed lanthanide chelate complex. Lanthanide luminescence is thereafter measured without a wash step from the spots by scanning in time-resolved mode. The homogeneous solid-phase array-based method resulted in quantitative detection of synthetic target oligonucleotides with 0.32 nM and 0.60 nM detection limits in a single target and multiplexed assay, respectively, corresponding to 3× SD of the background. Also qualitative detection of PCR-amplified target from Escherichia coli is described.
authors.affiliation: Division of Biotechnology, University of Turku, Turku, Finland. umskar@utu.fi
authors.forename: Ulla
authors.initials: U
authors.lastname: Karhunen
authors.forename: Minna
authors.initials: M
authors.lastname: Soikkeli
authors.forename: Susanne
authors.initials: S
authors.lastname: Lahdenperä
authors.forename: Tero
authors.initials: T
authors.lastname: Soukka
mesh_terms: D056831:Coordination Complexes / Q000737:chemistry*; D004247:DNA / Q000032:analysis*; D027081:DNA Gyrase / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D028581:Lanthanoid Series Elements / Q000737:chemistry*; D049449:Luminescence; D008163:Luminescent Measurements; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods* / Q000592:standards; D015345:Oligonucleotide Probes / Q000737:chemistry*; D009841:Oligonucleotides / Q000032:analysis*; D016133:Polymerase Chain Reaction
chemical_list: D056831:Coordination Complexes; D004269:DNA, Bacterial; D028581:Lanthanoid Series Elements; D015345:Oligonucleotide Probes; D009841:Oligonucleotides; D004247:DNA; D027081:DNA Gyrase