Quantitative detection of well-based DNA array using switchable lanthanide luminescence

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.

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► Oligonucleotide hybridization switches on lanthanide luminescence.
► The binary probe technology enables wash-free nucleic acid array.
► Detection of both synthetic targets and PCR-amplified E. coli DNA is demonstrated.