Homogeneous FRET immunoassay based on lanthanides to quantum dots energy transfer
Abstract
Homogeneous FRET (Forster Resonance Energy Transfer) fluoroimmunoassays (FIA) allow fast, inexpensive and highly sensitive monitoring of biochemical processes occurring on the nanometer scale. The technique is widely applied in high throughput screening (HTS) for in vitro diagnostics (IVD). Quantum dots (qdots) are usually applied as energy donors for FRET experiments in solution. In this contribution we show that commercially available biotinylated CdSe/ZnS core/shell qdots (Qdot 665 Biotin conjugate, Invitrogen Corp., USA) are excellent FRET acceptors in a time-resolved FIA based on interaction with lanthanide complex-labeled streptavidin as energy donor. The energy transfer experiments were performed on a modified commercial FIA reader system (KRYPTOR, Cezanne SA, France) using three di.erent lanthanide chelates (1 of terbium and 2 of europium). All three FRET donors showed efficient energy transfer to the qdots, evidenced both by nanocrystals emission sensitization and by a thousand fold increase of the qdot luminescence decay time, reaching some hundreds of microseconds. In a control experiment the unlabeled donors were used to rule out dynamic energy transfer between lanthanides and qdots. Due to the very high qdot absorption extremely large Forster radii of 104 A for terbium and 96 A for europium were achieved. FRET efficiency was up to 67 % and sub-picomolar detection limits were obtained for qdots in this type of homogeneous FIA. The use of qdots as energy acceptors…
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