Time-Resolved Analysis of a Highly Sensitive Förster Resonance Energy Transfer Immunoassay Using Terbium Complexes as Donors and Quantum Dots as Acceptors

CdSe/ZnS core/shell quantum dots (QDs) are used as efficient Förster Resonance Energy Transfer (FRET) acceptors in a time-resolved immunoassays with Tb complexes as donors providing a long-lived luminescence decay. A detailed decay time analysis of the FRET process is presented. QD FRET sensitization is evidenced by a more than 1000-fold increase of the QD luminescence decay time reaching ca. 0.5 milliseconds, the same value to which the Tb donor decay time is quenched due to FRET to the QD acceptors. The FRET system has an extremely large Förster radius of approx. 100 A and more than 70% FRET efficiency with a mean donor-acceptor distance of ca. 84 A, confirming the applied biotin-streptavidin binding system. Time-resolved measurement allows for suppression of short-lived emission due to background fluorescence and directly excited QDs. By this means a detection limit of 18 attomol QDs within the immunoassay is accomplished, an improvement of more than two orders of magnitude compared to commercial systems.