Time-Gated FRET from terbium labeled antibodies to quantum dot acceptors for broad ADP sensing

摘要

Time-gated Förster resonance energy transfer (TR-FRET) introduces a time-gate before the detection of the fluorescencespectra or photon count. If the donor is sufficiently long-lived TR-FRET allows for any initial acceptor sensitization todecay before the measurement. TR-FRET in the μs range is particularly advantageous for small molecule assays as iteliminates background fluorescence from screening compounds, which typically have ns lifetimes. The sensor wedeveloped utilizes Terbium (Tb)-labeled antibodies (Ab) that selectively recognizes adenosine diphosphate (ADP). TheTb emitters have fluorescence lifetimes on the ms scale, making them excellent candidates for TR-FRET donors. In anattempt to increase the FRET signal we utilized a semiconductor quantum dot (QD) as an acceptor. The QD presented anADP modified His6-peptide conjugated to its surface via self-assembly metal-affinity coordination, which bound the Tblabeled Ab to the QD surface. QDs have large extinction coefficients, broad absorption, brightness, and sharp emissionpeaks, optimal for sensitive and multiplexed detection. By using a QD acceptor the Förster radius was increased byapproximately 2 nm as compared to traditional organic dyes. We were able to demonstrate a Tb-to-QD based TR-FRETbioassay for broadly applicable ADP sensing, working at nM concentrations for sensor, analyte, and enzyme.Quantitative values were obtained for the kinetics of a model enzyme (glucokinase). The specific sensor was alsocapable of discriminating enzyme inhibitor capabilities of structurally similar compounds. The strategy of usingmodified peptides to present antibody epitopes on QD surfaces is readily transferable to other assays.