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Time-gated Förster resonance energy transfer (TR-FRET) introduces a time-gate before the detection of the fluorescence spectra or photon count. If the donor is sufficiently long-lived TR-FRET allows for any initial acceptor sensitization to decay before the measurement. TR-FRET in the μs range is particularly advantageous for small molecule assays as it eliminates background fluorescence from screening compounds, which typically have ns lifetimes. The sensor we developed utilizes Terbium (Tb)-labeled antibodies (Ab) that selectively recognizes adenosine diphosphate (ADP). The Tb emitters have fluorescence lifetimes on the ms scale, making them excellent candidates for TR-FRET donors. In an attempt to increase the FRET signal we utilized a semiconductor quantum dot (QD) as an acceptor. The QD presented an ADP modified His6-peptide conjugated to its surface via self-assembly metal-affinity coordination, which bound the Tb labeled Ab to the QD surface. QDs have large extinction coefficients, broad absorption, brightness, and sharp emission peaks, optimal for sensitive and multiplexed detection. By using a QD acceptor the Förster radius was increased by approximately 2 nm as compared to traditional organic dyes. We were able to demonstrate a Tb-to-QD based TR-FRET bioassay 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 also capable of discriminating enzyme inhibitor capabilities of structurally similar compounds. The strategy of using modified peptides to present antibody epitopes on QD surfaces is readily transferable to other assays.
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