Time-Gated Fluorescent Aptamer Sensors Eliminate Autofluorescence during Continuous Molecular Detection in Complex...

Background autofluorescence presents a major obstacle to fluorescence-based sensors in complex biological environments, creating substantial interference that undermines the sensitivity of detection. Here, we introduce a strategy for autofluorescence-free monitoring of biomarkers using time-gated fluorescent aptamer switches. The switches are labeled with a europium chelate donor and an acceptor fluorophore, and the Förster resonance energy transfer (FRET) between the dyes is modulated upon target binding. The long (∼1 ms) luminescence lifetime of the europium chelate enables the selective detection of the switch fluorescence while filtering out short-lived background autofluorescence. Furthermore, we present a generalizable framework for designing time-gated fluorescence aptamer switches and apply it to three clinically relevant biomarkers: glucose, lactate, and cortisol. By tuning the design of our switches, we can optimize the time-gated FRET signal change with target binding. These switches detect their targets across physiologically relevant concentration ranges and exhibit reversible responses with minute-scale temporal resolution, making them well suited for continuous monitoring applications. Finally, we develop a compact sensor platform that integrates our switches with custom hardware for monitoring biomarkers in undiluted human serum. Overall, our approach offers a simple, compact solution for biosensing in complex biological samples.