A Generalizable Fluorescence Sensor Platform for Sample Preparation‐Free Protein Detection

Abstract Modern molecular detection assays such as enzyme‐linked immunosorbent assays (ELISAs) offer excellent sensitivity and specificity, but typically require multiple reagents and extensive sample preparation, limiting their usefulness as rapid diagnostics. A generalizable biosensor platform is introduced that enables single‐step, sample preparation‐free detection of protein analytes with high sensitivity in complex samples. The NanoFluor system employs Janelia Fluor dyes coupled to a nanobody via HaloTag conjugation with a flexible glycine‐serine linker, where the dye undergoes a switch from a non‐fluorescent to a fluorescent state when the coupled nanobody binds to its target. It is demonstrated that the NanoFluor design achieves detection limits as low as picomolar concentrations across diverse protein targets. Molecular dynamics simulations, coupled with quantum mechanics/molecular mechanics computational models, reveal the mechanistic basis for the fluorescence change, and demonstrate the feasibility of multiplexed detection in complex samples including undiluted serum. This versatile, simple biosensor design can prove valuable for point‐of‐care diagnostics and other molecular detection applications.