Affinity-Activated Protein Switches for Modular and Signal-ON Detection of Small Molecules

Protein switches offer powerful strategies for detecting small molecules, yet current designs often suffer from limited dynamic range, lack of reversible control, and poor adaptability to nonfluorescent platforms. Here, we report a semisynthetic affinity-activated protein switch strategy that enables Signal-ON detection of small molecules across diverse environments, including live cells, Drosophila brain, and lateral flow assays. The switch consists of a self-labeling protein fused to a sensing protein and a synthetic probe containing a small-molecule ligand, a sterically shielded N'-3 urea nitrogen biotin derivative (N3B), and a tagging moiety. Ligand binding induces a conformational change that exposes the N3B for streptavidin-based signal output. This design achieves high dynamic range (up to 142-fold), low background, and nanomolar sensitivity for detecting sulfonamide, while demonstrating the transferability of the platform to trimethoprim detection. Reversible switching is enabled by the reduced N3B-streptavidin affinity, allowing signal reset via competitive displacement. In Drosophila brain, the protein switch enables spatially resolved ex vivo imaging of drug exposure. Furthermore, conjugation to gold nanoparticles allows robust Signal-ON colorimetric detection on lateral flow test strips. This work establishes a generalizable framework for building programmable protein switches with tunable output modes and potential diagnostic applicability.