Affinity‐Switchable Fluorescent Probes for Lateral Flow Detection of Small Reactive Molecules and Enzymatic Activities

ABSTRACT Lateral flow assays (LFAs) are powerful diagnostic tools for point‐of‐care testing, but their applications are largely constrained by reliance on antibody‐based capture and nanoparticle signal reporters. Here, we present an affinity‐switchable caged‐biotin fluorescent probe strategy (ASBF‐LFA) that expands the detectable target space to include small reactive molecules and enzymatic activities. Unlike conventional LFAs that depend on nanomaterials, this approach employs structurally defined small‐molecule probes. These probes integrate a fluorophore with a caged biotin moiety that remains inactive until triggered by a specific analyte, thereby inducing affinity switching to activate biotin–avidin binding and generate a localized signal at the test line. The modular platform was demonstrated using probes responsive to hydrogen peroxide, alkaline phosphatase, and esterase, achieving high sensitivity and selectivity in complex biological matrices. In addition, coupling the H 2 O 2 ‐responsive probe with glucose oxidase enabled indirect detection of glucose. Compared to solution‐based probes and nanomaterial‐enhanced LFAs, ASBF‐LFA offers improved sensitivity through spatial signal confinement, reduced background, and low probe consumption. This chemistry‐driven strategy establishes a generalizable LFA framework for targeting functional biomarkers beyond traditional antibody recognition.