Cavity Filling-Based Enzyme Activatable Luminophores Leveraged In Vivo Bioimaging

Enzymes are essential biocatalysts in living organisms, with their dysregulation linked to various human and plant diseases. Recent advancements in enzyme detection and imaging have primarily focused on fluorescent sensors due to their superior sensitivity. However, achieving a balance between high sensitivity, specificity, and excellent catalytic efficiency remains a major challenge in the design of enzyme-activated fluorescent sensors. Herein, we propose a novel cavity filling-based design strategy (CFRD) to optimize enzyme-activated fluorescent sensors. Using nitroreductase as an example, we computationally designed and experimentally validated six fluorescent sensors, with HC-SF exhibiting an impressive catalytic efficiency (k_cat/K_m) of 435.54 μM^-1·min^-1, demonstrating a substantial improvement in catalytic performance. Fluorescence imaging of HepG2 cells and zebrafish confirmed the NTR detection capability of HC-SF in living organisms. Most importantly, it enabled real-time, noninvasive monitoring of environmental stresses in plants. This strategy holds great potential for the design of enzyme-activated fluorescent sensors and offers a promising pathway for bioimaging applications.