Non-fluorescent benzothiadiazole probe with protein-triggered turn-on fluorescence for imaging SARS-CoV-2 spike protein in biological systems.

Herein, we report a non-fluorescent 2,1,3-benzothiadiazole (BTD) derivative, BTD-PhCOOH, as a protein-triggered fluorogenic platform for SARS-CoV-2 spike protein detection. Direct coupling under mild refrigerated conditions afforded the BTD-Spike conjugate, producing an intense green fluorescence signal and demonstrating efficient fluorescence turn-on after protein ligation. Solvent-accessible surface area analysis (SASA) identified Lys529 as the most accessible lysine residue, providing a structural rationale for conjugate formation. In MCF-7 cells, BTD-Spike enabled time-dependent visualization of spike-associated cellular interactions, with membrane-associated fluorescence after 30 min and a more defined peripheral and intracellular punctate pattern after 60 min. In mice, systemic intravenous administration established the current sensitivity limits of visible-range whole-body and ex vivo organ fluorescence imaging, as treated animals could not be clearly distinguished from controls under these acquisition conditions. Importantly, higher-resolution confocal analysis of fixed brain tissue revealed localized fluorescence differences between treated and control samples. Additionally, direct intracerebroventricular administration enabled brain-level detection of both BTD-Spike and BTD-labeled amyloid-β, generating qualitatively distinct fluorescence patterns in the central nervous system. Overall, BTD-PhCOOH establishes a protein-activated fluorescence platform strongly supported by chemical, photophysical, computational, and cellular validation, while the brain-detection experiments highlight its potential for probing protein-associated signals in complex biological environments.