Synergistic Dual-Enzyme Activation Boosts In Situ D-Luciferin Generation for High-Contrast Early Tumor...

Activity-based sensing (ABS) bioluminescence (BL) probes hold great potential for tumor imaging. However, current ABS BL probes, primarily based on "caged luciferin" or "in situ luciferin generation" strategies, suffer from intrinsic background BL signals or suboptimal D-luciferin (D-LH₂) generation efficiency, leading to low imaging contrast in early-stage tumors with low biomarker expression. Herein, we report CNET, a rationally designed ABS BL probe and D-LH₂ precursor for high-contrast BL imaging of early-stage tumors through synergistic dual-enzyme activation. CNET enhances tumor imaging contrast through three key features: (1) eliminating background BL; (2) codelivering D-cysteine (D-Cys) and 6-hydroxy-2-cyano-benzothiazole (HCBT) within a single molecule, enabling synchronized intracellular release of both substrates for efficient D-LH₂ generation in tumor cells; and (3) synergistic dual-enzyme activation by nitroreductase (NTR) and esterase, which boosts in situ D-LH₂ generation and amplifies the BL signal in tumors. In vitro results showed that CNET is highly sensitive and selective for both NTR and esterase, with detection limits of 0.11 μg/mL for NTR and 4.732× 10^-5 U/mL for esterase. Notably, due to the synergistic activation effect of NTR and esterase on CNET, CNET enabled high-contrast bioluminescence imaging of low-density (2.5 × 10³) 4T1-Luc cells and early-stage 4T1-Luc tumors (approximately 20 mm³), with signal enhancements of 285.8-fold and 9.5-fold, respectively. We anticipate that CNET could serve as a promising tool for facilitating early tumor diagnosis in clinical practice in the near future.