A unimolecular near-infrared fluorescent probe for <i>in vivo</i> imaging of enzymes with minimized false-negative signals

Accurate imaging of specific enzymes is critical for the early diagnosis of ovarian cancer; however, conventional imaging agents often produce false-negative signals due to the heterogeneous expression of individual biomarkers, leading to delayed treatment and increased risk of disease progression. To address this limitation, we present Cy-GGT-β-gal, a unimolecular near-infrared (NIR) fluorescent probe engineered for the simultaneous detection of two key ovarian cancer biomarkers: γ-glutamyl transpeptidase (GGT) and β-galactosidase (β-gal). This dual-enzyme-responsive probe is designed to activate NIR fluorescence upon interaction with either GGT or β-gal through an intramolecular elimination mechanism, thereby significantly minimizing the likelihood of false-negative results. Compared to single-enzyme-targeting probes, Cy-GGT-β-gal demonstrates superior diagnostic reliability, enabling high-contrast discrimination (signal-to-noise ratio, SNR > 6.1) between multiple ovarian cancer cell lines (SHIN3, OVCAR3, OVCAR5, A2780, SKOV3) and normal cells. Furthermore, the probe successfully visualizes tumor lung metastases and nodular tissues with high specificity (SNR ∼ 11.2), underscoring its potential for precise <i>in vivo</i> imaging. This study not only introduces a robust unimolecular NIR fluorescent probe to mitigate false-negative diagnoses in ovarian cancer but also establishes a versatile design strategy for developing multi-enzyme-responsive imaging agents, offering a promising approach to enhance diagnostic accuracy in clinical settings.