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Enzyme-Activated Dual-Locked Probes for Detecting Nitroreductase and Carboxylesterase
Xiaodan Chong1, Zimeng Luo2,3, Junhong Liu2,4
1Clinical Cancer Institute, Translational Medicine Center, Naval Military Medical University, Shanghai 200433, China.
Chemical & Biomedical Imaging
|March 27, 2026
Summary
Researchers developed a novel fluorescent probe, Flu-NitroCa-4, to simultaneously detect nitroreductase (NTR) and carboxylesterase (CarE) in lung cancer. This tool aids in rapid, sensitive diagnosis and intraoperative margin mapping.
Area of Science:
- Biochemistry
- Molecular Biology
- Medical Imaging
Background:
- Lung cancer is a leading cause of cancer mortality globally.
- Precise diagnostic tools targeting the tumor microenvironment are crucial.
- Hypoxia-driven nitroreductase (NTR) and carboxylesterase (CarE) are promising lung cancer biomarkers.
- Simultaneous detection of NTR and CarE using fluorescent probes is currently unavailable.
Purpose of the Study:
- To develop and characterize a novel dual-locked fluorescent probe for simultaneous detection of NTR and CarE.
- To evaluate the probe's performance in vitro and in vivo for lung cancer diagnosis.
- To assess the probe's utility in distinguishing cancerous from adjacent tissues.
Main Methods:
- Synthesis and optimization of fluorescein-based dual-locked probes.
- Characterization of probe activation by NTR and CarE, including fluorescence enhancement, response kinetics, and detection limits.
- In vitro validation using A549 cells and patient-derived lung cancer organoids under hypoxic conditions.
- In vivo evaluation in subcutaneous and orthotopic murine lung tumor models.
- Assessment of probe's ability to differentiate human lung cancer tissues from adjacent normal tissues.
Main Results:
- Flu-NitroCa-4 demonstrated a 48-fold fluorescence enhancement upon activation by both CarE and NTR.
- The probe exhibited rapid response kinetics (<30 min) and low detection limits (NTR: 0.18 μg/mL; CarE: 0.032 U/mL).
- In vitro studies accurately visualized CarE and NTR activity in lung cancer cells and organoids under hypoxia.
- In vivo studies successfully delineated tumors in murine models.
- The probe effectively distinguished lung cancer samples from adjacent tissues based on NTR/CarE activity.
Conclusions:
- Flu-NitroCa-4 is an optimal dual-locked fluorescent probe for simultaneous NTR and CarE detection.
- The probe offers a rapid, sensitive, and low-radiation tool for lung cancer diagnosis.
- Flu-NitroCa-4 shows significant potential for intraoperative tumor margin mapping and clinical diagnosis.
