博文
Fluorescent sensor visualizes energy metabolism pathways
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Volume 37, Issue 5, May 2026, 111334
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aState Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Collaborative Innovation Center of Advanced Microstructures, Nanjing University, Nanjing 210023, China
bCollege of Pharmacy, Jiangsu Joint International Laboratory of Animal-Derived Chinese Medicine and Functional Peptides, Nanjing University of Chinese Medicine, Nanjing 210023, China
cKey Laboratory of Haikou Trauma, Key Laboratory of Hainan Trauma and Disaster Rescue, Key Laboratory of Emergency and Trauma, Ministry of Education, The First Affiliated Hospital of Hainan Medical University, Hainan Medical University, Haikou 571199, China
Received 8 March 2025, Revised 13 May 2025, Accepted 15 May 2025, Available online 16 May 2025, Version of Record 18 February 2026.
https://doi.org/10.1016/j.cclet.2025.111334
AbstractThe rapid proliferation of tumor cells is driven by metabolic reprogramming and redox regulation. Real-time monitoring of glutathione (GSH)/adenosine-5′-triphosphate (ATP) provides a dynamic perspective for tumor metabolism and is crucial for guiding precision treatment. We report a dual-site activatable fluorescent probe M901 for simultaneously detecting GSH and ATP without spectral overlap, and the detection range (GSH: 0–7 mmol/L, ATP: 0–6.5 mmol/L) matching the physiological concentration range. Based on this, M901 visualizes a bidirectional regulatory relationship between ATP synthesis↓ (energy imbalance) ↔ electron transport chain dysfunction ↔ reactive oxygen species (ROS)↑ ↔ GSH↓ (oxidative stress). Additionally, M901 reveals for the first time the dynamic compensatory mechanism between GSH and ATP in cellular oxidative stress induced by the inhibition of solute carrier family 7 member 11 (SLC7A11) or glutathione peroxidase 4 (GPX4). In vivo imaging further confirms oxidative stress and mitochondrial dysfunction are core pathological mechanisms leading to liver injury, with treatment efficacy positively correlated with GSH/ATP levels. Importantly, the dynamic visualization of GSH/ATP by M901 enables real-time evaluation of the anti-tumor effects of ferroptosis inducers and cisplatin, guiding successful precision resection of invasive malignant tumors (negative margins <0.2 mm). This study confirms the potential of M901 as a clinical visualization tool for diagnosing, treating and monitoring a variety of diseases.
Graphical abstractThis study proposes a multifunctional visualization tool named M901, which integrates liver injury diagnosis, anti-tumor therapeutic efficacy evaluation, and guidance for precise surgical resection
Dual activation
Fluorescence imaging
Ferroptosis
Anti-tumor evaluation
Surgical navigation
Malignant tumors, characterized by uncontrolled cell proliferation and invasion, posing a significant threat to human survival [[1], [2], [3]]. Chemotherapy and surgical resection remain the primary treatment modalities for solid tumors [[4], [5], [6]]. Accurate monitoring of the tumor's status is crucial for assessing treatment efficacy and guiding optimization of treatment plans to improve survival rates [7]. In recent years, clinical imaging methods including X-rays, ultrasound (US), magnetic resonance imaging (MRI), and positron emission tomography-computed tomography (PET-CT) have made significant advancements in tumor localization [[8], [9], [10]]. However, these methods cannot provide real-time monitoring tumor progression or accurately distinguish tumor boundaries. Puncture biopsy is widely recognized as the clinical diagnostic "gold standard", but it carries a potential risk of tumor metastasis [[11], [12], [13]]. Therefore, there is an urgent need to develop a non-invasive method based on specific biomarkers for real-time monitoring of tumor progression.
Cancer cells sustain rapid proliferation through metabolic reprogramming, while their abnormally active biosynthetic demands significantly enhance their reliance on ATP and antioxidant systems [[14], [15], [16]]. Mitochondria, as the main site of ATP synthesis, generate substantial byproducts reactive oxygen species (ROS) during the process of energy production through oxidative phosphorylation [[17], [18], [19]]. Cancer cells maintain redox homeostasis to counteract the cytotoxicity of ROS by synthesizing glutathione (GSH) through solute carrier family 7 member 11 (SLC7A11)-mediated cystine (Cys) uptake, and utilizing glutathione peroxidase 4 (GPX4) to eliminate lipid peroxides [[20], [21], [22]]. Tumor cells effectively avoid programmed cell death pathways such as ROS induced ferroptosis while maintaining high proliferation activity [[23], [24], [25]]. Therefore, GSH and ATP could serve as potential biomarkers for detecting and evaluating the development status of tumors. Based on this, targeting the tumor antioxidant defense system (such as inhibiting SLC7A11 or GPX4) could disrupt the redox homeostasis, accurately monitor the specific compensation pathways of GSH and ATP, not only for studying the specific regulatory relationship between oxidative damage and energy blockade, but also provide new ideas for overcoming tumor drug resistance. Small molecule fluorescent probes have attracted wide-spread attention due to their advantages of excellent permeability, biocompatibility, non-invasiveness and real-time tracking of endogenous species [[26], [27], [28]]. The activatable sensor with multi-recognition sites eliminates false signals through specific multi biomarker activation and spatiotemporal consistency characteristics, enhancing detection accuracy and sensitivity, which is suitable for dynamic analysis of pathological mechanisms in complex diseases [[29], [30], [31]]. Currently, the development of several probes capable of simultaneously detecting ATP and reactive sulfur species (RSS) for synchronized imaging has provided critical tools for studying related pathological processes (Table S1 in Supporting information). These probes exhibit higher sensitivity and lower detection limits, but their detectable ranges are mostly confined to the micromolar range (µmol/L) [[32], [33], [34], [35], [36], [37]]. The reported literatures indicate that the concentration of GSH/ATP in tumor cells are in mmol/L range [[38], [39], [40]]. Therefore, achieving mmol-level visualization imaging of ATP/GSH in biological systems is crucial for disease diagnosis and evaluation of therapeutic efficacy.
To meet the above requirements, the sulfonamide group was introduced into 1,8-naphthalimide and incorporated into the modified rhodamine lactam skeleton to synthesize a dual-sites probe M901 for independent detection of GSH and ATP without spectral crosstalk (peak separation is 155 nm) (Scheme 1). Upon activation by GSH and ATP, M901 exhibited of 15- and 700-fold fluorescence responses, respectively, the response range of M901 for GSH (0–7 mmol/L) and ATP (0–6.5 mmol/L) matching the physiological concentration fluctuations [38,39]. Based on this, the bidirectional regulatory relationship between energy imbalance induced by carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and oxidative stress induced by l-buthionine sulfoximine (BSO) in tumor cells was visualized by M901. ATP synthesis inhibition↓ (energy imbalance) ↔ dysfunction of the electron transport chain ↔ ROS↑ ↔ depletion of GSH↓ (oxidative stress). Targeting the tumor antioxidant defense system (such as inhibiting SLC7A11 or GPX4) to disrupt redox homeostasis, the differential compensation mechanism of GSH/ATP was revealed for the first time. In addition, M901 revealed GSH depletion and ATP reduction are direct results of liver injury, and the therapeutic effect is positively correlated with the content of GSH/ATP. More importantly, M901 directly correlates metabolic regulation mechanisms with therapeutic effects by dynamically visualizing GSH/ATP in tumors. It could predict and evaluate the anti-tumor effects of different ferroptosis agents and cisplatin combination treatments, and the results showed that the therapeutic effect was positively correlated with the decrease in GSH/ATP concentration. Through dual-channel imaging, M901 guided precise resection of invasive malignant tumors (negative margin 0.2 mm). The M901 provides a multifunctional visualization tool for high-accuracy disease diagnosis, treatment efficacy evaluation, and precise surgical resection.
Designing GSH/ATP dual-responsive fluorescent probes must adhere to the following core principles: Spectrally resolvable dual-emissive fluorophores; non-emissive or weakly emissive in the absence of biomarkers; precise mitochondrial targeting capability. Rhodamine and 1,8-naphthalimide were selected as fluorophores. N,N-Dimethylbenzene was incorporated into the rhodamine skeleton to extend emission to the near-infrared range and avoid spectral crosstalk. A sulfonamide group was integrated into the 1,8-naphthalimide skeleton and combined with cyclorhodamine via diethylene triamine to construct probe M901 for GSH/ATP detection. The sulfonamide group suppresses the intramolecular charge transfer (ICT) of 1,8-naphthalimide, while spirocyclization disrupts the rhodamine π-conjugated region, placing M901 in a fluorescence-off state for both GSH and ATP channels. GSH cleaves the sulfonamide bond to generate a thiol substitute, restoring electron transfer in 1,8-naphthalimide and activating green fluorescence at 495 nm. ATP triggers a reversible non-conjugated-to-conjugated ring-opening transformation in the rhodamine derivative via synergistic hydrogen bonding and π-π stacking, activating near-infrared fluorescence at 650 nm. The two-channel fluorescence emission peaks differ by ∼155 nm, eliminating spectral crosstalk. M901 was synthesized according to the synthetic route shown in Scheme S1 (Supporting information). M901 and its corresponding intermediates were well characterized by high-resolution mass spectrometry (electron spray ionization) (HRMS (ESI)) and 1H, 13C nuclear magnetic resonance spectra (NMR) (Figs. S31–S35 in Supporting information).
We systematically investigated the dual-channel optical response characteristics of M901 to GSH and ATP. In a phosphate buffered saline (PBS) containing 10 % ethanol, the absorption spectra and concentration-dependent fluorescence spectra of M901 were measured by adding varying concentrations of GSH. As shown in Fig. 1A, a new absorption peak emerged at 398 nm. Simultaneously, under 450 nm excitation, the fluorescence intensity at 495 nm gradually increased with increasing GSH concentration (0–7 mmol/L), achieving a 15-fold fluorescence enhancement at 495 nm, which was attributed to the formation of nucleophilic substitution products mediated by GSH (Fig. 1B). The green fluorescence intensity at 495 nm exhibited a good linear relationship with GSH concentration (0.5–6.0 mmol/L), and the detection limit for GSH was calculated as 0.2 mmol/L (Fig. 1C). Subsequently, we evaluated the specificity of M901 for GSH recognition. As shown in Fig. S1A (Supporting information), in the presence of metal ions, ROS/RNS, or energy metabolism-related molecules alone, the fluorescence signal at 495 nm exhibited negligible enhancement. Notably, significant fluorescence enhancement effect was observed only when GSH was added, and this activation effect remained highly specific even after adding 1 mmol/L Cys (Fig. S2 in Supporting information).
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