An ɑ-ketoamide-based fluorescent probe via aggregation induced emission for ONOO− detection in vivo

Miao Yan a 1,Miaomiao Yu a 1,Zhenkai Wang b c,Fabiao Yu c,Weiwei Fang a

• aJiangsu Co-Innovation Center of Efficient Processing and Utilization of Forest Resources, International Innovation Center for Forest Chemicals and Materials, College of Chemical Engineering, Nanjing Forestry University, 159 Longpan Road, 210037, Nanjing, China

• bClinical Laboratory Department, Hainan General Hospital (Hainan Affiliated Hospital of Hainan Medical University), Hainan Medical University, Haikou, 570311, China

• cKey Laboratory of Hainan Trauma and Disaster Rescue, The First Affiliated Hospital of Hainan Medical University, Hainan Medical University, Haikou, 571199, China

Received 18 February 2025, Revised 28 March 2025, Accepted 31 March 2025, Available online 1 April 2025, Version of Record 6 April 2025.

https://doi.org/10.1016/j.talanta.2025.128064

Highlights

• •Novel turn-on probe merges α-ketoamide and AIE for selective ONOO− detection.

  •Real-time ONOO− tracking in cells and mice with high sensitivity and stability.

• •Reveals ONOO− fluctuation during inflammation progression via imaging.

• •Enables oxidative stress studies for early diagnosis and therapy development.

• Abstract

Peroxynitrite (ONOO−) plays a pivotal dual role in the inflammatory process. As a signaling molecule, it modulates the immune response, while its excessive production leads to oxidative stress and cellular damage, thereby exacerbating inflammation. Accurate detection of ONOO− dynamics in inflammatory process is crucial for elucidating its pathological roles and developing targeted therapeutic strategies. In this study, we report a novel α-ketoamide-based fluorescent probe, MeOTPE-NO2, enabling a highly selective detection of ONOO−. Upon reaction with ONOO−, the probe is converted into MeOTPE-NH2, featuring with aggregation-induced emission (AIE) property, which emits a strong fluorescence at 505 nm. This probe exhibits high specificity, sensitivity, and rapid fluorescence response, along with excellent pH stability across a broad range. Furthermore, MeOTPE-NO2 shows outstanding specificity for ONOO− over other reactive oxygen and nitrogen species, minimizing potential interference. Owing to its low cytotoxicity, MeOTPE-NO2 has been successfully applied for real-time imaging of ONOO− in live cells and in a mouse model of inflammation.

Graphical abstract

A Novel Fluorescent Probe for Real-Time Detection of Peroxynitrite (ONOO−) Dynamics in Inflammation.

Fig. 6Fig. 6. Fluorescent imaging of mice to monitor ONOO− during bacterial infection-induced arthritis caused by Staphylococcus aureus. Inactivated Staphylococcus aureus (1.0–1.4 × 107 CFU) was injected into the mouse knee joints, and after a) 0, b) 1, c) 2, d) 3, e) 5, and f) 7 days, MeOTPE-NO2 (10 μM) was injected. 450ch: λem = 450–550 nm, λex = 405 nm.

5. Conclusion

In this study, we developed MeOTPE-NO2, a “turn-on” fluorescent probe with an α-ketoamide unit for selective ONOO− detection. The α-ketoamide unit ensures specificity while regulating the AIE process. Upon reaction with ONOO−, strong fluorescence was induced by the in-situ generated MeOTPE-NH2. Cell experiments demonstrated the probe's high sensitivity and selectivity in the RAW 264.7 macrophage model. It remained stable across different pH values and time points, ensuring reliable performance in biological environments. In a mouse arthritis model, the probe successfully visualized ONOO− fluctuations, revealing an initial surge at inflammation onset, followed by a gradual decline.These results confirm that MeOTPE-NO2 is a powerful tool for real-time ONOO− imaging in vitro and in vivo. Its successful application provides a valuable platform for studying oxidative stress-related inflammation and holds potential for early disease diagnosis, therapeutic monitoring, and biomedical research.

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