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En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

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Published on: February 25, 2016

Real-Time Detection of Reduced Nitroreductase with a Reversible Fluorescent Probe.

Sourav Sarkar¹, Anushree Shil¹, Yong Woong Jun²

¹Department of Chemistry, Pohang University of Science and Technology (POSTECH), Gyeongbuk, 37673, Republic of Korea.

Advanced Science (Weinheim, Baden-Wurttemberg, Germany)

|August 26, 2025

Summary

Researchers developed a novel reversible probe to detect active nitroreductase (NTR) in real-time. This fluorescent tool monitors enzyme levels in hypoxic tumors and cells, offering dynamic insights into redox homeostasis.

Keywords:

bio‐imaging fluorescence probe nitroreductase reversible binding senescence tumor tissue

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Published on: February 16, 2022

Area of Science:

Biochemistry
Molecular Biology
Chemical Biology

Background:

Nitroreductase (NTR) is a key biomarker for hypoxic tumors.
Current fluorescent probes for NTR are reaction-based, providing only static enzyme activity data.
There is a need for reversible probes to monitor NTR levels dynamically.

Purpose of the Study:

To develop the first reversible binding probe for selective detection of active, reduced nitroreductase (red-NTR).
To enable real-time monitoring of NTR levels in biological systems.
To investigate dynamic biological processes involving NTR under redox homeostasis.

Main Methods:

A novel benzocoumarin dye probe functionalized with a (nitrobenzyl)pyridinium moiety was synthesized.
The probe utilizes hydrogen bonding with the reduced flavin mononucleotide (FMNH2) cofactor to stabilize red-NTR.
The probe exhibits a fluorescence turn-on response upon binding to red-NTR, distinguishing it from ox-NTR.

Main Results:

The probe selectively detects the active, reduced form of NTR (red-NTR) with a fluorescence turn-on response.
Hydrogen bonding interaction suppresses enzymatic reduction and photoinduced electron transfer quenching.
The probe successfully monitored active NTR levels in hypoxic cells, mouse tumor tissues, and cells undergoing premature senescence.

Conclusions:

The developed probe is the first reversible binding probe for selective detection of red-NTR.
This probe enables real-time observation of active NTR levels, overcoming limitations of static probes.
It serves as a valuable tool for studying dynamic biological processes related to NTR and redox homeostasis.