Nitric oxide (NO) is an important biologically active gas that is synthesized in the human body through the catalytic action of nitric oxide synthase using l-arginine as the starting material [1]. It is considered an essential high-activity molecule in living organisms, and various diseases such as tumors and immune system disorders are closely associated with NO dysregulation [2]. This is because excessive NO can react with molecular oxygen or O2 to produce highly oxidative compounds such as N2O3 or peroxynitrite (ONOO−) in the biological system [3], leading to serious cell damage and thus easily inducing the development of tumors, inflammation, and other diseases [4]. In addition to its oxidizing abilities, NO is also a type of active molecule that plays an important role in physiological signaling processes such as DNA, lipids, and enzymes functions[5]. Specifically, some research works have shown that patients with periodontitis have greater levels of NO amounts in their saliva than healthy people, proving that NO is strongly linked to the destruction and inflammation of oral tissues and the development of periodontal disease [6]. Therefore, the advance of reliable methods for precisely detecting NO fluctuation in vivo and in vitro is quite crucial, which will also help us better recognize the pathogenesis of NO-related diseases and enable scientists to design therapeutic interventions that target NO dysregulation [7].

Although the basic functions of NO in biology have been investigated and established [8], there are plenty of unknown effects remaining [9], due to the hindrance of current available approaches to detect and image NO in biological systems and samples [10]. In recent years, multiple methods have been reported to determine NO concentration [11], such as electrochemistry, mass spectrometry, atomic absorption/emission spectroscopy, etc [12]. However, there are still some challenges remaining to directly evaluate and image NO in vitro and in vivo because of its short lifetime and high reactivity [13]. In contrast, fluorescent probes have emerged to be an important tool for studying biological reaction-based molecules due to their fast response and non-invasive detection capabilities [14]. However, most fluorescent probes for NO sensing suffer from long responsive time(>60 min) [15], relatively low sensitivity(micro-molar level) [16], and poor selectivity [17], limiting their application in biological fluorescence imaging and in vitro detection area [18], hampering the high-sensitive monitoring of NO during various physiological process related to NO generation in biosystem [19].

To overcome these issues, this article proposed and described a single-molecule “turn-on” fluorescent probe ZPS-NO, composed of coumarin and o-phenylenediamine through a double bond connection (Scheme 1). The hypothetical characteristics of this probe are: 1. The double bond extends the π orbit of the fluorophore scaffold, resulting in an effective red shift compared to coumarin. 2. The strong electron-donating property of o-phenylenediamine causes the molecule to undergo the PET effect (photoinduced electron transfer), quenching the fluorescence. When incubating with NO, the PET effect of ZPS-NO is inhibited, thus causing a powerful fluorescence “turn-on” effect. 3. The probe ought to have a low detection limit due to the effective sensing mechanism based on the fast chemical formation of the five-member ring on the structure and can efficiently detect the NO content in live cells and zebrafish models. 4. The fluorescent probe was able to effectively differentiate nitric oxide variances between saliva samples from periodontitis patients and healthy persons. All the studies below indicate that ZPS-NO is a powerful tool for clinical-based NO imaging and diagnosis purposes. To the best of our knowledge, it is the first time that the coumarin-based fluorescent probe has been introduced to the clinical samples detection and periodontitis diagnosis field.