Inflammation is an innate protective response to pathological signs arising from tissue injury or pathogen-associated infection, which can promote the restoration of physiological homeostasis [1]. While excessive and sustained inflammatory state could induce persistent disorders of body metabolism and immune function, ultimately leading to acute or chronic inflammatory diseases (arthritis, atherosclerosis, cancers, etc.) [2], [3], [4], It is responsible that inflammation is almost associated with every major disease, making the timely diagnosis and management of inflammation considerable for delaying and preventing the progression of inflammatory diseases [5].

Peroxynitrite (ONOO−) is a typical type of the metabolic reactive nitrogen species (RNS), which can react with a wide array of bioactive species (proteins, lipids, and nucleic acids), owing to the innate property of strong oxidizability/nucleophilicity, and thus plays an important physiological function in living systems [6], [7]. Importantly, more and more evidence has demonstrated that the aberrant expression of ONOO− is implicated in various inflammation-related diseases [8], [9], [10]. Therefore, ONOO− can be served as an in-situ biomarker for inflammation, and the construction of reliable methods to monitor ONOO− fluctuation is very conducive to better diagnose and understand the pathological processes of inflammation.

Very recently, the prosperous area of optical imaging technology has received increasing attention in fluorescent probe which has become an indispensable tool for the research of various physiological/pathological processes and the progression of diseases, owing to its inherent advantages of simple operation, high spatial/temporal resolution, good sensitivity [11], [12], [13]. Various fluorescent probes composed of a fluorophore and one reaction site have been developed for ONOO− detection via employing various ONOO−-mediated oxidation mechanisms [14], [15], [16], [17], [18], [19], [20], [21]. Whereas most probes are still subject to limitations concerning the fact that the reaction moieties are also responsive to other competing reactive oxygen species (ROS) [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], and thus could lead to poor selectivity and false-positive result for ONOO− detection in the complex living systems. In consideration of the complicatedness of biological organisms that express plenty of competing ROS which may interfere the response performance of the ONOO−-activatable probes equipped with one recognition group, we envisioned that the constructed probe consists of two reaction sites of ONOO− could solve the above-mentioned issue by greatly promoting the specificity and sensitivity in complicated living organisms. Therefore, it is worth developing a fluorescent probe that combines two ONOO− reaction triggers into a single molecule for the assessment of inflammation progression through sensitively and selectively monitoring pathological ONOO− activity variation.

Some traditional Chinese medicines (TCMs) and their active ingredients have been broadly used successfully for anti-inflammatory therapy on account of desired pharmacological activity, and still remained an invaluable source for the creation of new medications [32], [33], [34], [35]. Nevertheless, testing the anti-inflammatory efficacy and safety of the ingredients in disease models still presents challenges due to the time-consuming and costly process by using the complicated pharmacological methods [36]. Therefore, rapid detecting and assessing the therapeutical effectiveness of TCMs ingredients for inflammation treatment is still highly desirable but challenging target.

Taking these into concentration, in this work, a new ONOO− activatable probe SZP was rationally developed for timely diagnosing acute inflammation and evaluating the TCMs ingredients effectiveness via detecting in situ ONOO− activity for the first time. As shown in Scheme 1, The key point of the design strategy is to equip probe SZP with a fluorescence signal site that determine the detection intensity characteristic and two recognition sites (diphenyl phosphinate moiety, CC unsaturated bond) that could response ONOO− [21]. As a result, only the ONOO−-mediated cleavage reaction of the two reaction triggers in probe SZP could liberate the free fluorophore 6-hydroxy-2-naphthaldehyde, which possesses a bright fluorescent signal with a large stokes shift (150 nm), thus procuring the ONOO− detection with outstanding sensitivity (LOD = 8.03 nM) and high selectivity. The effectiveness of probe SZP was manifested by direct evaluation of ONOO− fluctuation in pathological inflammation modes. Probe SZP was also successfully applied to visually evaluate the anti-inflammatory effect of TCMs ingredients for the remediation of inflammation in cells and in vivo.