Development of a Near-Infrared Fluorescent Probe Based on Nile Red for ONOO– and Its Imaging Applications

doi:10.6023/A23050225

Abstract

The production of peroxynitrite (ONOO^–) in situations of oxidative stress and inflammation has a profound impact on cellular damage, inflammatory responses, and immune regulation. Thus, the advancement of highly sensitive and selective techniques to detect ONOO^– is of great significance. These methods are crucial for gaining insights into disease mechanisms, improving early disease diagnosis, and enhancing treatment effectiveness. In this study, we have designed and synthesized a novel fluorescent probe, NR-Pro, specifically for the selective detection of ONOO^–. The probe incorporates a 2-hydroxy Nile red derivative (NR-OH) that is organically bound to a 4,4'-azabediyldiphenol group via an alkyl chain. Upon interaction with ONOO^–, the probe releases the near-infrared dye NR-OH, leading to a significant “OFF-ON” fluorescence signal response at 658 nm. The mechanism involves the initial attack of ONOO^– on the 4,4'-aza-diyl diphenol group, resulting in the formation of an alkylamine-modified Nile red derivative and the release of two 1,4-benzoquinone molecules. Subsequently, the alkylamine group undergoes further oxidation by ONOO^–, leading to the liberation of the near-infrared fluorophore NR-OH and acrolein through a 1,3-elimination reaction with the involvement of water. Experimental investigations have demonstrated that the probe exhibits a favorable linear relationship with ONOO^– concentration in the range of 0 to 10 μmol/L, with an exceptionally low detection limit of 17.7 nmol/L. Furthermore, the fluorescence emission of NR-Pro remained unaffected by various biological species, including reactive oxygen species (ROSs), metal ions, enzymes, anions, and amino acids. NR-Pro also exhibited minimal cytotoxicity and very high photostability, suggesting its potential suitability for cell imaging applications. Moreover, our study confirms the remarkable imaging capability of the NR-Pro probe in detecting both exogenous and endogenous ONOO^– in RAW264.7 cells. These findings present novel insights for the early diagnosis and treatment of diseases, paving the way for potential advancements in the field.

Key words: near-infrared, fluorescent probe, peroxynitrite, Nile red, cell imaging

Cite this article

Xiaomeng He, Fang Yuan, Suya Zhang, Jianjian Zhang. Development of a Near-Infrared Fluorescent Probe Based on Nile Red for ONOO^– and Its Imaging Applications[J]. Acta Chimica Sinica, 2023, 81(11): 1515-1521.

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Fig. & Tab. 8

Figure 1. Fluorescence spectra of NR-Pro (5 μmol/L) and ONOO– (0~50 μmol/L) in PBS (pH 7.4, 20 mmol/L, 20% MeCN) for 30 min (a); the embedded graph (b) shows the relationship between fluorescence intensity and ONOO– concentration of the reaction system at 658 nm

Figure 2. Fluorescence kinetic curves (a) and photostability curves (b) of NR-Pro (5 μmol/L) and ONOO– (20 μmol/L) in PBS (pH 7.4, 20 mmol/L, 20% MeCN)

Figure 3. Effect of pH on fluorescence intensity of NR-Pro (5 μmol/L) and ONOO– (20 μmol/L) reaction systems

Figure 4. Fluorescence intensity of NR-Pro (5 μmol/L) and different analytes (50 μmol/L) after 30 min reaction in PBS (pH 7.4, 20 mmol/L, 20% MeCN). Among them, analytes 1~25: Blank, ONOO–, H2O2, $\mathop{{O}}_{2}^{{\mathop{}^{-·}}}$, ClO–, TBO?, TBHP, NO, $NO_{2}^{-}$, Na2S, GSH, Lys, Cys, Na+, K+, Mg2+, Fe3+, Ca2+, Fe2+, Zn2+, ?OH, 1O2, β-galactosidase, trypsin, L-ascorbic acid Fluorescence intensity of NR-Pro (5 μmol/L) and different analytes (50 μmol/L) after 30 min reaction in PBS (pH 7.4, 20 mmol/L, 20% MeCN). Among them, analytes 1~25: Blank, ONOO–, H2O2, $\mathop{{O}}_{2}^{{\mathop{}^{-·}}}$, ClO–, TBO?, TBHP, NO, $NO_{2}^{-}$, Na2S, GSH, Lys, Cys, Na+, K+, Mg2+, Fe3+, Ca2+, Fe2+, Zn2+, ?OH, 1O2, β-galactosidase, trypsin, L-ascorbic acid

Figure 5. Possible mechanism of ONOO– detection by probe NR-Pro

Figure 6. High performance liquid chromatogram of the reaction of probe NR-Pro with ONOO–. (I) NR-Pro; (II, III) NR-Pro (5 μmol/L) and ONOO– (25 and 50 μmol/L) in PBS (pH 7.4, 20 mmol/L, 20% MeCN); (IV) NR-OH. Mobile phase: MeCN/H2O (70/30, V/V), flow rate (1 mL/min) High performance liquid chromatogram of the reaction of probe NR-Pro with ONOO–. (I) NR-Pro; (II, III) NR-Pro (5 μmol/L) and ONOO– (25 and 50 μmol/L) in PBS (pH 7.4, 20 mmol/L, 20% MeCN); (IV) NR-OH. Mobile phase: MeCN/H2O (70/30, V/V), flow rate (1 mL/min)

Figure 7. Fluorescence imaging of exogenous ONOO– in RAW264.7 cells. NR-Pro (10 μmol/L) was incubated with cells for 30 min, washed with PBS and treated differently: (a) control group; (b) add 20 μmol/L ONOO– and incubate for 30 min; (c) add 50 μmol/L ONOO– and incubate for 30 min; (d) average fluorescence intensity of cells in images a~c. Scale bar: 20 μm

Figure 8. Fluorescence imaging of endogenous ONOO– in RAW264.7 cells. (a) Incubate the cells with NR-Pro (10 μmol/L) for 30 min; (b) cells were first stimulated with 1 μg/mL LPS for 4 h, then 1 μg/mL PMA for 30 min, and finally incubated with NR-Pro (10 μmol/L) for 30 min; (c) after 100 μmol/L UA pretreated cells for 1 h, incubate with LPS (1 μg/mL) for 4 h, PMA (1 μg/mL) for 30 min, and NR-Pro (10 μmol/L) for 30 min; (d) average fluorescence intensity of cells in images a~c. Scale bar: 20 μm

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基于尼罗红类ONOO^–近红外荧光探针的开发及其成像应用