高灵敏线粒体靶向近红外二氧化硫荧光探针的开发及细胞、小鼠成像研究

doi:10.6023/cjoc202012049

有机化学 ›› 2021, Vol. 41 ›› Issue (3): 1108-1116.DOI: 10.6023/cjoc202012049 上一篇下一篇

研究论文

高灵敏线粒体靶向近红外二氧化硫荧光探针的开发及细胞、小鼠成像研究

李芳¹, 唐永和¹, 郭锐¹, 林伟英¹^,^*()

1 广西电化学能源材料重点实验室广西大学光功能材料与化学生物学研究院广西大学化学化工学院有色金属及材料加工新技术教育部重点实验室广西有色金属及特色材料加工重点实验室南宁 530004

收稿日期:2020-12-29 修回日期:2021-02-14 发布日期:2021-02-26
通讯作者: 林伟英
基金资助:
国家自然科学基金(21672083); 国家自然科学基金(21877048); 国家自然科学基金(22077048); 广西大学启动基金(A3040051003); 广西自然科学基金(2019GXNSFBA245068)

Development of an Ultrasensitive Mitochondria-Targeted Near Infrared Fluorescent Probe for SO₂ and Its Imaging in Living Cells and Mice

Fang Li¹, Yonghe Tang¹, Rui Guo¹, Weiying Lin¹^,^*()

1 Guangxi Key Laboratory of Electrochemical Energy Materials, Institute of Optical Materials and Chemical Biology, School of Chemistry and Chemical Engineering, MOE Key Laboratory of New Processing Technology for Nonferrous Metals and Materials, Guangxi Key Laboratory of Processing for Non-ferrous Metals and Featured Materials, Guangxi University, Nanning 530004

Received:2020-12-29 Revised:2021-02-14 Published:2021-02-26
Contact: Weiying Lin
About author:
* Corresponding author. E-mail: weiyinglin2013@163.com
Supported by:
Project supported by the NSFC(21672083); Project supported by the NSFC(21877048); Project supported by the NSFC(22077048); startup fund of Guangxi University(A3040051003); Guangxi Natural Science Foundation(2019GXNSFBA245068)

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摘要/Abstract

二氧化硫(SO₂)及其衍生物(亚硫酸盐、亚硫酸氢盐等)作为活性硫物种的重要组成部分, 与生命体的生理过程有着密切的关系. 此外, 摄入过量的SO₂会对呼吸系统造成不可修复的损伤, 例如呼吸系统损伤、心脑血管和神经疾病等. 近红外发射荧光探针具有较强的组织穿透能力, 可以有效地检测活体内的生物分子. 因此, 设计了一种基于迈克尔加成(Michael Addition)机理的线粒体靶向近红外SO₂荧光探针XA-SO₂. 溶液光谱实验表明, 该探针XA-SO₂对SO₂有着显著的选择性和灵敏度, 并且具有快速检测SO₂的能力. 值得注意的是, 该荧光探针XA-SO₂具有较好的细胞膜渗透能力, 成功检测到了细胞外/内源性SO₂. 更重要的是, 该探针XA-SO₂能够有效地在细胞的线粒体上富集, 有望实现细胞线粒体内SO₂的动态检测.

关键词: 二氧化硫, 荧光探针, 生物成像, 迈克尔加成, 线粒体

Sulfur dioxide (SO₂) and its derivatives (sulfites, bisulfites, etc.), as important components of active sulfur species, are closely related to the physiological processes of living organisms. In addition, excessive intake of SO₂ may cause irreparable damage to the respiratory system, such as lung cancer, tracheitis, asthma, etc. Near-infrared emission fluorescent probes have strong penetrating ability and can detect biomolecules in vivoeffectively. Therefore, in this paper, a mitochondrial targeted near-infrared SO₂ fluorescent probe based on the Michael addition mechanism was designed. The solution spectroscopic experiments show that XA-SO₂ has significant selectivity and sensitivity to SO₂, and has the ability to rapidly detect SO₂. It is worth noting that XA-SO₂ has good membrane permeability and has successfully detected extracellular/endogenous levels. More importantly, XA-SO₂ could be enriched effectively on the mitochondria of cells, which is promising for realizing the dynamic detection of SO₂ in the mitochondria of living cells.

Key words: sulfur dioxide, fluorescent probe, bioimaging, Michael addition, mitochondria

引用此文

李芳, 唐永和, 郭锐, 林伟英. 高灵敏线粒体靶向近红外二氧化硫荧光探针的开发及细胞、小鼠成像研究[J]. 有机化学, 2021, 41(3): 1108-1116.

Fang Li, Yonghe Tang, Rui Guo, Weiying Lin. Development of an Ultrasensitive Mitochondria-Targeted Near Infrared Fluorescent Probe for SO₂ and Its Imaging in Living Cells and Mice[J]. Chinese Journal of Organic Chemistry, 2021, 41(3): 1108-1116.

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图/表 11

图式1. 探针XA-SO2对SO2的识别机制

Scheme 1. Proposed response mechanism of probe XA-SO2 to SO2

图式2. 探针XA-SO2的合成路线

Scheme 2. Synthetic route of probe XA-SO2

图1. 探针XA-SO2 (5×10–6 mol/L)与不同浓度的NaHSO3 (0~8×10–4 mol/L)反应的紫外可见吸收曲线

Figure 1. UV-Vis absorption curves of probe XA-SO2 (5×10–6 mol/L) reacted with different concentrations of NaHSO3 (0~8×10–4 mol/L) UV-Vis absorption curves of XA-SO2 (5×10–6 mol/L) reacted with different concentrations of NaHSO3 (0~8×10–4 mol/L) in 1×10–2 mol/L PBS buffer (pH 7.4, 0.5% DMSO) for 10 min

图2. 探针XA-SO2 (5×10–6 mol/L)与不同浓度的NaHSO3 (0~8×10–4 mol/L)反应的荧光滴定曲线

Figure 2. Fluorescent curves of probe XA-SO2 (5×10–6 mol/L) reacted with different concentrations of NaHSO3 (0~8×10–4 mol/L) Fluorescent curves of XA-SO2 (5×10–6 mol/L) reacted with different concentrations of NaHSO3 (1×10–6~1.5×10–5 mol/L) in 1×10–2 mol/L PBS buffer (pH 7.4, 0.5% DMSO) for 10 min. λ ex=670 nm

图3. 探针XA-SO2在755 nm 处荧光强度与不同浓度的NaHSO3 (1×10–6~1.5×10–5 mol/L)的线性关系

Figure 3. Linear relationship between the fluorescence intensity of the probe XA-SO2 and the concentration of NaHSO3 (1×10–6~1.5×10–5 mol/L) at 755 nm Linear relationship between the fluorescence intensity of the probe XA-SO2 (5×10–6 mol/L) and the concentration of NaHSO3 (1×10–6~1.5×10–5 mol/L) at 755 nm

图4. 探针XA-SO2与SO2的测试体系在755 nm处的荧光强度随时间变化曲线

Figure 4. Fluorescence intensity (755 nm) curve of probe XA-SO2 in the presence SO2 at different time (0~60 s). Time-dependent fluorescent intensities (F755 nm) of probe XA-SO2 (5×10–6 mol/L) in the presence SO2

图5. 探针5×10–6 mol/L XA-SO2与各种分析物的反应体系在755 nm处的荧光强度

Figure 5. Fluorescence intensity of the reaction system of probe 5×10–6 mol/L XA-SO2 with various analytes at 755 nm The numbers represent: (1) probe, (2) glucose, (3) DL-Hcy, (4) H2O2, (5) FA, (6) L-Cys, (7) L-Ala, (8) L-Arg, (9) Ascorbic Acid, (10) NaClO, (11) NaHS, (12) KI, (13) NaF, (14) Gly, (15) GSH, (16) Na2S, (17) NaSCN, (18) Na2SO4, (19) TBHP, (20) KCl, (21) MgCl2, (22) CaCl2, (23) SNP, (24) NaNO2, (25) Na2SO3, (26) NaHSO3. λ ex=670 nm

图6. 不同pH溶液中探针XA-SO2与SO2的反应体系在755 nm的荧光强度

Figure 6. Fluorescence intensity of probe XA-SO2 and SO2 in different pH solutions at 755 nm The fluorescence intensity changes of XA-SO2 (5×10–6 mol/L) treated or untreated with SO2 (1.2×10–5 mol/L) at different pH (6.0~10.0) solutions for 10 min

图7. 探针XA-SO2在HeLa细胞中检测外加NaHSO3的荧光图像

Figure 7. Fluorescent images of probe XA-SO2 in HeLa cells for detecting added NaHSO3 (A) The HeLa cells were incubated with 1×10–6 mol/L probe XA-SO2 for 5 min; (B) The HeLa cells were pre-cultured with 1×10–5 mol/L NaHSO3 for 5 min, and then cultured with 1×10–6 mol/L probe XA-SO2 for another 5 min; (C) The HeLa cells were pre-cultured with 1×10–4 mol/L NaHSO3 for 5 min, and then cultured with 1×10–6 mol/L probe XA-SO2 for another 5 min. λ ex=640 nm, λ em=650~750 nm, scale bar=30 μm

图8. 探针XA-SO2在HeLa细胞中检测内源SO2的荧光图像

Figure 8. Fluorescent images of probe XA-SO2 in HeLa cells for detecting endogenous SO2 (A) The HeLa cells were incubated with 1×10–6 mol/L probe XA-SO2 for 5 min; (B) The HeLa cells were pre-cultured with 2×10–5 mol/L Cys for 20 min, and then cultured with 1×10–6 mol/L probe XA-SO2 for another 5 min; (C) The HeLa cells were pre-cultured with 5×10–5mol/L Cys for 20 min, and then cultured with 1×10–6 mol/L probe XA-SO2 for another 5 min. λ ex=640 nm, λ em=650~750 nm, scale bar=30 μm

图9. 探针XA-SO2与商业定位染料(Mito Tracker Green)的共定位成像实验

Figure 9. Co-location imaging experiment of probe XA-SO2 and commercial dye (Mito Tracker Green) Co-location imaging experiments between probe XA-SO2 and Mito Tracker Green. The green channel: λ ex=488 nm, λ em=500~550 nm; The red channel: λ ex=640 nm, λ em=650~750 nm. Scale bar: 30 μm

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高灵敏线粒体靶向近红外二氧化硫荧光探针的开发及细胞、小鼠成像研究

Development of an Ultrasensitive Mitochondria-Targeted Near Infrared Fluorescent Probe for SO₂ and Its Imaging in Living Cells and Mice

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高灵敏线粒体靶向近红外二氧化硫荧光探针的开发及细胞、小鼠成像研究

Development of an Ultrasensitive Mitochondria-Targeted Near Infrared Fluorescent Probe for SO2 and Its Imaging in Living Cells and Mice

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Development of an Ultrasensitive Mitochondria-Targeted Near Infrared Fluorescent Probe for SO₂ and Its Imaging in Living Cells and Mice