基于硝基还原机理的一氧化碳荧光探针的开发及细胞成像研究

doi:10.6023/cjoc202009016

有机化学 ›› 2021, Vol. 41 ›› Issue (3): 1200-1206.DOI: 10.6023/cjoc202009016 上一篇下一篇

研究论文

基于硝基还原机理的一氧化碳荧光探针的开发及细胞成像研究

陈恩庆^a, 唐永和^a, 王蕾^b, 任江波^a, 林伟英^a^,^*()

a 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南宁 530004
b 南宁市食品药品检验所南宁 530007

收稿日期:2020-09-07 修回日期:2020-10-06 发布日期:2020-10-28
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); 广西省自然科学基金(2019GXNSFBA245068)

Development of a New Carbon Monoxide Fluorescent Probe Based on Nitro Reduction and Its Bioimaging Research in Living Cells

Enqing Chen^a, Yonghe Tang^a, Lei Wang^b, Jiangbo Ren^a, Weiying Lin^a^,^*()

a Guangxi Key Laboratory of Electrochemical Energy Materials, Institute of Optical Materials and Chemical Biology, School of Chemistry and Chemical Engineering, Guangxi University, Nanning 530004
b Nanning Institute for Food and Drug Control, Nanning 530007

Received:2020-09-07 Revised:2020-10-06 Published:2020-10-28
Contact: Weiying Lin
About author:
* Corresponding author. E-mail: weiyinglin2013@163.com
Supported by:
National Natural Science Foundation of China(21672083); National Natural Science Foundation of China(21877048); National Natural Science Foundation of China(22077048); Startup Fund of Guangxi University(A3040051003); Natural Science Foundation of Guangxi Province(2019GXNSFBA245068)

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摘要/Abstract

一氧化碳(CO)作为一种重要的细胞气体信号传递分子, 参与生物体内重要的生理和病理功能. 选用经典的荧光平台——萘酰亚胺荧光平台, 以硝基基团作为识别位点, 通过严谨的设计和两步合成的方法, 开发了一种新型用来检测一氧化碳的荧光增强型荧光探针NaLy-CO. 光谱测试数据表明, 该荧光探针对一氧化碳具有较好的选择性和灵敏度. 利用荧光共聚焦成像技术, 成功地对细胞外源性和内源性一氧化碳进行了检测. 此外, 共定位成像实验表明, 该荧光探针可以用于检测细胞溶酶体中的一氧化碳.

关键词: 一氧化碳, 荧光探针, 生物成像, 硝基, 萘酰亚胺

Carbon monoxide (CO), as an important cell signaling molecule, is involved in important physiological and pathological functions in organism. A new fluorescence enhanced CO fluorescent probe NaLy-CO was developed by using naphthalimide dye as fluorescence platform and nitro group as the recognition site. The results of spectrum test in solution showed that the fluorescent probe NaLy-CO had good selectivity and sensitivity to CO. The fluorescent probe NaLy-CO was successfully used to detect exogenous and endogenous CO in living HeLa cells by using confocal fluorescence imaging technology. In addition, the colocation imaging experiments showed that the fluorescent probe NaLy-CO could detect CO in the lysosome of living cells.

Key words: carbon monoxide, fluorescent probe, bioimaging, nitro, naphthalimide

引用此文

陈恩庆, 唐永和, 王蕾, 任江波, 林伟英. 基于硝基还原机理的一氧化碳荧光探针的开发及细胞成像研究[J]. 有机化学, 2021, 41(3): 1200-1206.

Enqing Chen, Yonghe Tang, Lei Wang, Jiangbo Ren, Weiying Lin. Development of a New Carbon Monoxide Fluorescent Probe Based on Nitro Reduction and Its Bioimaging Research in Living Cells[J]. Chinese Journal of Organic Chemistry, 2021, 41(3): 1200-1206.

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图/表 12

图式1. 探针NaLy-CO对CO的识别机制

Scheme 1. Sensing mechanism of probe NaLy-CO to CO

图式2. 探针NaLy-CO的合成路线

Scheme 2. Synthetic route of probe NaLy-CO

图1. 探针NaLy-CO (1×10–5 mol/L)与CORM-2 (0~5×10–4 mol/L)相互作用的荧光曲线

Figure 1. Fluorescent curves of probe NaLy-CO (1×10–5 mol/L) encountering with CORM-2 (0~5×10–4 mol/L) In 1×10–2 mol/L PBS buffer (pH 7.4, 10 vol% DMSO) for 40 min. λ ex=430 nm

图2. 探针NaLy-CO在523 nm处荧光强度与不同浓度的CORM-2 (0~1×10–5 mol/L)的线性关系

Figure 2. Linear relationship between the fluorescence intensity of the probe NaLy-CO and the concentration of CORM-2 (0~1×10–5 mol/L) at 523 nm

图3. 探针NaLy-CO与CORM-2相互作用的荧光强度随时间变化曲线

Figure 3. Time-dependent fluorescent of probe NaLy-CO intensities at F523 nm treated with CORM-2 ▼ 2.50×10–4 mol/L, ▲ 2×10–4 mol/L, ● 1×10–4 mol/L, ■ 5×10–5 mol/L. λ ex=430 nm

图4. 探针NaLy-CO (1×10–5 mol/L)的光稳定性能

Figure 4. Photo-stability profiles of probe NaLy-CO (1×10–5 mol/L) Photo-stability profiles of the probe NaLy-CO (1×10–5 mol/L) in the absence (■) or presence of UV-irradiated (●) (365 nm)

图5. 探针NaLy-CO与各种分析物相互作用后在523 nm处的荧光强度变化

Figure 5. Fluorescent intensity changes of the probe NaLy-CO interacts with various analytes at 523 nm The numbers represent: (1) probe (1×10–5 mol/L), (2) glucose (5×10–4 mol/L), (3) L-Cys (1×10–3 mol/L), (4) ascorbic acid (5×10–4 mol/L), (5) acetone (5×10–4 mol/L), (6) pyruvic acid (5×10–4 mol/L), (7) pyruvic acid sodium (5×10–4 mol/L), (8) NaClO (5×10–4 mol/L), (9) Gly (5×10–4 mol/L), (10) DL-Hcy (5×10–4 mol/L), (11) GSH (5×10–3 mol/L), (12) H2O2(5×10–4 mol/L), (13) FA (5×10–4 mol/L), (14) Na2S (5×10–4 mol/L), (15) NaHS (5×10–4 mol/L), (16) NaSCN (5×10–4 mol/L), (17) TBHP (5×10–4 mol/L), (18) SNP (5×10–4 mol/L), (19) Na2SO3(5×10–4 mol/L), (20) NaHSO3(5×10–4 mol/L), (21) NaNO2 (5×10–4 mol/L), (22) GO (5×10–4 mol/L), (23) CORM-2 (1×10–4 mol/L). λ ex=430 nm

图6. 探针NaLy-CO和CORM-2在不同pH溶液里发生反应后在523 nm的荧光强度

Figure 6. Fluorescence intensity of probe NaLy-CO and CORM-2 in different pH solutions at 523 nm Fluorescence intensity changes of the probe NaLy-CO (1×10–5 mol/L) at different pH values in the absence (●) or presence (■) of CO (1×10–4 mol/L) for 40 min

图7. 探针NaLy-CO在HeLa细胞中检测外源性CO的荧光图像

Figure 7. Fluorescent images of probe NaLy-CO in HeLa cells for detecting exogenous CO A~C: the HeLa cells were incubated with 1×10–5 mol/L probe NaLy-CO for 30 min; D~F: the HeLa cells were incubated with 1× 10–4 mol/L CORM-2 for 30 min; G~I: the HeLa cells were pre-cultured with 1×10–4 mol/L CORM-2 for 30 min, then cultured with 1×10–5 mol/L probe NaLy-CO for another 30 min. λ ex=405 nm, λ em= 500~550 nm, scale bar=50 μm.

图8. 探针NaLy-CO在HeLa细胞中检测内源性CO的荧光图像

Figure 8. Fluorescent images of probe NaLy-CO in HeLa cells for detecting endogenous CO A~C: the HeLa cells were incubated with 1×10–4 mol/L Heme for 2 h; D~F: the HeLa cells were pre-cultured with 1×10–4 mol/L Heme for 2 h, then cultured with 1×10–5 mol/L probe NaLy-CO for another 30 min; G~I: the HeLa cells were pre-cultured with 1×10–4 mol/L Heme for 4 h, then cultured with 1×10–5 mol/L probe NaLy-CO for another 30 min. λ ex=405 nm, λ em=500~550 nm. scale bar=50 μm

图9. 定位染料与外源性CO测试体系(探针NaLy-CO和CORM-2)的共定位成像实验

Figure 9. Co-location imaging experiment of localized dyes and exogenous CO test system (probe NALy-CO and CORM-2) Co-location imaging experiments between Lyso Tracker Deep Red and probe NALy-CO; (B) Co-location imaging experiments between Mito Tracker Deep Red and probe NALy-CO. The green channel: λ ex=405 nm, λ em=500~550 nm; The deep red channel: λ ex=647 nm, λ em=670~720 nm. Scale bar: 50 μm

	Lyso Tracker Deep Red	Mito Tracker Red
Pearson’s correlation	0.85	0.48

表1. 探针NALy-CO与商业定位染料的共定位系数

Table 1. Co-localization coefficient of probe NALy-CO and commercial localized dye

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基于硝基还原机理的一氧化碳荧光探针的开发及细胞成像研究

Development of a New Carbon Monoxide Fluorescent Probe Based on Nitro Reduction and Its Bioimaging Research in Living Cells

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