基于萘酰亚胺的次氯酸荧光探针的合成及细胞成像性能研究

doi:10.6023/cjoc202008005

有机化学 ›› 2021, Vol. 41 ›› Issue (2): 719-725.DOI: 10.6023/cjoc202008005 上一篇下一篇

研究论文

基于萘酰亚胺的次氯酸荧光探针的合成及细胞成像性能研究

王欣宇^a^,^c, 戚少龙^a^,^c, 杜建时^a^,^c^,^*(), 李强^b^,^c, 祝录宝^b^,^c,
薛龙启^b^,^c, 赵晴^a^,^d, 杨清彪^b^,^c^,^*(), 李耀先^b, 丛宪玲^a

a 吉林大学中日联谊医院长春 130033
b 吉林大学化学学院长春 130061
c 吉林大学中日联谊医院吉林省淋巴外科重点实验室吉林省淋巴外科工程实验室长春 130033
d 吉林大学中日联谊医院吉林省记忆与认知障碍疾病工程实验室长春 130031

收稿日期:2020-08-06 修回日期:2020-09-01 发布日期:2020-09-22
通讯作者: 杜建时, 杨清彪
作者简介:
* Corresponding authors. E-mail: dujs@jlu.edu.cn; yangqb@jlu.edu.cn
† 共同第一作者(These authors contributed equally to this work).
基金资助:
吉林省重点实验室建设项目(20190901002JC); 吉林省自然科学基金(20200201169JC); 吉林省创新能力建设(2019C007)

A Naphthalimide-Based Hypochlorous Acid-Selective Fluorescent Probe and Its Application in Cell Imaging

Xinyu Wang^a^,^c, Shaolong Qi^a^,^c, Jianshi Du^a^,^c^,^*(), Qiang Li^b^,^c, Lubao Zhu^b^,^c, Longqi Xue^b^,^c, Qing Zhao^a^,^d, Qingbiao Yang^b^,^c^,^*(), Yaoxian Li^b, Xianling Cong^a

a China-Japan Union Hospital of Jilin University, Changchun 130061
b College of Chemistry, Jilin University, Changchun 130061
c Key Laboratory of Lymphatic Surgery Jilin Province, Engineering Laboratory of Lymphatic Surgery Jilin Province, China-Japan Union Hospital of Jilin University, Changchun 130031
d Engineering Laboratory of Memory and Cognitive Impairment Disease Jilin Province, China-Japan Union Hospital of Jilin University, Changchun 130031

Received:2020-08-06 Revised:2020-09-01 Published:2020-09-22
Contact: Jianshi Du, Qingbiao Yang
Supported by:
the Key Laboratory Construction Project of Jilin Province(20190901002JC); the Natural Science Foundation of Jilin Province(20200201169JC); the Innovation Building Projects of Jilin Province(2019C007)

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摘要/Abstract

以萘酰亚胺结构为荧光发色团, 设计开发了一种含C=C双键的、具有分子内电荷转移(ICT)效应的新型水溶性优化的次氯酸荧光探针3-(2-氰基丙烯酸乙酯基)-4-羟基-N-正丙基-1,8-萘酰亚胺(NAEC). 添加次氯酸后, 探针分子NAEC中的C=C双键被氧化, 生成醛基, 探针NAEC原有的ICT效应被破坏, 产生荧光信号. 经核磁、质谱、荧光发射光谱和UV-Vis吸收光谱对其结构和检测性能进行了研究. 结果表明, 在pH=7.4的N,N-二甲基甲酰胺(DMF)/磷酸缓冲盐溶液(PBS) (V∶V=1∶19)缓冲体系中, 探针NAEC可在10 s内完成对次氯酸的检测, 荧光分析检测限为2.4 nmol/L, 斯托克斯位移为100 nm; 探针NAEC显示出较强的抗干扰性, 能在其他活性氧、小分子生物硫醇及常见阴离子等22种干扰物存在下完成次氯酸的专一检测. 同时, 该探针分子的膜透性与生物相容性良好, 具备较好的活体内源性ClO^–荧光成像能力, 在生物检测及环境监控等领域具有良好的应用前景.

关键词: 萘酰亚胺, 次氯酸, 荧光探针, 细胞成像

A novel naphthalimide-based fluorescence probe ethyl (E)-3-(2-butyl-6-hydroxy-1,3-dioxo-2,3-dihydro-1H-benzo- [de]isoquinolin-5-yl)-2-cyanoacrylate (NAEC) containing C=C double bond group with intramolecular charge transfer (ICT) effect for hypochloric acid detection was synthesized, which could selectively and sensitively respond to hypochloric acid within 10 s in phosphate buffer saline (PBS) buffer solution [N,N-dimethylformamide (DMF)/PBS (V∶V=1∶19), pH=7.4]. The mechanism is that the C=C double bond in the probe can be oxidized and turned to aldehyde group, then the original ICT effect of probe is destroyed, and the fluorescence of naphthalimide restores. The spectroscopic properties of the probe NAEC towards ClO^– were studied through UV-vis absorption and ?uorescence emission spectroscopies. The results showed that the probe NAEC possessed a large pseudo Stokes shift (100 nm) and the limit of detection (LOD) for NAEC has been estimated to be 2.4 nmol/L. In addition, the probe exhibited good response and anti-interference performance for hypochloric acid in the presence of 22 kinds of interferences, such as other reactive oxygen, small molecule biosulphol and common anions. And probe NAEC exhibited good cell permeability and was successfully applied in living cells by fluorescence imaging. The method is expected to be extended to the diagnosis of diseases and the detection in environmental samples, etc.

Key words: naphthalimide, hypochlorous acid, fluorescence probe, cellular imaging

引用此文

王欣宇, 戚少龙, 杜建时, 李强, 祝录宝,
薛龙启, 赵晴, 杨清彪, 李耀先, 丛宪玲. 基于萘酰亚胺的次氯酸荧光探针的合成及细胞成像性能研究[J]. 有机化学, 2021, 41(2): 719-725.

Xinyu Wang, Shaolong Qi, Jianshi Du, Qiang Li, Lubao Zhu, Longqi Xue, Qing Zhao, Qingbiao Yang, Yaoxian Li, Xianling Cong. A Naphthalimide-Based Hypochlorous Acid-Selective Fluorescent Probe and Its Application in Cell Imaging[J]. Chinese Journal of Organic Chemistry, 2021, 41(2): 719-725.

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图/表 8

图式1. 探针NAEC合成路线

Scheme 1. Synthetic route of the probe NAEC

图1. (A)探针NAEC (5.0×10–6 mol/L)溶液在加入ClO–前 (a)、后(b)荧光光谱随溶液pH值的变化情况[DMF/PBS (V∶ V=1∶19), λex=420 nm, λ em=520 nm, slit: 5/5 nm]和(B)加入ClO–后荧光强度随时间变化情况[DMF/PBS (V∶V=1∶19), pH=7.4, λex=420 nm, λ em=520 nm, slit: 5/5 nm]

Figure 1. (A) Fluorescence spectra of NAEC (5.0×10–6 mol/L) before (a) and after (b) addition of ClO– in different pH conditions [DMF/PBS (V∶V=1∶19), λex=420 nm, λ em=520 nm, slit: 5/5 nm] and (B) time-dependent fluorescence intensity changes of NAEC (5.0×10–6 mol/L) after addition of ClO– [DMF/PBS (V∶V=1∶19), pH=7.4, λex=420 nm, λ em=520 nm, slit: 5/5 nm]

Figure 2. 探针NAEC (5.0×10–6 mol/L)在添加不同浓度的ClO– (0~9 equiv.)前后的UV-Vis吸收光谱[DMF/PBS (V∶V=1∶19), pH=7.4] UV-Vis absorption spectra of probe NAEC (5.0×10–6 mol/L) upon addition of different concentrations of ClO– (0~9 equiv.) [DMF/PBS (V∶V=1∶19), pH=7.4]

. Insert: emission color changes of probe NAEC (5.0×10–6 mol/L) before and after addition of ClO– (9 equiv.) under a 365 nm light

图3. (A)探针NAEC溶液(5.0×10–6 mol/L)在添加不同浓度的ClO– (0~9 equiv.)后的荧光光谱[DMF/PBS (V∶V=1∶19), pH=7.4, λex=420 nm, slit: 5/5 nm]和(B)探针NAEC的荧光强度与ClO–浓度的线性拟合曲线[DMF/PBS (V∶V=1∶19), pH=7.4, λex=420 nm, λ em=520 nm, slit: 5/5 nm]

Figure 3. (A) Fluorescence spectra of probe NAEC (5.0×10–6 mol/L) upon addition of different concentrations of ClO– (0~9 equiv.) [DMF/PBS (V∶V=1∶19), pH=7.4, λex=420 nm, slit: 5/5 nm] and (B) Linear fit curve of fluorescence intensity of NAEC vs ClO– concentration [DMF/PBS (V∶V=1∶19), pH=7.4, λex=420 nm, λ em=520 nm, slit: 5/5 nm]

图4. 向存在不同干扰离子(90 equiv.)的探针NAEC (5.0× 10–6 mol/L)溶液中加入ClO– (9 equiv.)前后的荧光变化[DMF/PBS (V∶V=1∶19), pH=7.4]

Figure 4. Variation in the fluorescence of NAEC (5.0×10–6 mol/L) before and after adding 9 equiv. ClO– in the presence of 90 equiv. of various interferents [DMF/PBS (V∶V=1∶19), pH=7.4]

图5. (A)分子4和探针NAEC反应后的荧光光谱; (B)探针NAEC对ClO–的检测机理示意图

Figure 5. (A) Fluorescence spectra of molecule 4 and NAEC and (B) proposed reaction mechanism of probe NAEC towards ClO–

图6. 四甲基偶氮唑盐(MTT)法测得HUVEC细胞与探针NAEC共孵育24 h后的细胞存活率(对照组为100%, C为对照)

Figure 6. Viability of HUVEC cell after incubated with probes NAEC by methyl thiazolyl tetrazolium (MTT) method (C represents control sample,, and the viability is 100%)

图7. HUVEC细胞与探针NAEC (5.0×10–6 mol/L)共孵育30 min (A, B, C), HUVEC细胞经PMA (1 mg/mL)共孵育2 h后加入探针NAEC (5.0×10–6 mol/L)后继续孵育30 min的荧光共聚焦显微图像(D, E, F); HUVEC细胞与探针NAEC (5.0×10–6 mol/L)共孵育30 min, 继续加入9.0 equiv. ClO–共孵育30 min后的荧光共聚焦显微镜图像(G, H, I) (左: 荧光图像; 中: 明场图像; 右: 重叠图像)

Figure 7. Optical confocus microscopic images of HUVEC cells incubated 30 min with the NAEC probe (5.0×10–6 mol/L) (A, B, C); pretreated for 2 h with PMA (1 mg/mL) and incubated with the NAEC probe (5.0×10–6 mol/L) for 30 min (D, E, F) and incubated 30 min with the NAEC probe (5.0×10–6 mol/L), followed by treatment with 9.0 equiv. ClO– for another 30 min (G, H, I) (left: fluorescence images, middle: bright-field images, right: merge images)

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基于萘酰亚胺的次氯酸荧光探针的合成及细胞成像性能研究

A Naphthalimide-Based Hypochlorous Acid-Selective Fluorescent Probe and Its Application in Cell Imaging

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