CN108191881A - 一种识别苯硫酚的荧光探针 - Google Patents

一种识别苯硫酚的荧光探针 Download PDF

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CN108191881A
CN108191881A CN201810046954.4A CN201810046954A CN108191881A CN 108191881 A CN108191881 A CN 108191881A CN 201810046954 A CN201810046954 A CN 201810046954A CN 108191881 A CN108191881 A CN 108191881A
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CN108191881B (zh
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宋相志
耿娅妮
苏远安
杨雷
张帆
韩金良
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Central South University
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    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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    • C09K2211/1025—Heterocyclic compounds characterised by ligands
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Abstract

本发明涉及一种识别苯硫酚的荧光探针，属于荧光探针领域。其分子结构如下：该探针分子最大发射波长在610nm，探针分子与苯硫酚(PhSH)作用后发出红色荧光，且具有较低的检测限，灵敏度好；而且发射波长在红光区能够减少探针检测过程中的背景荧光和活细胞的光损伤，增强生物对组织的穿透能力。本发明所述的探针分子在一定时间与浓度范围具有良好线性，对苯硫酚(PhSH)识别能力强，选择性好、抗干扰能力强，能够在纯水溶液和活细胞内实现苯硫酚(PhSH)的可视化检测，该类探针在生物化学等领域具有重要的应用价值。

Description

一种识别苯硫酚的荧光探针
本发明属于荧光探针领域，具体涉及一种苯硫酚荧光探针的制备及其在活细胞内部检测苯硫酚(PhSH)的应用。
背景技术
在农药工业和制药工程中，苯硫酚经常被作为有机合成的原料用于制备各种农用和医用产品，但是同时具有很高的毒性，排放到环境中则会造成很严重的污染问题。例如，苯硫酚对鱼和白鼠的半致死量(LD50)分别为0.01-0.4mM和2.15-46.2mg/kg。长时间接触被苯硫酚污染的水或者土壤会引起一系列的健康问题，包括呼吸系统，中枢神经系统损伤，肌无力，呕吐，昏迷甚至是死亡因此，所以有必要设计一种有效的方法，实现环境和生物体内的苯硫酚可视化检测。
发明内容
本发明目的之一在于提供一种简便高效的荧光探针合成方法；本发明之另一目的是提供一种选择性好，抗干扰能力强，具有红光发射波长，能够实现对体外或者活细胞内部苯硫酚(PhSH)检测的荧光探针。
本发明解决问题采取的技术方案为，一种识别苯硫酚(PhSH)的新型荧光探针，其分子结构式如下：
合成路线如下：
具体合成方法如下：(a)将化合物2(255.3mg，1.0mmol)和对氨基苯酚(130.8mg，1.2mmol)溶于5mL DMF中，再加入碘化亚铜(57.1mg，0.3mmol)，油浴升温至110℃，回流反应12h。冷却至室温，加入50mL饱和食盐水中稀释，二氯甲烷萃取(25.0mL×4)，收集有机层，然后用饱和食盐水溶液洗三次，无水硫酸钠干燥，柱层析分离得到棕黄色固体产物。产量：68.9mg。产率：20％。(b)于20mL厚壁耐压瓶中，加入化合物3(180.1mg，0.52mmol)，用5mL无水二氯甲烷溶解，然后加入2mL碘甲烷，并用锡箔纸包裹耐压瓶90℃避光反应12h，反应完成，冷却至室温，柱层析分离得到染料4，红色固体。产量：219.3mg。产率：86.7％。(c)将染料4(111.9mg，0.23mmol)，2,4-二硝基氟苯(93.1mg，0.5mmol)溶于10mL无水DMF中，加入无水碳酸钾(110.6mg，0.7mmol)，油浴升温至53℃搅拌过夜。反应完成，冷却至室温，加入50mL饱和食盐水稀释，乙酸乙酯萃取(25.0mL×4)，收集有机层，饱和食盐水溶液洗涤三次，无水硫酸钠干燥。柱层析分离得到探针1，褐色固体。产量：34.2mg，产率：25.4％。
本发明的荧光探针的作用机理如下，甲基化吡喃喹啉类染料4由于分子内的电荷转移(ICT)过程发射红光并且具有良好的水溶性。我们以2,4-二硝基氟苯作为识别集团取代染料4中羟基的氢，合成探针1。染料4与识别基团之间的光诱导电子转移过程(PET)使荧光团染料4的荧光淬灭。然后苯硫酚能与探针发生亲核取代反应使探针脱去识别基团，释放染料4并发射红色荧光。探针1是一个典型的开-关型检测苯硫酚的荧光探针。
探针分子的响应过程如下：
本发明的荧光探针具有红光发射，其与苯硫酚(PhSH)作用前几乎不发射荧光，作用后荧光发射峰在610nm处。
本发明的荧光探针能够实现在实际水样和活细胞内对苯硫酚(PhSH)的检测。
本发明的荧光探针选择性好。探针分子的测试体系是PBS缓冲溶液(10mM，pH＝7.4)纯水体系，室温下测试。探针分子本身几乎不发射荧光，随着加入的苯硫酚浓度的增加，在610nm处出现新的发射峰，并且强度逐渐增加，在加入30倍当量苯硫酚(PhSH)之后，在最大发射波长处荧光强度增强了28倍。而在加入其他各种氨基酸(Cys，Hcy，GSH，His，Met)，阳离子(K+，Mg2+，Na+)，阴离子(HS-，I-，F-，Cl-，NO2 -，S2-，S2O3 2-，SCN-，SO3 2-，SO4 2-)以及活性氧(ClO-，H2O2)后，荧光发射峰没有变化。
本发明的荧光探针抗干扰能力强，其他各种氨基酸(Cys，Hcy，GSH，His，Met)，阳离子(K+，Mg2+，Na+)，阴离子(HS-，I-，F-，Cl-，NO2 -，S2-，S2O3 2-，SCN-，SO3 2-，SO4 2-)以及活性氧(ClO-，H2O2)的存在不影响探针分子与苯硫酚(PhSH)的作用。
本发明的荧光探针在加入苯硫酚(PhSH)作用后，随着加入的苯硫酚浓度的增加，在610nm处出现新的发射峰，并且强度逐渐增加，在加入30倍当量苯硫酚(PhSH)之后，在最大发射波长处荧光强度增强了28倍。
本发明的荧光探针表现出良好的动力学现象，探针分子与苯硫酚(PhSH)选择性识别后，荧光强度比值与时间表现出良好的线性关系。
本发明的荧光探针能够实现实际水样与细胞内检测苯硫酚(PhSH)，通过分别检测矿泉水和自来水中苯硫酚的加标回收率，以及细胞内外源性苯硫酚(PhSH)，表明该探针能实现实际水样与细胞内检测苯硫酚(PhSH)。
本发明所述的探针分子对苯硫酚(PhSH)响应前几乎没有荧光，响应后发射在610nm而呈现红色荧光并且对其他各种氨基酸、阴离子、阳离子以及活性氧具有良好的选择性和抗干扰能力，并且具有好的灵敏度，具有较宽的应用范围。长的发射波长使其有强的组织穿透能力，该荧光探针在生物与化学等领域具有实际的应用价值。
附图说明
图1为本发明的荧光探针1(5.0μM)在PBS(10mM，pH＝7.4)中与递增浓度苯硫酚(PhSH)(0.0-30.0倍当量.)响应后的荧光光谱图，横坐标为波长，纵坐标为荧光强度。λex＝500nm，激发和发射狭缝宽度：5.0nm/5.0nm。
图2为本发明的荧光探针1(5.0μM)在PBS(10mM，pH＝7.4)中与递增浓度苯硫酚(PhSH)响应后在610nm处荧光强度散点图。插图：610nm处荧光强度与苯硫酚(PhSH)浓度线性关系图。横坐标为苯硫酚(PhSH)浓度，纵坐标为荧光强度。
图3为本发明的荧光探针1(5.0μM)在其他干扰离子存在下与30.0倍量苯硫酚(PhSH)作用后荧光强度比值图。λex＝500nm，激发和发射狭缝宽度：5.0nm/5.0nm。(1)PhSH，(2)Cl-，(3)ClO-，(4)Cys，(5)F-，(6)GSH，(7)H2O2，(8)Hcy，(9)His，(10)HS-，(11)I-，(12)K+，(13)Met，(14)Mg2+，(15)Na+，(16)NO2-，(17)S2-，(18)S2O3 2-，(19)SCN-，(20)SO3 2-，(21)SO4 2-。
图4为本发明的荧光探针1(5.0μM)在PBS缓冲液(10mM，pH＝7.4)中与各种干扰离子作用后荧光强度比，横坐标为波长，纵坐标为荧光强度。
图5为本发明的荧光探针1(5.0μM)和探针1(5.0μM)与30.0倍量苯硫酚(PhSH)反应后在610nm处荧光强度随时间变化点状图，横坐标为时间，纵坐标为荧光强度。
图6为本发明的荧光探针1(5.0μM)及探针1(5.0μM)与30.0倍量苯硫酚(PhSH)作用后在pH等于2.0到13.0范围内610nm处荧光强度变化，横坐标为Ph，纵坐标为荧光强度。
图7为本发明的荧光探针1(5.0μM)在PBS缓冲溶液(10mM，pH＝7.4)中加入30.0倍量苯硫酚(PhSH)前后的紫外吸收光谱，横坐标为波长，纵坐标为吸收强度。
图8为本发明的荧光探针1在细胞内成像情况，上：细胞先用苯硫酚(PhSH)(1mM)预处理再用探针1(5.0μM)在37℃培养20分钟(a,b,c)。下：细胞用探针1(5.0μM)在37℃培养20分钟(d,e,f)。左：荧光图像；中：明场图像；右：明场与荧光叠加图像。细胞放大了60倍。
图9为本发明的荧光探针1(5.0μM)在自来水中的荧光强度与PhSH浓度(0.0-8.0μM)的线性关系图，横坐标为苯硫酚(PhSH)浓度，纵坐标为荧光强度。
图10为本发明的荧光探针1(5.0μM)在矿泉水中的荧光强度与PhSH浓度(0.0-8.0μM)的线性关系图，横坐标为苯硫酚(PhSH)浓度，纵坐标为荧光强度。
具体实施实例
实施例1：探针的合成
取染料4(111.9mg，0.23mmol)，2,4-二硝基氟苯(93.1mg，0.5mmol)溶于10mL无水DMF中，加入无水碳酸钾(110.6mg，0.7mmol)，油浴升温至53℃搅拌过夜。反应完成，将反应液冷却至室温，加入50mL饱和水盐水稀释，乙酸乙酯萃取(25.0mL×4)，收集有机层，饱和食盐水溶液洗三次，无水硫酸钠干燥。经柱层析分离得探针1，褐色固体。产量：34.2mg，产率：25.4％。HRMS(ESI)m/z：计算C29H25N4O6[M+1]+，525.1769；发现，525.1754。1H NMR(400MHz，DMSO-d6)δH 8.99(d，J＝2.8Hz，1H)，8.55(d，J＝9.2，2.8Hz，1H)，8.46(s，1H)，8.36(d，J＝9.5Hz，1H)，8.04(d，J＝2.8Hz,1H)，7.96(d，J＝9.4，2.8Hz，1H)，7.56(s，1H)，7.40(d，J＝9.2Hz，1H)，5.77(s，1H)，5.26(s，2H)，4.39(s，3H)，3.43(t，J＝10.4Hz，4H)，2.83-2.76(m，2H)，2.68(t，J＝6.1Hz，2H)，1.97-1.86(m，4H)。13C NMR(100MHz,DMSO-d6)δC 158.03，154.31，152.79，150.19，149.77，142.87，140.51，138.67，135.09，131.69，130.32，128.94，127.40，126.10，122.59，122.40，121.24，118.31，117.93，107.37，103.42，67.70，55.39，50.33，49.67，45.00，27.30，21.17，20.52，20.13。
实施例2：本发明的荧光探针的应用
为了探究探针1的实用性，测定了矿泉水和的自来水中苯硫酚(PhSH)的加标回收率。测试过程中我们首先用0.22μm的滤膜过滤待测水样，然后配成PBS缓冲溶液(10mM，pH＝7.4)，加入的苯硫酚(PhSH)的浓度分别为0.0、2.0、4.0、6.0、8.0μM，探针溶液的浓度为5.0μM。在两种水样中，探针溶液在610nm处的荧光强度与所加入苯硫酚的浓度都有良好的线性关系，其线性系数分别是R自来水＝0.99643，R矿泉水＝0.99418。结果表明，两种水样中添加的苯硫酚都有良好的回收率。这就说明探针1可以在实际水样中检测苯硫酚，具有潜在的实用价值。

Claims (1)

1.一种识别苯硫酚(PhSH)的荧光探针，其结构为：
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CN109761978A (zh) * 2019-01-18 2019-05-17 商丘师范学院 一种检测苯硫酚的近红外荧光探针及其合成方法与应用
CN114989184A (zh) * 2022-05-17 2022-09-02 郑州大学 一种吡喃喹啉杂化香豆素类的荧光染料
CN114989184B (zh) * 2022-05-17 2023-09-08 郑州大学 一种吡喃喹啉杂化香豆素类的荧光染料

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