CN115197204B - 基于噻吩-氧杂蒽染料的硫化氢荧光探针的制备和应用 - Google Patents
基于噻吩-氧杂蒽染料的硫化氢荧光探针的制备和应用 Download PDFInfo
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Abstract
本发明涉及基于噻吩‑氧杂蒽染料的硫化氢荧光探针的制备和应用，该荧光探针的结构式为：本发明提供了以噻吩‑氧杂蒽染料、2,4‑二硝基氟苯、三乙胺等为原料合成该荧光探针的制备方法；该荧光探针是一种具有近红外发射、高选择性的硫化氢荧光探针。首先，该荧光探针对H2S表现出很高的灵敏度，探针与H2S反应之后荧光显著增强；其次，该荧光探针对H2S表现出很好的选择性，不受其他常见无机离子、活性氧、活性氮、氨基酸以及生物硫醇的干扰；并且，该荧光探针与H2S作用迅速，响应时间在3min以内；此外，该荧光探针已成功用于细胞中的H2S成像，可以检测细胞中的H2S水平。
Description
技术领域
本发明属于荧光探针技术领域，具体涉及一种基于噻吩-氧杂蒽染料的硫化氢荧光探针的制备和应用。
背景技术
硫化氢(H2S)是生命中不可缺少的气体递质(R.Wang,Antioxid.RedoxSignaling.2003,5,493-501.)。这种气体小分子可在体内通过酶催化的半胱氨酸(Cys)及其衍生物的代谢产生，在许多生理过程中发挥重要作用(B.Xu,H.Zhou,Q.Mei,W.Tang,Y.Sun,M.Gao,C.Zhang,S.Deng,Y.Zhang,Anal.Chem.2018,90,2686-2691.)。同时，细胞内H2S水平异常与多种疾病有关，比如：糖尿病、唐氏综合征、肝硬化和阿尔茨海默病(C.Szabo.Nat.Rev.Drug Discovery.2007,6,917-935；M.Lavu,S.Bhushan.Clin.Sci.2011,120,219-229；A.Martelli,L.Testai,M.C.Breschi,C.Blandizzi,Res.Rev.2012,32,1093-1130.)。因此，开发一种方便可靠的实时检测活细胞和体内H2S水平的方法至关重要。
荧光方法具有灵敏度高、操作简单、无创原位和实时空间成像的优点而显示出很大的潜力(W.Xuan,C.Sheng,Y.Cao,W.He,W.Wang,Angew.Chem.Int.Ed.2012,51,2282-2284；V.S.Lin,C.J.Chang,Curr.Opin.Chem.Biol.2012,16,595-601.)。到目前为止，已经开发了一些检测H2S的荧光探针，用于实时监测细胞内的H2S浓度(P.Ou,R.L.Zhang,Z.J.Liu,X.T.Tian,G.M.Han,B.H.Liu,Z.J.Hu,Z.P.Zhang,Angew.Chem.Int.Ed.2019,58,2261-2265；Z.Qian,H.Y.Zhang,K.W.Wang,Y.R.Zhang,Talanta.2019,195,850-856；L.Yuan,Q.P.Zuo,Sensors andActuators B.2014,196,151-155.)。但是，这些H2S探针分析波长较短，没有达到近红外范围，容易被活体内生物分子产生的自发荧光信号干扰，且组织穿透能力较弱，从而限制了在生物体内的应用。
噻吩-氧杂蒽作为一种新型的荧光染料，具有斯托克斯位移大，灵敏度高等优点。特别是，由于其染料具有近红外发射，因此具有较深的组织穿透深度，不易受到生物自体荧光的干扰，对生物成像更有利。二硝基苯基作为一种H2S识别团，已被成功用于构建荧光探针来特异性检测H2S(Z.P.She,W.X.Wang,W.L.Jiang,Z.Q.Wang,G.J.Mao,J.J.Fei,Y.F.Li,C.Y.Li,Anal.Chem.2021,93,11826-11835；L.Yan,Q.S.Gu,W.L.Jiang,M.Tan,Z.Ke.Tan,G.J.Mao,F.Xu,C.Y.Li,Anal.Chem.2022,94,5514-5520.)。但是，到现在为止，还没有基于噻吩-氧杂蒽染料作为荧光探针来检测H2S。因此，设计和合成一种基于噻吩-氧杂蒽染料的荧光探针来检测H2S是非常有必要的。
发明内容
根据所提出的要求，本发明人对此进行了深入研究，在付出了大量创造性劳动后，提供了一种基于噻吩-氧杂蒽染料的硫化氢近红外荧光探针。
本发明的技术方案是，一种硫化氢近红外荧光探针，其结构式如下：
一种硫化氢近红外荧光探针的制备方法。步骤如下：
将1.0当量的TX-OH，1.0当量的2,4-二硝基氟苯和0.4～0.6mL的三乙胺分别加入到25mL圆底烧瓶中，然后加入6～10mL丙酮将其溶解。将反应混合液回流搅拌0.5～1小时，反应完成后，减压蒸发除去丙酮。在所得混合物中加入8～12mL 5％的HCl溶液，过滤沉淀物，并用蒸馏水洗涤数次。将粗产物在丙酮中重结晶纯化，得到深绿色固体TX-H2S，即为所述的荧光探针。
本发明的有益效果是，一种基于噻吩-氧杂蒽染料的H2S荧光探针的良好的光谱响应性能。首先，研究该探针的荧光光谱性质。探针本身在715nm处没有明显的近红外发射；加入H2S后，在715nm处出现了明显的近红外发射。并且，随着H2S浓度的增大，探针的近红外荧光强度不断增强。当加入50μM的H2S时，荧光强度大约增强9倍。该探针的检测范围从0.5μM到50μM，检测限为0.24μM，这说明该探针可以高灵敏的检测H2S。接着，研究探针的紫外吸收光谱。探针本身在530nm附近有吸收带，加入H2S后，530nm附近的吸收峰明显减小，在590nm附近出现新的吸收峰。然后，研究探针的选择性。考察了探针与无机离子(K+、Na+、Ca2+、Mg2+、Cu2+、Cl-、Br-、S2O3 2-、SO4 2-)，活性氧(ClO-、H2O2)，活性氮(NO2 -、NO)，氨基酸(Lys、Phe、Met、Thr、Ile、Val)，生物硫醇(Cys、Hcy、GSH)以及检测物(H2S)的荧光响应情况。结果发现，只有H2S引起荧光光谱的改变，其他检测物对探针的荧光光谱没有明显的影响。最后，研究了pH值对荧光探针测定H2S的影响，当pH值在5.0到8.0之间时，不影响荧光探针对H2S的测定。此外，该荧光探针响应比较迅速，响应时间在3分钟以内。
一种硫化氢近红外荧光探针的应用。在对照组肝癌细胞HepG2中观察不到明显的荧光，当细胞中加入荧光探针后，可以观察到较强的荧光，这说明细胞中的H2S含量较高。在HepG2细胞中加入NaHS，再和荧光探针共同孵育时，可以观察到更强的荧光，这是因为细胞中的H2S含量增加。而用N-乙基马来酰亚胺(NEM)预处理细胞，再用探针共同孵育时，只能在细胞中观察到微弱的荧光，这是因为NEM清除了细胞内硫醇。这些结果说明荧光探针能检测到细胞内产生的H2S，这为监控人体内和硫化氢相关病变提供一种可靠的手段。
附图说明
图1为荧光探针的合成路线。
图2为荧光探针与不同浓度的H2S作用后的荧光光谱图。
横坐标为波长，纵坐标为荧光强度。荧光探针的浓度为10μM，H2S的浓度分别为：0,0.5,1.0,5.0,10.0,15.0,20.0,25.0,30.0,35.0,40.0,45.0,50.0μM。发射波长范围为650～850nm，对应的激发波长为590nm。
图3为荧光探针对不同H2S浓度的荧光线性响应图。
图4为荧光探针与H2S作用后的紫外可见吸收光谱图。
横坐标为波长，纵坐标为吸光度。荧光探针的浓度为10μM，H2S浓度为50μM。
图5为荧光探针的选择性图。
荧光探针的浓度为10μM，H2S浓度为50μM，GSH，Cys和Hcy的浓度为5mM，其它分析物浓度均为200μM。
图6为pH对荧光探针的影响图。
图7为荧光探针与H2S作用后荧光强度随时间变化的关系曲线图，H2S浓度为0,10,20,30,40,50μM。
图8为细胞毒性实验图。横坐标为荧光探针的浓度，纵坐标为细胞的存活率。
图9为荧光探针与H2S作用的细胞成像图，以及细胞的相对荧光强度图。(a)细胞成像图，(b)相对荧光强度图。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明，但不限于此。
实施例1：
荧光探针的合成
合成路线如图1。将TX-OH(110mg，0.30mmol)，2,4-二硝基氟苯(60mg，0.30mmol)，0.5mL的三乙胺分别加入到25mL圆底烧瓶中，然后加入10mL的丙酮使其溶解。将反应混合物回流搅拌0.5小时。蒸发除去丙酮，然后加入10mL 5％的HCl溶液，将沉淀物抽滤，并用蒸馏水洗涤数次。将粗产物在丙酮中重结晶纯化，得到深绿色固体TX-H2S(150mg，产率93％)，即为所述的荧光探针。1H NMR(400MHz,DMSO-d6,δ,ppm)δ8.87(d,J＝4.0Hz,1H),8.42(d,J＝8.0Hz,1H),8.02(d,J＝4.0Hz,1H),7.57(d,J＝4.0Hz,1H),7.44(d,J＝8.0Hz,1H),7.30(t,J＝8.0Hz,3H),7.15(s,1H),7.06(d,J＝8.0Hz,1H),6.92(s,1H),6.67(d,J＝12.0Hz,1H),2.82(s,2H),2.73(s,2H).13C NMR(100MHz,DMSO-d6,δ,ppm)δ161.7,159.5,154.9,154.8,153.0,142.4,140.8,140.1,138.8,133.4,133.2,130.2,129.1,122.4,121.5,120.8,117.9,117.2,115.8,115.0,108.6,74.3,55.4,25.8,24.6.MS(TOF):536.2.
实施例2：
荧光探针和H2S溶液配制
称取一定量荧光探针TX-H2S固体溶解在DMSO中来制备1.0×10-4mol·L-1的TX-H2S储备溶液。H2S储备溶液(1.0×10-3mol·L-1)是通过将一定量的NaHS固体溶解在蒸馏水中所制备，然后逐步稀释至5.0×10-4～5.0×10-6mol·L-1。将1.0mL TX-H2S储备溶液、2.0mLDMSO和1.0mL不同浓度的H2S储备溶液加入10mL的容量瓶，通过PBS缓冲溶液定容，然后进行荧光检测。
实施例3：
荧光探针与H2S作用的荧光光谱的测定
图2为荧光探针与H2S作用的荧光光谱，荧光探针的浓度为10μM，H2S的浓度依次为0,0.5,1.0,5.0,10.0,15.0,20.0,25.0,30.0,35.0,40.0,45.0,50.0μM。实验所用激发波长为590nm，发射波长范围为650～850nm。狭缝宽度为10.0nm/10.0nm，所用的荧光测定仪器为日立F4600荧光分光光度计。从图2可以看出，加入H2S之前，由于二硝基苯醚基团的淬灭作用，探针本身几乎没有发射峰；随着H2S的加入，在715nm处发射峰大幅度的增强，并且随着H2S浓度的增大，探针的荧光强度不断增强。图3为探针对不同H2S浓度的线性响应图。荧光强度跟H2S的浓度呈现线性关系，该探针的检测范围从0.5μM到50μM，检测限为0.24μM。这说明该探针可以高灵敏的检测H2S。
实施例4：
荧光探针与H2S作用的紫外可见吸收光谱的测定
图4为荧光探针与H2S作用后的紫外可见吸收光谱图，荧光探针的浓度为10μM，H2S的加入量为50μM。紫外可见吸收光谱测定用的仪器为安捷伦Cary60紫外可见分光光度计。从图4中可以看出，探针本身在530nm处有吸收带；加入H2S之后，530nm处的吸收峰红移，在590nm附近出现新的强吸收峰。
实施例5：
荧光探针对H2S测定的选择性
图5为荧光探针对H2S测定的选择性图。考察在浓度为10μM的荧光探针中加入H2S(50μM)以及无机离子(K+、Na+、Ca2+、Mg2+、Cu2+、Cl-、Br-、S2O3 2-、SO4 2-)，活性氧(ClO-、H2O2)，活性氮(NO2 -、NO)，氨基酸(Lys、Phe、Met、Thr、Ile、Val)，生物硫醇(Cys、Hcy、GSH)的荧光响应情况。从图5可以看出，只有H2S能引起荧光光谱的明显增强，其他检测物对探针的荧光光谱没有明显的影响。这些结果表明，荧光探针对H2S有良好的选择性。
实施例6：
溶液pH值对荧光探针测定H2S的荧光性质的影响
考察pH值对荧光探针测定H2S的荧光光谱的影响，其结果如图6。我们研究的pH范围为3.0～12.0，荧光探针的浓度为10μM，H2S的浓度为50μM。从图中可以看出，荧光探针随着pH的变化，荧光强度基本不变，说明pH对探针本身没有影响。然而，加入H2S之后，在pH在5.0～8.0范围内，荧光强度比值显著增强。综上所述，当pH值在5.0到8.0之间时，不影响荧光探针对H2S的测定，是比较合适的pH值范围，这非常有利于该探针用于实际样品中H2S的测定。
实施例7：
荧光探针与H2S作用的响应时间的测定
我们研究了荧光探针对H2S的响应时间，其结果如图7。从图中可以看出，该探针对H2S的响应时间为3min，这能够满足在实际样品中进行实时监测的要求。从图7还可以看出，荧光强度达到最大值后，在之后的时间里，荧光强度不再发生变化，这表明此荧光探针光稳定性较好。
实施例8：
荧光探针在活细胞中的应用
首先，我们做了细胞毒性实验，如图8所示。当加入0～30μM探针，肝癌细胞HepG2的存活率在90％以上。这说明该荧光探针毒性较小，可应用于检测活细胞内的H2S。然后，我们研究荧光探针在活细胞中的应用，选择肝癌细胞HepG2进行共聚焦显微成像，结果如图9(a)所示。在对照组细胞中，几乎没有观察到荧光。细胞用探针孵育后，观察到荧光增强。当在细胞中加入NaHS后，再加入探针，观察到荧光明显增强。而在细胞中加入H2S清除剂NEM后再加入探针，发现细胞内的荧光几乎消失。图9(b)为相对荧光强度图。这些结果说明该探针可以高灵敏性的检测细胞内的H2S。
Claims (3)
1.一种基于噻吩-氧杂蒽染料的硫化氢荧光探针，即TX-H2S，其特征在于，其结构如下：
2.根据权利要求1所述的一种基于噻吩-氧杂蒽染料的硫化氢荧光探针的制备方法，其特征在于，反应步骤如下：
将1.0当量的TX-OH，1.0当量的2,4-二硝基氟苯和0.4～0.6mL的三乙胺分别加入到25mL圆底烧瓶中，然后加入6～10mL丙酮将其溶解；将反应混合液回流搅拌0.5～1小时，反应完成后，减压蒸发除去丙酮，在所得混合物中加入8～12mL 5％的HCl溶液，过滤沉淀物，并用蒸馏水洗涤数次；将粗产物在丙酮中重结晶纯化，得到深绿色固体TX-H2S，即为所述的荧光探针，其中，TX-OH的结构如下：
3.根据权利要求1所述的一种基于噻吩-氧杂蒽染料的硫化氢荧光探针的应用，其特征在于，所述荧光探针用于制备检测细胞内硫化氢含量的产品。
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