CN115073435B - 一种检测硫化氢的近红外荧光探针及其制备方法 - Google Patents
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Abstract
本发明公开了一种检测硫化氢的近红外荧光探针及其制备方法，具体为：将6‑羟基‑1‑四氢萘酮与浓硫酸混合并冷，加入4‑(二乙胺基)水杨醛，进行加热搅拌，柱层析分离纯化，得到荧光团FR‑OH；将2‑巯基吡啶溶于氯仿中，再加入2‑巯基水杨酸和氯化亚砜，回流搅拌，抽滤，得到2‑(2‑吡啶基二硫代)苯甲酸PBA；将无水二氯甲烷溶液、荧光团FR‑OH、PBA、EDU和DMAP进行混合搅拌，柱层析分离即可。本发明的探针分子水溶性好，在580nm波长激发下对硫化氢有特异性响应，且选择性和抗干扰性优异，实现了细胞内源性硫化氢的可视化检测。
Description
技术领域
本发明属于分析检测技术领域，具体涉及一种检测硫化氢的近红外荧光探针，还涉及该近红外荧光探针的制备方法。
背景技术
硫化氢一直被认为是一种有臭鸡蛋味的有毒气体，但随着科学技术的发展，硫化氢被看作第三种气体信号分子，它在人体内的浓度异常与多种疾病相关。内源性硫化氢在生命体中主要是通过内源性酶催化途径产生，即以L-半胱氨酸为底物，分别在三种酶胱硫醚-β-合成酶CBS，胱硫醚-γ-裂解酶CSE以及3-巯基并酮酸巯基转移酶3-MST催化下产生硫化氢。这三种酶在人体的大脑、心脏、肺、肝、肾、血管胰腺、肠道中均有分布。而硫化氢的浓度异常会引发相关疾病，比如：糖尿病、心脏病、高血压、肝硬化等。
近年来科学研究者们也构筑了许多检测硫化氢的荧光探针。但由于探针的专一性差，尤其是复杂生物环境中硫醇的存在导致探针对硫化氢的识别响应具有干扰性。因此，设计一例能特异性识别硫化氢、且具有水溶性好的特性、还可以对内源性硫化氢进行细胞成像的近红外荧光探针具有重要的意义。
发明内容
本发明的目的在于提供一种检测硫化氢的近红外荧光探针，该荧光探针分子对硫化氢的检测具有良好的选择性和抗干扰能力。
本发明另一的目的在于提供上述检测硫化氢的近红外荧光探针的制备方法。
本发明所采用的技术方案是，一种检测硫化氢的近红外荧光探针，该荧光探针具有如下式(Ⅰ)所示的结构式：
本发明所采用的另一技术方案是，一种检测硫化氢的近红外荧光探针的制备方法，具体按以下步骤实施：
步骤1，将6-羟基-1-四氢萘酮与浓硫酸混合并冷却到0℃，之后加入4-(二乙胺基)水杨醛，搅拌均匀，在氮气保护条件下，进行加热搅拌反应，待冷却后倒入-5℃～0℃的冰水浴中，使反应物完全融化，柱层析分离纯化，得到荧光团FR-OH；
步骤2，将2-巯基吡啶溶于氯仿中，再加入2-巯基水杨酸和氯化亚砜，室温条件下回流搅拌1.0h，再用布氏漏斗进行抽滤，得到淡黄色固体即为产物2-(2-吡啶基二硫代)苯甲酸PBA；
步骤3，将无水二氯甲烷溶液、荧光团FR-OH、PBA、EDU和DMAP进行混合，室温搅拌，柱层析分离得到产物即为检测硫化氢的近红外荧光探针FR-H2S。
步骤1中，6-羟基-1-四氢萘酮、浓硫酸与4-(二乙胺基)水杨醛的质量比为1:9.38:1；反应温度为90℃，反应时间为6h。
步骤2中，2-巯基吡啶、氯仿、2-巯基水杨酸和氯化亚砜的质量比为2:67:1:1。
步骤3中，无水二氯甲烷溶液、荧光团FR-OH、PBA、EDU和DMAP的质量比为3900:10:10:10:1。
本发明的有益效果是，本发明设计的探针分子水溶性好，在580nm波长激发下对硫化氢有特异性响应，且选择性和抗干扰性优异，实现了细胞内源性硫化氢的可视化检测。
附图说明
图1是本发明方法制备近红外荧光探针的原理图；
图2是在不同有机溶剂与水体积比1:1条件下荧光探针FR-H2S(5μmol/L)的荧光发射光谱图；
图3是在不同有机溶剂与水体积比1:1条件下荧光探针FR-H2S(5μmol/L)与硫化氢响应的荧光发射光谱图；
图4是含有1％DMSO的溶液中探针分子(5μmol/L)在不同pH下与硫化氢响应荧光变化图；
图5是含有1％DMSO的溶液中探针FR-H2S分子(5μmol/L)荧光强度随时间变化的光谱图；
图6是含有1％DMSO的溶液中探针FR-H2S分子(5μmol/L)荧光强度随时间变化的趋势图；
图7是含有1％DMSO的溶液中探针FR-H2S分子(5μmol/L)与不同浓度硫化氢响应紫外吸收光谱图；
图8是含有1％DMSO的溶液中探针FR-H2S分子(5μmol/L)与不同浓度硫化氢响应荧光发射光谱图；
图9是含有1％DMSO的溶液中探针FR-H2S分子(5μmol/L)响应前后荧光强度与硫化氢浓度的线性拟合图；
图10是含有1％DMSO的溶液中探针FR-H2S分子(5μmol/L)对多硫化物和其它小分子氨基酸选择性响应的荧光发射光谱图；
图11是含有1％DMSO的溶液中探针FR-H2S分子(5μmol/L)与硫化氢及其他不同种氨基酸的竞争响应的荧光强度柱状图；
图12是探针FR-H2S分子对细胞毒性实验图；
图13是探针FR-H2S分子在A549细胞中对硫化氢检测细胞成像图。
具体实施方式
下面结合具体实施方式和附图对本发明进行详细说明。
本发明一种检测硫化氢的近红外荧光探针，该荧光探针具有如下式(Ⅰ)所示的结构式：
本发明一种检测硫化氢的近红外荧光探针，具体按以下步骤实施：
步骤1，将6-羟基-1-四氢萘酮与浓硫酸混合并冷却到0℃，之后加入4-(二乙胺基)水杨醛，搅拌均匀，在氮气保护条件下，进行加热搅拌反应，待冷却后倒入-5℃～0℃的冰水浴中，使反应物完全融化，柱层析分离纯化，得到荧光团FR-OH，其结构式如式(Ⅱ)所示；
6-羟基-1-四氢萘酮、浓硫酸与4-(二乙胺基)水杨醛的质量比为1：9.38:1；
反应温度为90℃，反应时间为6h；
步骤2，将2-巯基吡啶溶于氯仿中，再加入2-巯基水杨酸和氯化亚砜，室温条件下回流搅拌1.0h，再用布氏漏斗进行抽滤，得到淡黄色固体即为产物2-(2-吡啶基二硫代)苯甲酸PBA，其结构式如式(Ⅲ)所示；
2-巯基吡啶、氯仿、2-巯基水杨酸和氯化亚砜的质量比为2:67：1:1；
步骤3，将无水二氯甲烷溶液、荧光团FR-OH、PBA、4-二甲氨基吡啶EDU和1-(3-二甲基氨基丙基)-3-乙基碳二亚胺盐酸盐DMAP进行混合，室温搅拌，柱层析分离得到产物即为检测硫化氢的近红外荧光探针FR-H2S；
无水二氯甲烷溶液、荧光团FR-OH、PBA、EDU和DMAP的质量比为3900:10:10:10:1；
本发明方法制备的荧光探针的设计原理如图1所示，探针FR-H2S对硫化氢的识别过程如图大致分为三步：(1)硫化氢与探针FR-H2S发生亲核取代反应进攻二硫键；(2)探针FR-H2S结构中二硫键断裂，生成含有巯基的中间体；(3)中间体发生分子内环化反应，释放出荧光团FR-OH，产生红色的荧光信号。与硫化氢比，硫醇分子例如Cys、Hcy、GSH都可以与探针发生类似的亲核反应，但是不能进一步发生分子内环化反应，所以探针FR-H2S只能特异性对硫化氢进行识别响应。
实施例
本发明一种检测硫化氢的近红外荧光探针的制备方法，具体按照以下步骤实施：
反应式如下：
步骤1，以6-羟基-1-四氢萘酮和4-(二乙胺基)水杨醛为原料反应生成如下式(Ⅱ)所示荧光团FR-OH；
具体为：在5mL的圆底烧瓶中，将0.3g的6-羟基-1-四氢萘酮与1.0mL的浓硫酸放入其中，冷却到0℃。之后加入0.392g的4-(二乙胺基)水杨醛，充分搅拌，在氮气保护条件下，将所得混合物在90℃的加热条件下搅拌6h，冷却后倒入冰水中，等到完全融化后，柱层析分离纯化得到产物FR-OH；
其中，产物表征数据如下：
1H NMR(400MHz,DMSO-d6)δ11.12(s,1H),8.63(s,1H),8.16(d,J＝8.6Hz,1H),7.91(d,J＝9.4Hz,1H),7.41(d,J＝9.4Hz,1H),7.27(s,1H),6.94(d,J＝8.7Hz,1H),6.87(s,1H),3.69–3.64(m,5H),3.01(s,5H),1.24(t,J＝7.0Hz,8H).13C NMR(100MHz,DMSO-d6)δ164.8,164.3,158.3,155.3,148.4,146.2,132.1,129.5,120.7,118.0,117.7,116.2,96.2,45.8,27.0,25.2,12.9。
HRMS:Calcd.for C21H22NO2[M]+:320.1645；Found:320.1608.
步骤2，利用2-巯基吡啶与2-巯基水杨酸(2.31g，15.0mmol)反应生成2-(2-吡啶基二硫代)苯甲酸PBA；
具体为：将2-巯基吡啶(3.33g，30.0mmol)溶于氯仿(80.0mL)，加入250mL圆底烧瓶中，再加入2-巯基水杨酸(2.31g，15.0mmol)和氯化亚砜(1.10mL，15.0mol)，室温条件下回流搅拌1.0小时，用布氏漏斗抽滤，得到淡黄色固体即为产物PBA，无需进一步分离纯化；
其中，产物表征数据如下：
1H NMR(400MHz,DMSO-d6)δ13.55(s,1H),8.48(s,2H),8.03(d,J＝7.5Hz,2H),7.78(dd,J＝17.5,7.9Hz,3H),7.60(dd,J＝17.4,8.8Hz,3H),7.50(d,J＝8.0Hz,1H),7.35(dd,J＝15.6,7.8Hz,2H),7.30–7.23(m,2H).13C NMR(100MHz,DMSO-d6)δ168.48,158.24,150.54,139.77,138.83,134.11,132.27,128.52,126.91,125.73,122.55,120.25.HRMS:Calcd.forC12H9NO2S2[M+H]+:264.0147；Found:264.0124.
步骤3，荧光团FR-OH与PBA桥连合成近红外荧光探针FR-H2S；
具体为：取25mL无水二氯甲烷溶液于100mL圆底烧瓶中，加入FR-OH(0.32g)，PBA(0.263g)，EDU(0.192g)，DMAP(0.0122g)，室温搅拌，柱层析分离得到产物即为探针FR-H2S。
其中，产物表征如下：
1H NMR(400MHz,DMSO-d6)δ8.76(s,1H),8.54(s,1H),8.49(ddd,J＝8.0,4.0,3.2Hz,1H),8.36(t,J＝7.4Hz,1H),8.00(d,J＝9.5Hz,1H),7.91(d,J＝8.2Hz,1H),7.85–7.79(m,1H),7.79–7.74(m,1H),7.66–7.57(m,1H),7.57–7.53(m,1H),7.51(d,J＝7.4Hz,1H),7.39(s,1H),7.30(ddd,J＝7.5,4.9,0.8Hz,1H),3.76(dd,J＝24.7,18.3Hz,1H),3.22–3.03(m,1H),1.26(t,J＝7.0Hz,1H).13C NMR(100MHz,DMSO-d6)δ164.1,161.4,158.8,157.2,156.1,154.7,150.2,149.1,144.0,140.6,138.5,135.0,132.6,132.4,127.6,126.9,125.9,125.7,124.7,122.9,122.2,121.9,121.4,120.1,119.7,119.0,95.9,45.9,39.8,26.5,24.6.
HRMS:Calcd.for C33H29N2O3S2[M+H]+:565.1614；Found:565.1703.
对本实施例制备的荧光探针的荧光发射性能进行测试，具体如下：
不同有机溶剂中荧光性能测试：
激发波长570nm，测试了荧光探针FR-H2S(5μmol/L)在不同有机溶剂中的荧光发射，为了研究加入硫化氢前后探针FR-H2S在不同溶剂中的荧光强度值，选择较为合适的溶剂环境，选择七种常见的溶剂，其中包括：甲醇(MeOH)、乙腈(MeCN)、乙醇(EtOH)、二甲亚砜(DMSO)、N,N-二甲基甲酰胺(DMF)、丙酮(Acetone)、和四氢呋喃(THF)。分别在只有探针(5μmol/L)存在的情况下和探针(5μmol/L)和硫氢化钠(50μmol/L)同时存在的条件下测试了荧光强度，测试条件为有机溶剂比水相＝1:1。如图2和3所示，研究发现纯探针在DMSO中荧光强度最弱，发射波长630nm；在探针和识别物硫氢化钠共同存在的情况下，探针在DMSO中发射波长仍然是630nm，荧光强度较纯探针增加了将近5倍，因此选择DMSO作为测试溶剂。
荧光探针FR-H2S的pH稳定性测试：
在1％的DMSO溶剂体系中，对不同pH(1～12)范围内荧光探针的稳定性进行了测试，如图4所示，研究发现：纯探针在pH＝1～12的范围内的荧光强度均很弱。当加入硫化氢后，探针在pH＝6～9的范围内，荧光强度较为稳定；在极酸和极碱的条件下，探针FR-H2S的荧光被淬灭，说明探针可以用于生理条件对硫化氢的检测。
测试了加入600μmol/L的硫化氢条件下，探针FR-H2S(5μmol/L)在630nm处随着时间的推移荧光强度的变化，溶剂体系为水溶液(1％DMSO，pH＝7.4)。从图5和6可以看出，加入硫化氢后，前30min内探针FR-H2S的荧光强度增加地很迅速。30-120min时间段内，探针FR-H2S的荧光强度增加地较为平缓。在大约2h时，探针的荧光强度趋于稳定。
探针FR-H2S的荧光滴定实验：
在水溶液(1％DMSO，pH＝7.4)溶剂体系下，测试了探针FR-H2S分别随硫化氢浓度增加时紫外吸光度值以及荧光强度值的变化情况。如图7和8所示，纯探针FR-H2S在580nm处的吸收峰和630nm的发射峰都较低。当硫化氢浓度增加时，探针FR-H2S的紫外吸光度值和荧光强度值都随之增加。当硫化氢浓度达到600μmol/L，荧光强度增加了大约5倍达到最大值。如图9所示，当硫化氢的浓度在0～500μmol/L，随着硫化氢浓度的增加，FR-H2S的荧光强度值呈线性增加，探针FR-H2S的回归方程为y＝304.73+2.60x(R2＝0.9936)。根据检测限的计算公式3δ/k算出探针FR-H2S对硫化氢的检测限为8.66μM。由于探针的溶解性较好，可用于细胞中对硫化氢浓度的检测。
探针FR-H2S的选择性实验性能测试：
在水溶液(1％DMSO，pH＝7.4)的检测条件下，探针浓度为5μmol/L，待测物浓度为1000μmol/L，我们选取了Arg，Val，Thr，Lys，Hcy，GSH，Cys，Na2S2O3，Na2S2O4，Na2SO4，Na2SO3，NaHSO3，NaNO2，NaNO3，Na2CO3，NaHCO3，KSCN，H2O2，NaClO，NaF，NaBr，NaI作为待测物，测试了探针对其是否具有识别响应。如图10所示，在630nm发射波长处，只有硫化氢的加入导致探针FR-H2S的荧光信号显著增加，其它待测物的加入均没有导致探针的荧光信号发生明显变化，结果说明探针FR-H2S对硫化氢具有专一性识别响应。
探针FR-H2S的抗干扰性实验性能测试：
在水溶液(1％DMSO，pH＝7.4)的检测条件下，探针浓度为5μmol/L，硫化氢浓度为600μmol/L，同时加入了1000μmol/L的分析物作为干扰条件，测试了探针在630nm发射波长处的荧光强度。如图11所示，仅仅加入硫化氢时，探针FR-H2S的荧光强度有明显的增加。在其它分析物存在的干扰条件下，探针FR-H2S对硫化氢的检测能力没有发生明显的下降，大部分干扰物的存在都没有对探针FR-H2S的荧光强度造成影响。结果表明探针FR-H2S在复杂的环境中对硫化氢有专一性识别并且不受环境中其它分析物的干扰，具有良好的抗干扰能力。
探针FR-H2S的细胞毒性：
通过MTT实验来检测细胞毒性，配制了浓度依次为2.5μmol/L，5μmol/L，10μmol/L，20μmol/L，50μmol/L，100μmol/L的探针FR-H2S，选取了A549细胞，记录了不同浓度下的细胞存活率。如图12所示，当探针FR-H2S的浓度在100μmol/L时，细胞的存活率仍在90％以上，表明探针FR-H2S对细胞几乎没有毒性，可用于后续的细胞成像实验研究过程。
探针FR-H2S的荧光成像实验：
在细胞荧光成像实验中，选取A549细胞，将细胞分别用硝普化钠SNP和N-乙基马来酰亚胺NEM预处理，SNP可以刺激细胞产生更多的硫化氢，NEM可以抑制细胞内硫化氢以及硫醇物质的产生，设计实验组、抑制组、诱导组、空白组四组实验。如图13所示，A549细胞中仅仅加入探针FR-H2S时，几乎没有荧光信号；在外源性用硫氢化钠孵育后，共聚焦显微镜下可以观察到明亮的红色荧光；当用NEM孵育后再加入探针FR-H2S时，几乎观察不到荧光信号；当用SNP孵育后再加入探针FR-H2S时，共聚焦显微镜下再次呈现出明亮的红色荧光信号。结果表明探针FR-H2S可用于细胞内源性硫化氢的识别检测。
本发明以类香豆素为荧光团骨架，PBA为识别团，设计合成了发射波长在630nm的一例亲核反应激活型近红外荧光探针。通过一系列光学测试，表明探针FR-H2S仅仅对硫化氢识别响应，不会受到硫醇分子的干扰、水溶性好、灵敏度高、选择性强。在含有1％DMSO的溶剂体系中，硫化氢的浓度在0～500μmol/L的范围内，随着硫化氢浓度的增加，探针FR-H2S的荧光强度值呈线性增强。探针对细胞几乎没有毒性，可以用于细胞外源性和内源性对硫化氢的荧光成像实验。
Claims (3)
1.一种检测硫化氢的近红外荧光探针，其特征在于，该荧光探针具有如下式(Ⅰ)所示的结构式：
2.一种如权利要求1所述的检测硫化氢的近红外荧光探针的制备方法，其特征在于，具体按以下步骤实施：
步骤1，将6-羟基-1-四氢萘酮与浓硫酸混合并冷却到0℃，之后加入4-(二乙胺基)水杨醛，搅拌均匀，在氮气保护条件下，进行加热搅拌反应，待冷却后倒入-5℃～0℃的冰水浴中，使反应物完全融化，柱层析分离纯化，得到荧光团FR-OH；
6-羟基-1-四氢萘酮、浓硫酸与4-(二乙胺基)水杨醛的质量比为1:9.38:1；反应温度为90℃，反应时间为6h；
步骤2，将2-巯基吡啶溶于氯仿中，再加入2-巯基水杨酸和氯化亚砜，室温条件下回流搅拌1.0h，再用布氏漏斗进行抽滤，得到淡黄色固体即为产物2-(2-吡啶基二硫代)苯甲酸PBA；
2-巯基吡啶、氯仿、2-巯基水杨酸和氯化亚砜的质量比为2:67:1:1；
步骤3，将无水二氯甲烷溶液、荧光团FR-OH、PBA、EDU和DMAP进行混合，室温搅拌，柱层析分离得到产物即为检测硫化氢的近红外荧光探针FR-H2S。
3.如权利要求2所述的一种检测硫化氢的近红外荧光探针的制备方法，其特征在于，所述步骤3中，无水二氯甲烷溶液、荧光团FR-OH、PBA、EDU和DMAP的质量比为3900:10:10:10:1。
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