CN111040465A - 一种双模态检测二氧化硫的近红外荧光探针及其制备方法和用途 - Google Patents

一种双模态检测二氧化硫的近红外荧光探针及其制备方法和用途 Download PDF

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CN111040465A
CN111040465A CN201911298350.XA CN201911298350A CN111040465A CN 111040465 A CN111040465 A CN 111040465A CN 201911298350 A CN201911298350 A CN 201911298350A CN 111040465 A CN111040465 A CN 111040465A
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王振洋
杨林林
张忠平
刘变化
韩光梅
赵君
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Hefei Institutes of Physical Science of CAS
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Abstract

本发明公开了一种双模态检测二氧化硫的近红外荧光探针及其制备方法和用途，其中双模态检测二氧化硫的近红外荧光探针是半菁染料衍生物，其结构如下所示：
Figure DDA0002321199310000011
本发明荧光探针可用于比色法和比率法双模态检测水体中的二氧化硫，二氧化硫与探针反应后引起探针溶液颜色(深蓝‑浅粉)及荧光(近红外‑黄色区)两种信号的显著变化，从而实现对水体中二氧化硫的高选择和高灵敏的双重可视化检测。本发明荧光探针可实现活细胞内二氧化硫的双通道比率成像和定量化，该探针长波长的吸收和近红外发射，可有效避免生物背景荧光的干扰、减小对细胞造成的光损伤。

Description

一种双模态检测二氧化硫的近红外荧光探针及其制备方法和 用途
技术领域
本发明涉及合成与技术应用领域，具体涉及一种双模态检测二氧化硫的近红外荧光探针及其制备方法和用途。
背景技术
二氧化硫(SO2)是大气主要污染物之一，极易被生物体吸入并在血液或其他体液中转化为其衍生物-亚硫酸盐和亚硫酸氢盐，从而引发呼吸系统疾病、神经系统疾病，甚至肺癌。研究发生，生物体内也含有SO2，是含硫氨基酸或硫化氢经生物合成途径产生的，比如硫化氢的线粒体解毒、活性氧物种的非酶促反应等。生物体内正常水平的SO2具有降低血压、舒张血管、诱导心脏负性肌力等作用。然而，SO2含量水平的异常则与呼吸和心血管疾病、神经系统疾病、甚至肺癌等病理状态密切相关。因此，发展一种高灵敏荧光传感器实现SO2的快速、准确及原位检测具有重要意义。
目前，已发展的多数SO2的荧光探针主要存在以下问题：1)选择性低、灵敏性差，易受复杂检测体系中其他干扰物质的影响；2)信号输出模式单一，单一荧光强度或探针溶液颜色的变化以及探针在生物体分布不均等，容易造成信号输出的假阳性；3)短波长的吸收和发射，对细胞造成较大的光损伤。为了能够在复杂环境或生物体系中实现对SO2的高选择性、灵敏性及低损伤检测，亟需发展一种对SO2特异响应及具有多重信号输出的近红外荧光传感器。发展性能优越的双模态SO2荧光传感器，不仅能够在复杂的环境体系中准确识别SO2，同时也将为生物体内SO2及其衍生物生理功能的研究提供一种有力的工具。
发明内容
本发明针对现有SO2荧光探针中存在的技术问题，提供了一种双模态检测二氧化硫的近红外荧光探针及其制备方法和用途。本发明荧光探针具有水溶性好、光稳定性强和近红外发射等特点，对SO2具有高选择性和灵敏性，在水环境中实现对SO2的多重信号响应，以及对生物体系中SO2的双通道比率成像和定量化检测。
本发明双模态检测二氧化硫的近红外荧光探针，为半菁类近红外荧光染料，其结构如下式I所示：
Figure BDA0002321199290000011
Figure BDA0002321199290000021
其中R1为氢、C1-5烷基、苯基、烷氧基或卤素；R2为氢、C1-5烷基、苯基、氨基、N,N-二甲基胺或吗啉。
本发明双模态检测二氧化硫的近红外荧光探针的制备方法，首先由吲哚和碘乙烷通过取代反应得到中间体吲哚盐，然后中间体吲哚盐与醛在催化剂的存在下通过羟醛缩合反应得到目标化合物。包括以下步骤：
步骤1：将吲哚和碘乙烷溶于有机溶剂中，于30-100℃下搅拌反应1-36h；反应结束后冷却至室温，晶体析出，抽滤，滤饼用冰乙醇清洗1-5次，得中间体，其结构如下式II所示；
Figure BDA0002321199290000022
其中R1为氢、C1-5烷基、苯基、烷氧基或卤素。
步骤1中，吲哚和碘乙烷的摩尔比为5:1-1:8；所述有机溶剂为无水乙腈或丙酮。
步骤2：将步骤1获得的中间体和醛溶于有机溶剂中，加入几滴催化剂量的催化剂，于30-100℃下搅拌反应1-18h；反应结束后冷却至室温，真空旋转蒸发仪除去溶剂，得粗产品，柱层析纯化得目标产物。
步骤2中，所述催化剂为哌啶。
步骤2中，中间体和醛的摩尔比为1:15-8:1。
步骤2中，所述醛的结构如下式III所示：
Figure BDA0002321199290000023
其中R2为氢、C1-5烷基、苯基、氨基、N,N-二甲基胺或吗啉。
步骤2中，所述有机溶剂为甲醇或乙醇。
步骤2中，所述纯化是采用硅胶柱层析分离方法进行纯化，洗脱剂由二氯甲烷、乙酸乙酯和甲醇按体积比60:90:1-10:20:1的比例混合构成。
本发明近红外荧光探针的用途，是在通过比色或比率两种检测方法实现水体系中SO2的定性或定量检测过程中作为检测试剂使用，该探针对浓度范围在0-200μM的SO2，在溶液颜色及荧光发射上均呈现显著变化，并对浓度20-80μM范围的SO2具有良好线性关系。
所述水体系为pH＝7.4的PBS或HEPES缓冲溶液。
本发明近红外荧光探针的用途，是在细胞内SO2的双通道比率成像及定量化检测过程中作为检测试剂使用。
所述细胞为腺癌人类肺泡基底上皮细胞(A549细胞)。
本发明荧光探针可用于比色法和比率法双模态检测水体中的二氧化硫，二氧化硫与探针反应后引起探针溶液颜色(深蓝-浅粉)及荧光(近红外-黄色区)两种信号的显著变化，从而实现对水体中二氧化硫的高选择和高灵敏的双重可视化检测。探针可实现活细胞内二氧化硫的双通道比率成像和定量化，该探针长波长的吸收和近红外发射，可有效避免生物背景荧光的干扰、减小对细胞造成的光损伤，因而在生物成像及小分子检测领域具有广阔的应用前景。
与现有技术相比，本发明的有益效果体现在：
1、本发明合成的荧光探针先后通过取代和羟醛缩合反应获得的，操作简单、反应条件温和。
2、本发明合成的荧光探针对水环境中的SO2具有多重信号响应，探针溶液颜色由深蓝转变为浅粉色，以及荧光发射由近红外区蓝移到黄色可见光区，两种不同输出信号的显著变化，使探针能够通过比色和比率两种方法实现水体系中SO2的双模态检测。
3、本发明合成的荧光探针的近红外发射，能够有效增强探针的信噪比、降低对细胞产生的光损伤以及减小染料在细胞内分散不均导致的信号干扰。
4、本发明合成的荧光探针具有优良的生物兼容性，可快速穿越细胞膜实现细胞内SO2的双通道比率荧光成像和定量化检测。
附图说明
图1为本发明的荧光探针与SO2反应前后的紫外-可见吸收光谱及加入SO2前后探针溶液在自然光下的颜色变化。探针浓度为20μM，待测NaHSO3(SO2供体)的浓度范围为0-150μM，溶液为10mM，pH＝7.4的HEPES缓冲溶液，2％的乙醇作为共溶剂。
图2为本发明的探针对SO2的荧光响应及探针荧光强度比值与SO2浓度之间的线性关系。探针浓度为20μM，待测NaHSO3(SO2供体)的浓度范围为0-200μM，溶液为10mM，pH＝7.4的HEPES缓冲溶液，2％的乙醇作为共溶剂。激发光波长为520nm，狭缝宽度为10和10nm。
图3为本发明的探针对SO2的选择性。探针浓度为20μM，待测NaHSO3(SO2供体)的浓度范围为0-150μM，溶液为10mM，pH＝7.4的HEPES缓冲溶液，2％的乙醇作为共溶剂。其它阴离子、生物硫醇和还原性分子分别为F-、Cl-、Br-、I-,HCO3 -、AcO-、NO2 -、NO3 -、ppi、SO4 2-、HSO4 -、S2O3 2-、S2O8 2-、SCN-、Vc、Cys、Hcy、GSH、S2-、SO3 2-和HSO3 -，其中一组为空白对照。激发光波长为520nm，狭缝宽度为10和10nm。
图4为本发明的探针对细胞内SO2的双通道比率成像。以用100μM NaHSO3(SO2供体)预先孵育30min的A549细胞为实验组，未经任何处理的A549细胞为对照组。分别向两组细胞中加入5μM探针培养30min后对细胞进行双通道比率成像。标尺：20μm。
图5为本发明的探针对细胞内SO2的定量化检测。首先向A549细胞中加入不同浓度的NaHSO3(SO2供体)，孵育30min后，更换新鲜培养基，然后加入5μM探针继续培养30min后对细胞进行双通道比率成像。在细胞中选择相同面积的荧光区域并收集其两通道荧光强度的比值，并选择多个细胞求其平均值，然后作出两个通道荧光强度比的平均值与SO2浓度对应的柱状图。标尺：20μm。
具体实施方式
实施例1：中间体1的合成
Figure BDA0002321199290000041
将化合物2,3,3-三甲基-3H-吲哚(9.87g,0.062mol)与化合物碘乙烷(14.5g,0.093mol)加入到150mL的圆底烧瓶中，然后加入50mL无水乙腈，在85℃下搅拌反应24h。冷却至室温，待晶体析出后，抽滤，滤饼用3×10mL冰乙醇洗涤3次，得中间体1。
实施例2：近红外荧光探针的合成
Figure BDA0002321199290000042
将中间体1(1g,3.17mmol)和4-(二甲基氨基)肉桂醛(0.67g,3.8mmol)溶于30mL无水乙醇中，然后加入100μL(2滴)哌啶作催化剂，于80℃下搅拌反应8h，冷却至室温，真空旋转蒸发仪除去溶剂，得到的粗产品用硅胶柱层析法纯化，洗脱剂比例为二氯甲烷/乙酸乙酯/甲醇＝40/60/1(v/v)得目标产物。
注：探针通式中各个化合物的合成，可按照上述探针的合成步骤进行合成。
实施例3：荧光探针对水体系中SO2的目视比色法检测
取实施例2中制备的荧光探针溶于乙醇中，配置成浓度为1×10-3mol/L的储备液。量取2mL探针储存液加入到100mL的容量瓶中，用HEPES缓冲溶液(10mM，pH＝7.4)定容至100mL，使探针的最终浓度为20μM。向探针溶液中加入不同浓度的NaHSO3(SO2供体)溶液，使其最终浓度为0-150μM。测定加入不同浓度SO2后，探针吸收峰强度及波长的变化，并用数码相机记录探针与SO2反应前后溶液颜色的变化。紫外-可见吸收光谱及荧光溶液颜色变化的照片见图1。由图所示，探针在波长557nm处具有很强的吸收，随着SO2浓度的增加，其吸收强度逐渐减低，并且在330nm处出现了一个新的吸收峰。插图为加入SO2前后探针溶液颜色的变化，可以清晰地看出探针溶液由深蓝色变为浅粉红色，探针与SO2反应后颜色的显著变化可实现其对SO2的目视比色法检测。
实施例4：荧光探针对水体系中SO2的比率法检测
向实施例3制备的探针溶液中加入不同浓度的NaHSO3(SO2供体)溶液，使SO2最终浓度为0-200μM。加入SO2前后探针荧光光谱变化及其在557和695nm波长处发色峰强度比值与SO2浓度变化的线性关系见图2。由图所示，探针在波长695nm处的近红外区域的发射峰强度很强，而在557nm的可见光区仅有微弱的发射。当向探针溶液中加入不同浓度的SO2，随其浓度的增加，695nm处发射峰强度逐渐减低，而557nm处的发射峰强度逐渐增强，通过比较两个不同波长发射峰强度比值的变化，实现探针对SO2的比率法检测。另外，探针在557和695nm波长处荧光强度的比值与SO2浓度(20-80μM)之间具有很好的线性关系(R2＝0.9948)，在该浓度范围内可实现探针对SO2的定量化检测。
实施例5：荧光探针对SO2的选择性
向实施例3制备的探针溶液中加入浓度为1mM的其它阴离子、生物硫醇和谷胱甘肽(F-、Cl-、Br-、I-,HCO3 -、AcO-、NO2 -、NO3 -、ppi、SO4 2-、HSO4 -、S2O3 2-、S2O8 2-、SCN-、Vc、Cys、Hcy、GSH)以及100μM的S2-、SO3 2-和HSO3 -，其中一组为空白对照。加入不同离子、生物硫醇及谷胱甘肽前后，探针在557和695nm波长处荧光强度比值变化的柱状图见图3。由图可以看出，向探针溶液中加入其他离子、生物硫醇或谷胱甘肽后，探针在557和695nm荧光强度的比值没有发生变化，S2-仅引起探针荧光强度比值的微小变化，而SO2在缓冲溶液中两种存在形式SO3 2-和HSO3 -则使探针两个波长的荧光强度比值显著升高，上述结果表明探针对SO2具有高选择性。
实施例6：荧光探针对细胞内SO2的双通道比率成像
首先向A549细胞中加入100μM的NaHSO3，然后于37℃、5％CO2条件下培养30min，移除旧培养基，并用新鲜培养基(或PBS)洗涤1-2次除去残余的NaHSO3。其中，未加NaHSO3的细胞作为对照组。随后，向细胞中添加实施例3制备的储存液，使探针的最终浓度为5μM，继续培养30min后进行双通道比率成像。细胞荧光图像及双通道中荧光在细胞中的强度变化曲线见图5。由图可以看出，向对照组细胞中加入探针后，红色荧光通道中可观察到明亮的荧光，而黄色荧光通道中仅能观察到微弱的荧光。而向预先加入100μM SO2的A549细胞中加入探针后，红色荧光通道中的荧光变弱，黄色通道中的荧光明显增强。此外，细胞的比率图像及不同通道中荧光曲线也表明，加入外源性SO2后，细胞内两个通道荧光强度的比值(黄/红)明显增加了。上述结果表明探针能够通过双通道比率成像检测细胞内的SO2。
实施例7：荧光探针对细胞内SO2的定量化检测
向A549细胞中分别加入0、10、25、50、100和200μM的NaHSO3，然后于37℃、5％CO2条件下培养30min，移除旧培养基，并用新鲜培养基(或PBS)洗涤1-2次除去残余的NaHSO3。随后，向细胞中添加实施例3制备的储存液，使探针的最终浓度为5μM，继续培养30min后进行激光共聚焦成像。选择多个细胞中相同面积的细胞区域，计算两个通道强度比值的平均值，并做强度比值与SO2浓度对应关系的柱状图。细胞荧光图像及双通道荧光强度比的平均值的柱状图如图5所示。由图5A可以看出，随着细胞内SO2浓度的增加，黄色通道中的荧光亮度逐渐变明亮，红色通道中的荧光强度逐渐变暗。并且当细胞内SO2的浓度达到一定浓度时，两个通道中的荧光亮度不再发生明显变化。由图5B所示，随着SO2浓度增加，两个通道中荧光强度的比值逐渐增加，当细胞内SO2的浓度达到100μM时，其强度比值达到最大值，约增加了4倍。上述结果表明探针能够对活细胞内的SO2进行定量化检测。
上述虽然结合附图对本发明的具体实施方式进行了描述，但并非是对发明保护范围的限制。对本发明所属技术领域的技术人员来说，在不脱离本发明构思的前提下，所作出的若干简单的推演和替换，都应当视为属于本发明的保护范围。

Claims (10)

1.一种双模态检测二氧化硫的近红外荧光探针，其特征在于：
所述近红外荧光探针为半菁类近红外荧光染料，其结构如下式I所示：
Figure FDA0002321199280000011
其中R1为氢、C1-5烷基、苯基、烷氧基或卤素；R2为氢、C1-5烷基、苯基、氨基、N,N-二甲基胺或吗啉。
2.一种权利要求1所述的近红外荧光探针的制备方法，其特征在于：首先由吲哚和碘乙烷通过取代反应得到中间体吲哚盐，然后中间体吲哚盐与醛在催化剂的存在下通过羟醛缩合反应得到目标化合物。
3.根据权利要求2所述的制备方法，其特征在于包括以下步骤：
步骤1：将吲哚和碘乙烷溶于有机溶剂中，于30-100℃下搅拌反应1-36h；反应结束后冷却至室温，晶体析出，抽滤，滤饼用冰乙醇清洗1-5次，得中间体，其结构如下式II所示；
Figure FDA0002321199280000012
其中R1为氢、C1-5烷基、苯基、烷氧基或卤素。
步骤2：将步骤1获得的中间体和醛溶于有机溶剂中，加入催化剂，于30-100℃下搅拌反应1-18h；反应结束后冷却至室温，真空旋转蒸发仪除去溶剂，得粗产品，柱层析纯化得目标产物。
4.根据权利要求3所述的制备方法，其特征在于：
步骤1中，吲哚和碘乙烷的摩尔比为5:1-1:8。
5.根据权利要求3所述的制备方法，其特征在于：
步骤2中，所述催化剂为哌啶。
6.根据权利要求3所述的制备方法，其特征在于：
步骤2中，中间体和醛的摩尔比为1:15-8:1。
7.根据权利要求3所述的制备方法，其特征在于：
步骤2中，所述醛的结构如下式III所示：
Figure FDA0002321199280000021
其中R2为氢、C1-5烷基、苯基、氨基、N,N-二甲基胺或吗啉。
8.根据权利要求3所述的制备方法，其特征在于：
步骤2中，所述纯化是采用硅胶柱层析分离方法进行纯化，洗脱剂由二氯甲烷、乙酸乙酯和甲醇按体积比60:90:1-10:20:1的比例混合构成。
9.一种权利要求1所述的近红外荧光探针的用途，其特征在于：是在通过比色或比率两种检测方法实现水体系中SO2的定性或定量检测过程中作为检测试剂使用。
10.一种权利要求1所述的近红外荧光探针的用途，其特征在于：是在细胞内SO2的双通道比率成像及定量化检测过程中作为检测试剂使用；所述细胞为腺癌人类肺泡基底上皮细胞。
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