CN110423610B - 一种检测双光子汞离子荧光探针及其制备方法与使用方法 - Google Patents
一种检测双光子汞离子荧光探针及其制备方法与使用方法 Download PDFInfo
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Abstract
本发明公开了一种检测双光子汞离子荧光探针及其制备方法与使用方法。本发明利用苯并香豆素构筑具有双光子发射的荧光体系，并在酯羰基处引入硫原子作为汞离子的设别部分，使其能对汞离子进行特异性响应。探针本身由于硫代酯基的存在而在长波处有橙色荧光发射。但当存在汞离子的条件下，由于汞离子促进的水解脱硫反应，使得硫代酯基水解为酯羰基，探针分子发射出强的黄色荧光。本发明提供的苯并香豆素染料的“比率”型汞离子探针对汞离子溶液具有良好的响应，能够实现对样品内微量汞离子的灵敏定量检测，具有操作简便，成本低廉，响应灵敏，易于推广和应用等优点。
Description
技术领域
本发明属于有机小分子荧光探针领域，具体涉及一种作为双光子汞离子荧光探针使用的苯并香豆素类衍生物及其制备方法与使用方法。
背景技术
汞是一种具有严重生理毒性的广泛分布于空气，水和土壤的重金属。汞离子可以通过微生物作用转化成甲基汞，进一步通过各种途径进入食物链并在生物体内逐级积累最终进入到人体内，对人体的大脑、中枢神经等系统产生极大的破坏作用，可造成语言和记忆能力障碍等严重疾病，甚至导致有机体的死亡。近几年来，随着汞在工业生产中的频繁应用，汞已经成为全球性的环境污染物，由此引发的生物危害日益严重。因此，发展高灵敏度和高特异性的汞离子的检测方法，实现实时、原位、动态地检测水体中汞离子的含量，对于保障人民群众生命安全等具有重要的理论意义和实用价值。
荧光检测法由于其优秀的检测灵敏度和选择性，并能实现对生物样品的实时、在线检测而受到研究者的广泛关注。香豆素类荧光分子因其具有良好的光稳定性、高摩尔消光系数和量子产率等独特光学性质而成为该方法最重要的荧光母体之一，在多种待测分子的荧光检测中得到了广泛的应用。
目前已开发的用于检测汞离子的小分子荧光探针主要基于汞离子与硫原子之间的特异性结合力而设计的。当存在汞离子的条件下，探针分子中的硫缩醛或是硫代酰胺等官能团被汞离子选择性地水解并以HgS或是汞的二硫配体等形式脱除硫原子，导致探针分子的荧光性质发生变化，从而实现对汞离子的特异性识别。
然而，目前基于硫缩醛或是硫代酰胺为探针报告基团的探针(参见综述Mahato,P.；Saha,S.；Das,P.；Agarwalla H.；Das,Amitava.RSC Adv.,2014,4,36140–36174.)常以香豆素、荧光素、BODIPY、罗丹明等为荧光母团，波长主要集中在蓝-绿-黄等区域，在生物实验中往往难以穿透生物的深层组织，不利于其在复杂的生物体系内进行准确检测。此外，目前的汞离子荧光探针的检测限相对较高，难以对微量甚至极微量的含汞离子样品进行定量检测。
发明内容
本发明为了克服现有技术中的上述缺陷，本发明提出了一种定量检测汞离子的苯并香豆素类双光子荧光探针(TPM1)。利用本发明可对样品中极微量的汞离子进行定量检测。
本发明的核心在于利用苯并香豆素构筑具有双光子发射的荧光体系，并在酯羰基处引入硫原子作为汞离子的设别部分，使其能对汞离子进行特异性响应。探针本身由于硫代酯基的存在而在长波处有橙色荧光发射。但当存在汞离子的条件下，由于汞离子促进的水解脱硫反应，使得硫代酯基水解为酯羰基，探针分子发射出强的黄色荧光。通过上述方案，获得了“比率”型的荧光响应，实现了对汞离子的高灵敏、特异性检测。
本发明所述汞离子荧光探针命名为4-甲基-8-吗啉代-2H-苯并[g]色烯-2-硫酮，结构式如式(I)所示：
上述汞离子荧光探针的制备方法如下：将一定量的8-羟基-4-甲基-2H-苯并[g]苯并吡喃-2-酮(1)，和三氟甲磺酸酐溶于吡啶中，常温搅拌12小时，旋干溶剂，加入二氯甲烷，用水洗涤，干燥得化合物8-三氟甲磺酰氧基-4-甲基-2H-苯并[g]苯并吡喃-2-酮(2)。将一定量的8-三氟甲磺酰氧基-4-甲基-2H-苯并[g]苯并吡喃-2-酮(2)，醋酸钯，碳酸铯，2,2'-双(二苯基膦)-1,1'-联萘和吗啡啉溶于甲苯中，加热回流12小时，将反应液用硅藻土过滤，旋干溶剂，向混合物中加入水，用二氯甲烷萃取，干燥后得到4-甲基-8-吗啉代-2H-苯并[g]色烯-2-酮(3)。将一定量4-甲基-8-吗啉代-2H-苯并[g]色烯-2-酮(3)和劳森试剂溶于甲苯中，回流反应4小时后，旋干溶剂，向混合物中加入水，用二氯甲烷萃取，干燥后的固体用乙醇重结晶后得到4-甲基-8-吗啉代-2H-苯并[g]色烯-2-硫酮(4)，即：TPM1。
上述探针的制备反应式如下：
上述汞离子荧光探针的使用方法如下：
步骤1：向不同浓度汞离子的缓冲溶液中加入相同浓度的式(I)所示化合物，配置至少5种不同汞离子含量的含有式(I)所示化合物的标准溶液；
所示缓冲溶液可以是磷酸盐缓冲溶液、Tris-HCl缓冲溶液、HEPES缓冲溶液或硼酸-硼酸钠缓冲溶液；
所示标准溶液的pH值为5～11；
所示标准溶液中式(I)所示化合物的浓度为1nM～1μM；
所示标准溶液中汞离子的含量为0.1nM～1mM；
步骤2：分别测定所述标准溶液的荧光发射光谱，激发波长为485nm或800nm，以汞离子浓度为横坐标，以I553/I635为纵坐标，建立标准曲线；
I553表示所述标准溶液在波长为553nm处的荧光发射峰强度值；
I635表示所述标准溶液在波长为635nm处的荧光发射峰强度值；
步骤3：向待测样品中加入式(I)所示化合物，控制其浓度与所述标准溶液中式(I)所示化合物的浓度相等；测定其在激发波长为485nm的激发光下的荧光发射谱，即根据标准曲线计算得出待测样品的汞离子含量。
本发明具有如下特点：
1)本发明提供的荧光探针是黄色固体粉末，分子结构光学稳定性。
2)本发明提供的荧光探针，其溶液对汞离子的浓度敏感，随着汞离子浓度的增加，紫外灯下观察到其水溶液的荧光由橙色变为黄色。
3)本发明提供的荧光探针，其激发波长为485nm或800nm，为双光子探针，能大大增加其在生物领域的应用。
4)本发明提供的荧光探针，其发射波长为553nm/635nm，为荧光“比率”型响应，能大大消除检测时检测条件差异对结果的影响，提高检测的灵敏度。
5)本发明提供的荧光探针对汞离子浓度呈线性关系，可用于汞离子精确测量。
本发明提供的新型双光子苯并香豆素类“比率”型汞离子探针对汞离子溶液具有良好的响应，能够实现对样品内汞离子的灵敏定量检测，具有操作简便，成本低廉，响应灵敏，易于推广和应用等优点。
附图说明
图1：荧光探针TPM1的核磁共振氢谱。
图2：荧光探针TPM1对汞离子水溶液的颜色响应图。
图3：荧光探针TPM1对汞离子水溶液的荧光响应图。
图4：荧光探针TPM1在缓冲液中汞离子的紫外滴定曲线，其中探针浓度为1.0μM。
图5：荧光探针TPM1在缓冲液中汞离子的荧光滴定曲线，其中激发波长为485nm，探针浓度为1.0μM。
图6：荧光探针TPM1对常见共存离子的荧光响应图，其中激发波长为485nm，探针浓度为1.0μM，离子浓度为10.0μM。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均从商业途径得到。
实施例中的化合物号码对应上述化合物中的号码。
实施例1、化合物TPM1的合成
化合物8-三氟甲磺酰氧基-4-甲基-2H-苯并[g]苯并吡喃-2-酮(2)的合成
将10g 8-羟基-4-甲基-2H-苯并[g]苯并吡喃-2-酮(1)(44mmol)和9.6mL三氟甲磺酸酐(57mmol)溶于400mL吡啶中，室温反应12小时后，旋干溶剂，加入1L二氯甲烷，用1L水洗涤，干燥得化合物8-三氟甲磺酰氧基-4-甲基-2H-苯并[g]苯并吡喃-2-酮(2)，该化合物可直接用于下一步反应。
化合物4-甲基-8-吗啉代-2H-苯并[g]色烯-2-酮(3)的合成
将5g化合物8-三氟甲磺酰氧基-4-甲基-2H-苯并[g]苯并吡喃-2-酮(2)(14mmol)，188mg醋酸钯(0.8mmol)，3.8g碳酸铯(11.7mmol)，782mg 2,2'-双(二苯基膦)-1,1'-联萘(1.2mmol)和7mL吗啡啉(84mmol)溶于500mL甲苯中，加热回流12小时，将反应液用硅藻土过滤，旋干溶剂，向混合物中加入500mL水，用500mL二氯甲烷萃取，干燥后得到4-甲基-8-吗啉代-2H-苯并[g]色烯-2-酮(3)，该化合物可直接用于下一步反应。
化合物1-(3’-(二乙基氨基)-6’-((4-甲氧基苯基)乙炔基)-3-氧代螺[异二氢吲哚-1,9'-呫吨]-2-基)-3-苯基硫脲(5)的合成
将1.3g 4-甲基-8-吗啉代-2H-苯并[g]色烯-2-酮(3)(4.5mmol)和2.7g劳森试剂(6.7mmol)溶于200mL甲苯中，回流反应4小时后，旋干溶剂，向混合物中加入200mL水，用300mL二氯甲烷萃取，干燥后的固体用50mL乙醇重结晶后得到4-甲基-8-吗啉代-2H-苯并[g]色烯-2-硫酮(4)，即：TPM1，产率45％。核磁共振图谱如图1。
1H NMR(400MHz,Chloroform-d)δ7.95(s,1H),7.81(d,J＝9.2Hz,1H),7.60(s,1H),7.30(dd,J＝9.0,2.2Hz,1H),7.08(d,J＝1.3Hz,1H),7.03(d,J＝2.4Hz,1H),3.98–3.92(m,4H),3.42–3.36(m,4H),2.43(d,J＝1.2Hz,3H)。
实施例2、化合物TPM1对汞离子的颜色响应
配制浓度为1mM的本发明所述检测汞离子的荧光探针TPM1的二甲基亚砜(DMSO)的测试母液溶液待用。量取10μL的此母液滴加到一定浓度汞离子的磷酸盐缓冲液中，并用相应的磷酸盐缓冲液定容到10mL，使得测试液中，探针的浓度为1.0μM，汞离子浓度为10.0μM进行颜色响应测试。如图2和3所示，加入汞离子水溶液后，肉眼观察到溶液的颜色由浅黄色变为无色，同时溶液的荧光也由橙色荧光变为黄色荧光，表明探针TPM1对汞离子具有直观的显色响应。
实施例3、不同浓度汞离子对化合物TPM1的紫外滴定检测
配制浓度为1mM的本发明所述检测汞离子的荧光探针TPM1的二甲基亚砜(DMSO)的测试母液溶液待用。量取10μL的此母液分别滴加到不同浓度汞离子的磷酸盐缓冲液中，并用相应的磷酸盐缓冲液定容到10mL，使得测试液中，探针的浓度为1.0μM，汞离子浓度为0-3.0μM进行吸收光谱测试。得各体系中紫外吸收曲线，建立吸光度与汞离子浓度标准曲线。如图4所示，随着汞离子浓度的增加，370nm处吸光度逐渐增加而416nm处吸光度逐渐减弱，并且A370/A416的比值与汞离子浓度(0-3.0μM)呈良好的线性关系。
实施例4、不同浓度汞离子对化合物TPM1的荧光滴定检测
配制浓度为1mM的本发明所述检测汞离子的荧光探针TPM1的二甲基亚砜(DMSO)的测试母液溶液待用。量取10μL的此母液分别滴加到不同浓度汞离子的磷酸盐缓冲液中，并用相应的磷酸盐缓冲液定容到10mL，使得测试液中，探针的浓度为1.0μM，汞离子浓度为0-1.5μM进行荧光检测(λex＝485nm,λem1＝553nm，λem2＝635nm)。得各体系中荧光强度，建立荧光强度与汞离子浓度标准曲线。如图5所示，随着汞离子浓度的增加，553nm处荧光强度逐渐增加而635nm处荧光强度逐渐减弱，当汞离子浓度达到2.0μM时，反应体系荧光强度达到饱和状态。此外，在低浓度下，荧光强度和汞离子的浓度(0-0.8μM)之间呈现良好的线性关系(R2＝0.99)，探针TPM1对汞离子的检测限可达8.5nM。
实施例5、化合物TPM1对常见共存离子的选择性
配制浓度为1mM的本发明所述检测汞离子的荧光探针TPM1的二甲基亚砜(DMSO)的测试母液溶液待用。配制浓度为10mM的各种不同离子，小分子的溶液作为备用。量取10μL的此母液分别滴加到不同待测离子或小分子的磷酸盐缓冲液中，并用相应的磷酸盐缓冲液定容到10mL，使得测试液中，探针的浓度为1.0μM，待测离子或小分子的浓度为2.0μM进行荧光检测(λex＝485nm,λem1＝553nm，λem2＝635nm)。得各体系中荧光强度，建立荧光强度与各离子的柱状图。如图6所示，其他离子对探针TPM1的荧光几乎没有影响。
Claims (3)
1.一种检测双光子汞离子荧光探针,其特征在于：其分子式为C18H17NO2S，简称TPM1，结构式为式(I)：
2.如权利要求1所述的一种检测双光子汞离子荧光探针的制备方法，其特征在于，合成步骤如下：
1)将10g 8-羟基-4-甲基-2H-苯并[g]苯并吡喃-2-酮和9.6mL三氟甲磺酸酐溶于400mL吡啶中，室温反应12小时后，旋干溶剂，加入1L二氯甲烷，用1L水洗涤，干燥得化合物8-三氟甲磺酰氧基-4-甲基-2H-苯并[g]苯并吡喃-2-酮；
2)将5g化合物8-三氟甲磺酰氧基-4-甲基-2H-苯并[g]苯并吡喃-2-酮，188mg醋酸钯，3.8g碳酸铯，782mg 2,2'-双(二苯基膦)-1,1'-联萘和7mL吗啡啉溶于500mL甲苯中，加热回流12小时，将反应液用硅藻土过滤，旋干溶剂，向混合物中加入500mL水，用500mL二氯甲烷萃取，干燥后得到4-甲基-8-吗啉代-2H-苯并[g]色烯-2-酮；
3)将1.3g 4-甲基-8-吗啉代-2H-苯并[g]色烯-2-酮和2.7g劳森试剂溶于200mL甲苯中，回流反应4小时后，旋干溶剂，向混合物中加入200mL水，用300mL二氯甲烷萃取，干燥后的固体用50mL乙醇重结晶后得到4-甲基-8-吗啉代-2H-苯并[g]色烯-2-硫酮，即：TPM1。
3.根据权利要求1所述的一种检测双光子汞离子荧光探针的使用方法；其特征在于：
1)向不同浓度汞离子的缓冲溶液中加入相同浓度的式(I)所示化合物，配置至少5种不同汞离子含量的含有式(I)所示化合物的标准溶液；
所示缓冲溶液为磷酸盐缓冲溶液、Tris-HCl缓冲溶液、HEPES缓冲溶液或硼酸-硼酸钠缓冲溶液；
所示标准溶液的pH值为5～11；
所示标准溶液中式(I)所示化合物的浓度为1nM～1μM；
所示标准溶液中汞离子的含量为0.1nM～1mM；
2)分别测定所述标准溶液的荧光发射光谱，激发波长为485nm，以汞离子浓度为横坐标，以I553/I635为纵坐标，建立标准曲线；
I553表示所述标准溶液在波长为553nm处的荧光发射峰强度值；
I635表示所述标准溶液在波长为635nm处的荧光发射峰强度值；
3)向待测样品中加入式(I)所示化合物，控制其浓度与所述标准溶液中式(I)所示化合物的浓度相等；测定其在激发波长为485nm的激发光下的荧光发射谱，根据标准曲线计算得出待测样品的汞离子含量。
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