CN116496233A - 一种具有聚集诱导发光性质的pH荧光探针及其制备和应用 - Google Patents
一种具有聚集诱导发光性质的pH荧光探针及其制备和应用 Download PDFInfo
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Abstract
本申请公开了一种具有聚集诱导发光性质的pH荧光探针及其制备方法和应用，pH荧光探针结构式如式(I)所示：
Description
技术领域
本发明涉及荧光检测领域，具体涉及一种聚集诱导发光性质的pH荧光探针及其制备和应用。
背景技术
众所周知，pH是影响着环境水质和生物体生长发育的一项重要指标。一方面，天然水体的pH是处于一定范围内，其异常变化会抑制水中微生物的生长，若长期处于异常值下会破坏水的生态平衡，降低水中种群数量，甚至可导致鱼类绝迹。另一方面，pH在许多生理过程中起着重要的作用，具有调节细胞活动的功能，正常情况下细胞内pH一般为7.2～7.4，在阿尔兹海默症、肝损伤、糖尿病和肿瘤等疾病中，pH值往往都会偏离正常生理指标。
综上所述，在环境监测、生态保护和细胞内环境检测等领域实现pH值的定量检测具有十分重要的实际意义。检测pH值的方法有很多，如电位滴定法、酸碱滴定法等。但是这些方式具有操作不简便、价格高等缺点。和以上手段相比，荧光探针往往具有灵敏度高、选择性好、响应时间短、操作方便等优点，到目前为止，研究者们已开发多种pH荧光探针，但是多数探针有以下三个缺点：1)多数探针合成步骤多，制备成本高；2)多数荧光探针具有不良的聚集诱导猝灭效应，容易被光漂白；3)多数探针的斯托克斯位移小，容易收到仪器背景的干扰。因此，迫切需要开发具有易制备、聚集诱导发光性质和斯托克斯位移大等优点的pH荧光探针。
发明内容
本申请提供一种易制备、聚集诱导发光性质和斯托克斯位移大等优点的pH荧光探针及其制备方法和应用。
本申请的第一个目的是提供一种具有聚集诱导发光性质的pH荧光探针，所述pH荧光探针的结构式如式(I)所示：
可选的，所述pH荧光探针的pH值响应范围为5.33～8.72。
可选的，所述pH荧光探针的pKa为7.07。
本申请的第二个目的是提供一种所述pH荧光探针的制备方法，包括：
将化合物(II)、溶剂、碘化钾、碳酸铯和化合物(III)依次放入反应瓶中，并置于55～65℃条件下反应；待反应结束后，将反应液进行分离纯化，获得所述式(I)所示的pH探针。
反应式如下：
可选的，所用实际化合物(II)和化合物(III)均为公开的化合物，购买自试剂公司。
可选的，所述化合物(II)与化合物(III)、碘化钾、碳酸铯按物质的量比为1:1.5～2:0.1:2。
可选的，所述溶剂为乙腈。
可选的，反应时间为15-20小时。
进一步可选的，反应温度为60℃，反应时间为16小时。
可选的，所述反应液分离纯化为：先过滤，滤液减压旋蒸去除溶剂，取浓缩物进行柱层析分离，以体积比70:1的二氯甲烷甲醇混合液为展开剂，洗脱目标产物，获得式(I)所示的pH荧光探针。
本申请的第三个目的是提供所述的具有聚集诱导发光性质的pH荧光探针在pH值检测中的应用。
具体地，本申请提供一种所述pH荧光探针在制备检测pH值的试剂、试剂盒或试纸条中的应用。
本申请还提供一种检测pH值的试剂盒，包含所述的pH荧光探针。所述pH荧光探针按照本领域成熟的方法制成试剂盒。
本申请还提供一种所述pH荧光探针在检测生态环境中pH值或非诊断目的的体外检测活细胞内pH值中的应用。
本申请提供的具有聚集诱导发光性质的pH荧光探针，可对溶液中的pH值进行定量检测，因此，本申请还提供一种pH值定量检测方法，可用于生态环境中pH值的定量检测，包括：
将所述的pH荧光探针加入待测溶液中，混匀得检测液；在激发波长为320nm、发射波长为450nm下采集检测液的荧光强度，代入标准曲线计算得到待测溶液的pH值。
可选的，待测溶液中加入的具有聚集诱导发光性质的pH荧光探针的终浓度为0.01mM，与待测溶液的pH值为5.33～8.72时呈良好的线性关系，能够对该浓度范围内的待测溶液的pH值的定量检测。
可选的，标准曲线的制备如下：
将0.01mM荧光探针分别置于pH值为5.33～8.72内的溶液，激发波长为320nm，发射波长为450nm下采集待测溶液的荧光强度，以pH为纵坐标、Log[(Imax-I)/(I-Imin)]为横坐标，绘制即得线性标准曲线。
本申请还提供一种所述pH荧光探针在细胞成像或在制备细胞成像试剂中的应用。
可选的，所述细胞为肿瘤细胞。进一步地，所述细胞为人宫颈癌细胞Hela细胞。
本申请提出的荧光探针检测原理为：以萘环为荧光母核，吗啉基团通过光诱导电子转移机制使得探针的荧光淬灭，pH值的降低会使吗啉基团发生质子化，光诱导电子转移机制降低，荧光开启。
与现有技术相比，本申请至少具有如下有益效果之一：
(1)本申请提供的pH荧光探针具有聚集诱导发光性质，不易发生光漂白。
(2)本申请提供的pH荧光探针的制备方法简单，反应产率高，其信号不受离子的干扰，并可对溶液pH值进行精准的定量检测。
(3)本申请提供的pH荧光探针属于D-π-A类荧光探针，具有斯托克斯位移大的优点，不易受到仪器的干扰，具有良好的科研价值和应用价值。
(4)在检测pH过程中，探针的选择性好，斯托克斯位移大，可精准的定量检测溶液的pH值，在环境监测、生态保护和细胞内环境等领域在探测酸度有广泛的应用前景。
(5)本申请提供的pH荧光探针可用于生态环境中的pH定量检测。
(6)本申请提供的pH荧光探针可用于细胞成像，检测细胞内的pH变化。
附图说明
图1为实施例1制备的探针(I)的核磁氢谱。
图2中a为实施例1制备的探针(I)随着水/二甲基亚砜混合溶液中水体积的变化而产生的荧光光谱图(激发波长320nm)；图2中b为探针(I)随着水/二甲基亚砜混合溶液中水体积的变化而产生的荧光强度图(激发波长320nm，发射波长450nm)。
图3中a为实施例1制备的探针(I)加入到不同pH的DMSO/PBS缓冲液(v/v＝1/99)中的荧光发射光谱图，图3中b为pH与Log[(Imax-I)/(I-Imin)]的线性关系(激发波长320nm，发射波长450nm)；I为荧光强度，Imax是探针在pH＝5.03时的荧光强度，Imin是探针在pH＝9.87时的荧光强度。
图4为实施例1制备的探针(I)对常见离子的抗干扰性图(激发波长320nm，发射波长450nm)(横坐标的1～8依次为CO3 2-、CH3COO-、SO4 2-、I-、F-、K+、Zn2+、Sr2+)。
图5为实施例1制备的探针(I)在pH酸碱交替条件下的荧光强度图。(激发波长320nm，发射波长450nm)。
具体实施方式
下面将结合本申请实施例中的附图，对本申请实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。
除非另有定义，本文所使用的所有的技术和科学术语与属于本申请的技术领域的技术人员通常理解的含义相同。本文中在本申请的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本申请。
以下以具体的实施例进行说明：
实施例1：探针(I)的制备
将化合物(II)、溶剂(乙腈)、碘化钾、碳酸铯和化合物(III)依次放入圆底瓶中，化合物(II)与化合物(III)、碘化钾、碳酸铯按物质的量比1:1.6:0.1:2。其中化合物(II)为1mmol，乙腈用量为5mL。反应液置于60℃条件下反应16小时。待反应结束后，将反应液进行分离纯化，先过滤，滤液减压旋蒸去除溶剂，取浓缩物进行柱层析分离，以体积比70:1的二氯甲烷甲醇混合液为展开剂，洗脱目标产物，得到白色固体251.3mg，产率为72.1％。
其核磁氢谱参见图1。
1H NMR(400MHz,Chloroform-d)δ10.10(s,1H),8.26(s,1H),7.91(t,J＝8.8Hz,2H),7.79(d,J＝8.6Hz,1H),7.23(dd,J＝8.9,2.4Hz,1H),7.19(d,J＝2.3Hz,1H),4.19(t,J＝6.3Hz,2H),3.75(q,J＝4.4Hz,4H),2.61–2.56(m,2H),2.52(s,4H),2.07(d,J＝6.5Hz,2H).
实施例2：探针(I)(10μM)的聚集诱导发光性质的研究。
准确称取一定量的探针(I)(实施例1制备)，用二甲基亚砜配制成浓度为1mM的探针母液，移液枪吸取40μL探针溶液，加入到4mL不同比例DMSO/水混合溶液中，探针终浓度10μM，用荧光分光光度计测试荧光光谱。激发波长320nm、发射波长450nm。
图2中a为实施例1制备的探针(I)随着水/二甲基亚砜混合溶液中水体积的变化而产生的荧光光谱图(激发波长320nm)；图2中b为探针(I)随着水/二甲基亚砜混合溶液中水体积的变化而产生的荧光强度图(激发波长320nm，发射波长450nm)。由图2中a和b所示附图可知，在良性溶剂DMSO中，探针(I)几乎没有荧光，随着DMSO/水混合溶液中水含量比例的增加，即不良溶剂比例在混合溶剂中比例越来越高，探针(I)的荧光强度逐渐增强。因此，此结果表明探针(I)具有聚集诱导发光的性质。
实施例3：探针(I)(10μM)对pH响应的荧光光谱测试。
准确称取一定量的探针(I)(实施例1制备)，用二甲基亚砜配制成浓度为1mM的探针母液，移液枪吸取5μL加入到495μL PBS缓冲液中，配置成DMSO/PBS缓冲液(1:99，v:v，pH依次为5.03、5.33、5.81、6.65、6.97、7.44、7.73、8.72、9.87)，使探针终浓度10μM，摇匀后加入到96孔板中用多功能酶标仪测定探针(I)的荧光光谱，并做相关线性曲线。
图3中a所示附图为实施例1制备的探针(I)加入到不同pH的DMSO/PBS缓冲液(v/v＝1/99)中的荧光发射光谱图，由图3中a可知，随着pH的降低，探针(I)的荧光强度不断增强，在450nm处具有最强发射荧光强度，表明探针(I)的斯托克斯位移为130nm。
图3中b为pH与Log[(Imax-I)/(I-Imin)]的线性关系(激发波长320nm，发射波长450nm，其中，I为荧光强度，Imax是探针在pH＝5.03时的荧光强度，Imin是探针在pH＝9.87时的荧光强度。根据Henderson-Hasselbalch方程计算，得出探针(I)的pKa为7.07，且得到pH＝1.59Log[(Imax-I)/(I-Imin)]+7.07，从而实现探针对溶液中pH定量检测的能力。
实施例4：探针(I)(10μM)对pH的选择性研究
准确称取一定量的探针(I)(实施例1制备)，用二甲基亚砜配制成浓度为1mM的探针母液，移液枪吸取5μL加入到495μL含有相关离子(依次为CO3 2-、CH3COO-、SO4 2-、I-、F-、K+、Zn2+、Sr2+)，使最终的混合液体系为DMSO/PBS缓冲液(1:99，v:v，pH 7.4)，探针终浓度10μM，其中各种离子的终浓度为0.1mM，摇匀后加入到96孔板中，并用多功能酶标仪测定探针(I)的荧光强度。
荧光图谱见图4，其中横坐标的1～8依次为CO3 2-、CH3COO-、SO4 2-、I-、F-、K+、Zn2+、Sr2 +。数据表明，在pH为7.4的PBS缓冲液环境下，几个重要相关离子对探针(I)的荧光信号的影响不大。以上的实验数据表明探针(I)具有良好的pH专一性。
实施例5：探针(I)(10μM)对pH响应可逆性
准确称取一定量的探针(I)(实施例1制备)，用二甲基亚砜配制成浓度为1mM的探针母液，移液枪吸取10μL加入到990μL溶液中(pH＝4.0)，然后交替调节溶液pH从酸至碱再至酸，往返调节4次，激发波长320nm、发射波长450nm测试其荧光强度。
荧光图谱见图5，数据表明，探针(I)随着4次酸性-碱性的pH循环，其荧光强度随之变化，证明探针的pH响应可逆性良好。
以上所述实施例仅表达了本申请的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本申请构思的前提下，还可以做出若干变形和改进，这些都属于本申请的保护范围。因此，本申请专利的保护范围应以所附权利要求为准。
Claims (10)
1.一种具有聚集诱导发光性质的pH荧光探针，其特征在于，所述pH荧光探针的结构式如式(I)所示：
2.根据权利要求1所述的pH荧光探针，其特征在于，所述pH荧光探针的pH值响应范围为5.33～8.72。
3.根据权利要求1所述的pH荧光探针，其特征在于，所述pH荧光探针的pKa为7.07。
4.如权利要求1所述pH荧光探针的制备方法，其特征在于，包括：
将化合物(II)、溶剂、碘化钾、碳酸铯和化合物(III)依次放入反应瓶中，并置于55～65℃条件下反应；待反应结束后，将反应液进行分离纯化，获得如式(I)所示的pH荧光探针。
5.根据权利要求4所述的制备方法，其特征在于，所述化合物(II)与化合物(III)、碘化钾、碳酸铯按物质的量比为1:1.5～2:0.1:2；所述溶剂为乙腈；反应时间为15-20小时；
所述反应液分离纯化为：先过滤，滤液减压旋蒸去除溶剂，取浓缩物进行柱层析分离，以体积比70:1的二氯甲烷甲醇混合液为展开剂，洗脱目标产物，获得式(I)所示的pH荧光探针。
6.如权利要求1～3任一项权利要求所述pH荧光探针在制备检测pH值的试剂、试剂盒或试纸条中的应用。
7.一种检测pH值的试剂盒，其特征在于，包含如权利要求1～3任一项权利要求所述的pH荧光探针。
8.如权利要求1～3任一项权利要求所述pH荧光探针在检测生态环境中pH值或非诊断目的的体外检测活细胞内pH值中的应用。
9.一种非诊断目的的pH值定量检测方法，其特征在于，包括：
将如权利要求1所述的pH荧光探针加入待测溶液中，混匀得检测液；在激发波长为320nm、发射波长为450nm下采集检测液的荧光强度，代入标准曲线计算得到待测溶液的pH值。
10.根据权利要求9所述的pH值定量检测方法，其特征在于，所述待测溶液中加入的pH荧光探针的终浓度为0.01mM，待测溶液的pH值为5.33～8.72。
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