CN114835636A - 一种萘-乙烯基吡啶基双响应型荧光探针及其制备方法和用途 - Google Patents
一种萘-乙烯基吡啶基双响应型荧光探针及其制备方法和用途 Download PDFInfo
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Abstract
本发明公开了一种萘‑乙烯基吡啶基双响应型荧光探针及其制备方法和用途，其中荧光探针的结构式为：
本发明提供的萘‑乙烯基吡啶基双响应型荧光探针具有多功能性，可以通过荧光光谱法分别实现对ONOO‑离子和/或粘度的识别及定量检测，可用于制备ONOO‑浓度和/或粘度检测试剂，在其它分析物存在的情况下有很好的抗干扰性、高选择性和灵敏性；同时该探针能够用于细胞内的成像，有望在工业生产和临床医学中发挥作用，具有广阔的应用前景。
Description
技术领域
本发明涉及一种萘-乙烯基吡啶基双响应型荧光探针及其制备方法和用途，属于ONOO-离子和/或粘度的检测和荧光分子探针领域。
背景技术
过氧亚硝酸根(ONOO-)作为生物体内一种高活性的物质，与关节炎、糖尿病、癌症、心血管疾病及神经退行性疾病等疾病密切相关。同时，相关研究表明ONOO-在生命体中会起到非常积极的作用，如通过硝化酪氨酸残基可实现信号传导，在免疫反应中对抗病原体的入侵。所以，体内ONOO-实时的浓度变化及其在体内分布情况的分析对生物学和生命医学的研究具有重要参考意义。因此，建立一种可行的、灵敏度高、特异性强的过氧亚硝酸根检测方法显得尤为重要。
粘度是反映溶液粘稠程度的一种重要参数，其主要由物质在溶液中的扩散速度决定。无论在宏观还是微观领域，都具有十分重要的应用价值。在生物体系中，其内部所处环境的粘度水平在整个生命体系中发挥着不可替代的作用。当人体内的血浆粘度逐渐增加时，就会使人诱发各种疾病，例如高血压、高血脂、脑梗塞、心脏病等，危害人们的生命健康。
近年来，荧光探针法因其制备过程简单、生物相容性好、稳定性强及灵敏度高等良好的化学和物理性能而受到广泛关注。此外，荧光探针具有很强的实际应用价值，可以广泛用于自然环境水体资源中、有机体内监测金属离子及生命体细胞的显微成像等。因此，合成检测ONOO-和/或粘度荧光探针具有重要意义。经检索，有关ONOO-和/或粘度荧光双响应型探针及其应用的专利鲜见报道。
发明内容
本发明旨在提供一种萘-乙烯基吡啶基双响应型荧光探针及其制备方法和用途，所要解决的技术问题是通过分子设计合成能够识别检测ONOO-和/或粘度的双响应型荧光探针。
本发明萘-乙烯基吡啶基双响应型荧光探针，化学式为C26H23Cl2NO，结构式如下所示：
本发明萘-乙烯基吡啶基双响应型荧光探针的制备方法，包括如下步骤：
以25mL丙酮作溶剂，称取化合物ZL(435mg，1mmol)和1,4对二氯苄(193mg，1.1mmol)，滴加两滴DMF增加溶解性，60℃下回流反应8-12h，得到黄色沉淀，用甲醇过滤重结晶两次得到目标产物。
本发明合成过程如下：
本发明萘-乙烯基吡啶基双响应型荧光探针的用途，是在定性或定量检测ONOO-和/或粘度时作为检测试剂使用。
本发明萘-乙烯基吡啶基双响应型荧光探针作为检测试剂，在含水介质中进行荧光光谱测定，通过荧光强度的变化实现对ONOO-和/或粘度的定性或定量检测。
所述含水介质为DMF和水按体积比3：1构成的混合溶液。
所述双响应型荧光探针作为检测试剂用于检测ONOO-离子时，检测限为0.45μM。
本发明双响应型荧光探针，可用于ONOO-和/或粘度的识别和检测，对多种分析物有较强的抗干扰能力。
本发明的有益效果体现在：
本发明双响应型荧光探针具有多功能性，可以通过荧光光谱法实现对ONOO-离子和/或粘度的识别。本发明双响应型荧光探针可用于在含水的介质中对ONOO-离子的快速识别、定量检测，而且对ONOO-离子识别具有较高的选择性和较好的抗干扰能力。
本发明双响应型荧光探针可用于含水溶液中对ONOO-离子和/或粘度进行快速的识别和定量检测，并且明显的颜色变化现象可以实现裸眼识别。在生物应用方面对探针的实用性探究表明，该探针溶液可对细胞中外源性的ONOO-离子的进行检测。上述实验结果说明我们的探针在环境监测及生物体中有良好的应用潜质。
附图说明
图1为本发明双响应型荧光探针的核磁氢谱。
图2中a为本发明双响应型荧光探针在不同粘度值的溶液的荧光发射光谱图(λex＝400nm)；图2中b为该荧光探针在荧光发射波长550nm处的荧光强度与溶液粘度的线性关系。
图3中a为本发明双响应型荧光探针在DMF和水体积比为3：1的混合溶液中加入不同分析物的紫外-吸收光谱；图3中b为该荧光探针在DMF和水体积比为3：1的混合溶液中加入不同分析物的荧光发射光谱图(λex＝396nm)。
图4为本发明双响应型荧光探针在DMF和水体积比为3：1的混合溶液中加入不同分析物的抗干扰柱状图；
代表荧光探针在DMF和水体积比为3：1的混合溶液中加入不同分析物的荧光强度(λex＝396nm)；
代表荧光探针化合物在DMF和水体积比为3：1的混合溶液中加入不同分析物和ONOO-离子的荧光强度(λex＝396nm)。
图5中a为本发明双响应型荧光探针在DMF和水体积比为3：1的混合溶液中加入0-5.0equiv离子浓度的ONOO-后的荧光发射光谱(λex＝396nm)；图5中b为该荧光探针在荧光发射波长540nm处荧光强度与ONOO-浓度的线性关系。
图6为本发明双响应型荧光探针用于Hela细胞中ONOO-检测的荧光成像图。
具体实施方式
本发明可以通过以下的实施例进一步说明，但不仅仅局限于实施例。
实施例1：目标产物的合成
以25mL丙酮作溶剂，称取化合物ZL(261mg,1mmol)和1,4对二氯苄(193mg，1.1mmol),滴加两滴DMF增加溶解性，60℃下回流过夜，冷却室温，析出黄色沉淀，用甲醇过滤重结晶两次得到目标产物246mg,产率为56.55％。1H NMR(400MHz,DMSO-d6)δ9.02(d,J＝6.5Hz,2H),8.24(d,J＝6.5Hz,2H),8.16–8.08(m,2H),7.90–7.85(m,3H),7.59(d,J＝4.6Hz,1H),7.52(d,J＝18.9Hz,4H),7.36(d,J＝2.5Hz,1H),7.20(dd,J＝8.9,2.5Hz,1H),5.74(d,J＝6.9Hz,2H),4.74(s,2H),3.87(s,3H).
实施例2：本发明双响应型荧光探针对溶液粘度的特异性响应及探针的荧光强度与溶液粘度的相关性
准确称取一定量的萘-乙烯基吡啶基双响应型荧光探针，用二甲基亚砜配制成浓度为1mM的探针母液，移液枪吸取20μL加入到2.0mL不同粘度值的水-甘油混合液，经过混匀，超声、静置后，加入到荧光比色皿中，在激发波长为400nm处测定探针的荧光光谱，统计数据，并考察荧光强度值与粘度值两者的定量关系。不同粘度值的水-甘油混合液中探针的荧光图谱见图2a。数据表明，在激发波长为400nm处，随着混合液粘度η的升高，探针的荧光强度逐渐增加。以log(I/I0)为纵坐标(其中I为发射波长550nm处的荧光强度，I0为纯水中的荧光强度)、logη(η为溶液粘度)为横坐标做线性关系图(图2b)，可以发现，两者具有很好的线性关系(R2＝0.96)，证明了探针对溶液粘度(1.005～219CP之间)具有良好的定量检测效果。
实施例3：本发明双响应型荧光探针对ONOO-离子的特异性响应
准确称取一定量萘-乙烯基吡啶基双响应型荧光探针，溶解并配制成浓度为1.0×10-3mol/L的DMSO储备溶液；用DMF和水体积比为3：1的混合溶液将储备液稀释成浓度为1.0×10-5mol/L的待测溶液。取3mL浓度为1.0×10-5mol/L的待测溶液于石英比色皿中(石英比色皿的厚度为1cm)，然后分别加入30μL浓度为1.0×10-2mol/L的各种分析物(Blank，ONOO-，ClO-，Cys，H2O2，GSH，NO2 -，CO3 2-，HCO3 -，S2-，HSO3 -，SO3 2-，H2PO4 -，HPO4 2-，·OH)的水溶液，摇匀后测定溶液的紫外-可见吸收光谱(如图3a)。探针溶液的紫外-可见吸收光谱在410nm处，ONOO-的吸收峰明显降低，说明在紫外-可见吸收光谱的条件中，探针对ONOO-离子有明显的识别效果。加入ONOO-离子的探针溶液颜色从绿色变为无色且裸眼可见，加入其他分析物的探针溶液颜色没有明显变化。
准确称取一定量萘-乙烯基吡啶基双响应型荧光探针，溶解并配制成浓度为1.0×10-3mol/L的DMSO储备溶液；用DMF和水体积比为3：1的混合溶液将储备液稀释成浓度为1.0×10-5mol/L的待测溶液。取3mL浓度为1.0×10-5mol/L的待测溶液于石英比色皿中(石英比色皿的厚度为1cm)，然后分别加入30μL浓度为1.0×10-2mol/L的各种分析物(Blank，ONOO-，ClO-，Cys，H2O2，GSH，NO2 -，CO3 2-，HCO3 -，S2-，HSO3 -，SO3 2-，H2PO4 -，HPO4 2-，·OH)的水溶液，摇匀后测定溶液的荧光光谱光谱(图3b)。从图3b可以看出本探针可特异性识别ONOO-，对其他离子几乎不响应。结果表明，荧光探针对ONOO-具有高选择性。
实施例4：本发明双响应型荧光探针对ONOO-离子识别的抗干扰性
取3mL浓度为1.0×10-5mol/L的待测溶液于石英比色皿中，加入3μL浓度为1.0×10-2mol/L的ONOO-离子后，再分别加入3μL浓度为1.0×10-2mol/L的各种分析物(Blank，ONOO-，ClO-，Cys，H2O2，GSH，NO2 -，CO3 2-，HCO3 -，S2-，HSO3 -，SO3 2-，H2PO4-，HPO4 2-，·OH)溶液，摇匀，在λ＝396nm的激发波长下测定其荧光发射光谱，结果显示均对荧光探针的荧光强度几乎没有影响(如图4)，这就表明了荧光探针对ONOO-离子识别具有较好的抗干扰能力。在365nm的紫外灯下，探针溶液发出黄光；加入ONOO-离子的探针溶液不发光且裸眼可见，而加入其他分析物的探针溶液没有明显变化。
实施例5：本发明双响应型荧光探针的荧光强度与ONOO-浓度的相关性
将探针溶于DMF中，配置10μM的探针溶液。在每个试管滴入探针溶液，取ONOO-(0-5.0equiv)滴入上面试管中。在540nm处测定这些待测溶液的荧光光谱(如图5a所示)，随着ONOO-浓度的增加，该探针的荧光强度逐渐降低，荧光探针(10μM)与ONOO-(0-5.0equiv)存在良好的线性关系R2＝0.99474(如图5b所示)，这表明，荧光探针在荧光光谱中可以定量的检测ONOO-。根据LOD＝3σ/k，计算得出在荧光光谱中该荧光探针对ONOO-的检出限为0.45μM。其中σ是空白测量值的标准偏差，k是荧光强度对ONOO-浓度图的斜率。
实施例6：本发明双响应型荧光探针监测细胞内ONOO-离子
生长良好的Hela细胞用荧光探针(10μM)处理30min后，用激光共聚焦显微镜进行细胞成像测试，随后在培养皿中加入ONOO-离子(50μM)培养10min后，进行细胞成像测试。由图6可知，加入探针培养后，探针有良好的渗透性，可以观察到细胞有较强的绿色荧光。用探针培养后再加入ONOO-离子培养，可以观察到细胞荧光明显猝灭(如图6)。明显的荧光变化显示本探针能够监测ONOO-在生物系统中的存在。
Claims (6)
1.一种萘-乙烯基吡啶基双响应型荧光探针，其特征在于其结构式如下所示：
2.一种权利要求1所述的萘-乙烯基吡啶基双响应型荧光探针的制备方法，其特征在于：
由含醛基的萘荧光基团衍生物与1,4对二氯苄经缩合反应得到，包括如下步骤：
以丙酮作溶剂，将化合物ZL和1,4对二氯苄加入反应器中，滴加DMF增加溶解性，60℃下回流反应，反应结束后冷却室温，析出黄色沉淀，用甲醇过滤重结晶后得到目标产物；
反应路线如下所示：
3.一种权利要求1所述的萘-乙烯基吡啶基双响应型荧光探针的用途，其特征在于：
所述双响应型荧光探针在定性或定量检测ONOO-离子和/或粘度时作为检测试剂使用。
4.根据权利要求3所述的用途，其特征在于：
所述双响应型荧光探针作为检测试剂用于检测ONOO-离子时，检测限为0.45μM。
5.根据权利要求3所述的用途，其特征在于：
所述双响应型荧光探针作为检测试剂使用，在含水介质中进行紫外或荧光光谱测定，通过荧光强度的变化实现对ONOO-离子和/或粘度的定性或定量检测。
6.根据权利要求5所述的用途，其特征在于：
所述含水介质为DMF和水按体积比3：1构成的混合溶液。
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