CN113402655B - 一种可用于fret荧光探针供体的聚合物通用荧光平台及其应用 - Google Patents
一种可用于fret荧光探针供体的聚合物通用荧光平台及其应用 Download PDFInfo
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Abstract
一种可用于FRET荧光探针供体的聚合物通用荧光平台及其应用，属于有机化学和高分子材料化学技术领域。本发明将甲基丙烯酸甲酯与4‑烯丙氧基苯甲醛通过合理配比聚合，并进一步通过高效的希夫碱反应引入香豆素荧光团制备本发明中的通用聚合物蓝光平台MS‑1。MS‑1通过MS‑1和MS‑2分步制备得到。本发明利用香豆素荧光团制备得出的聚合物通用荧光平台原料易得，合成简单易行，成本低廉。当与成熟的长波长荧光探针组合后，可实现对目标分子的比例荧光检测，甚至可通过颜色实现肉眼直观检测。
Description
技术领域
本发明属于有机化学和高分子材料化学技术领域，具体涉及一种可用于FRET荧光探针供体的聚合物通用荧光平台及其应用。
背景技术
自21世纪以来，由于光纤传感器在工业分析、生物化学、药物检测等具有巨大的应用潜力，这类传感器一直被密切关注(Adv Optical Mater.2021,9,2001913)。设计出这种传感器需要具有高灵敏度的新型功能材料，并不容易实现。而荧光传感器聚合物材料由于检测性能优异、易于制备成聚合纤维材料已成为研究人员攻克的重点方向。但由于荧光传感器浓度过大易发生荧光猝灭使其检测灵敏度大大下降，如何在在聚合物基质中保持探针的传感特性是一个巨大的挑战。
相比于单纯依赖于荧光强度来进行目标分子识别，比例荧光探针用两个波长的荧光强度比值和颜色的双重变化来检测目标分子不仅适合于肉眼观测，更加直观，同时也避免了仪器损耗、溶剂以及温度变化等客观因素所引起的检测误差，使检测灵敏度大大提高(Chem Soc Rev.2016,45,2976)。但在合成上要将两个不同发射波长的荧光团组合于一个分子中，并且不影响比率荧光识别目标分子，则需要精密的分子设计和复杂的合成过程。相比之下，特异性识别目标的单波长荧光探针则在合成上更容易实现，且有很多成熟的分子可供选择。
因此，本发明预期实现开发一个通用的聚合物蓝光平台，将具有优异光物理性能的蓝光发射荧光团香豆素以适当配比组合到聚合纤维材料中，通过控制香豆素配比使其在聚合物中保持适当距离，避免聚集。在这个香豆素聚合物通用荧光平台中，香豆素即可作为荧光共振能量转移(FRET)的能量供体，亦可同时作为荧光内标。而在识别目标分子时，只需选择可以和香豆素具有良好能量匹配的成熟荧光探针，将其以适当配比与聚合物通用荧光平台组合，就可以实现对目标分子的双波长荧光检测，不仅避免单荧光检测时易受干扰的缺陷，同时也降低了分子设计的难度。这样的一个聚合物通用荧光平台的开发，也使荧光探针后期制备成柔性纳米织物成为了可能，这将大大扩展其应用领域。
发明内容
本发明的目的是将简单易得的蓝光发射团香豆素通过希夫碱反应与制备的带醛基活性位点的聚合物分子组合，制备可发射蓝光的聚合物通用荧光平台，并进一步将此平台与单纯荧光增强识别汞离子的荧光探针进行组合，其可实现对汞离子的高灵敏度的比率荧光检测，避免了单纯荧光增强所受的外界干扰。本发明的聚合物通用荧光平台一方面有利于荧光探针后期的成型加工(如可通过静电纺丝制备柔性膜)，另一方面可通过控制聚合物分子上醛基活性位点的浓度使香豆素在聚合物中浓度适宜，预期可避免固体材料中由于香豆素的聚集导致荧光猝灭。
本发明解决上述技术问题所采用的技术方案是：将甲基丙烯酸甲酯与4-烯丙氧基苯甲醛通过合理配比聚合，并进一步通过高效的希夫碱反应引入香豆素荧光团制备本发明中的通用聚合物蓝光平台MS-1，其具体结构式如下：
为了进一步考察其应用效果，本发明将该聚合物通用荧光平台与已发表的罗丹明荧光探针MS-4进一步组合，协同实现对目标分子的双波长比率荧光检测。
本发明通过以下具体步骤进行：对羟基苯甲醛与等摩尔的烯丙基氯和三倍摩尔量的碳酸钾在乙腈(1克对羟基苯甲醛/15毫升乙腈)溶液中加热回流二小时得到化合物MS-3，甲基丙烯酸甲酯与0.41倍摩尔量的MS-3在偶氮二异丁腈(0.4％)的引发下于二甲基甲酰胺(DMF)(1克甲基丙烯酸甲酯/4毫升)中加热24小时聚合得到聚合物MS-2，MS-2在二甲基甲酰胺(DMF)溶液中进一步与香豆素水合肼(MS-2质量的9.4％)加热反应24小时反应得到聚合物荧光平台MS-1。
制备上述结构的聚合物荧光平台MS-1的制备反应路线如下：
本发明中聚合物通用荧光平台MS-1的紫外吸收光谱中主要吸收峰来自组成它的香豆素在439纳米处的吸收峰，而发射波长为香豆素在478纳米的蓝色荧光。当与MS-4组合后，随着汞离子的加入，在吸收光谱中出现了属于罗丹明的新吸收峰，两个峰在基态时同时出现。汞离子加入后，荧光光谱中也出现了新的属于罗丹明开环的568纳米出的荧光峰，同时香豆素的478纳米处荧光峰有下降趋势。当汞离子达到一下浓度后，荧光不再进一步变化。说明聚合物通用荧光平台为此检测体系提供了蓝光波长作为内标，可实现对汞离子的双波长荧光检测。
本发明的有益效果是，利用香豆素荧光团制备得出的聚合物通用荧光平台原料易得，合成简单易行，成本低廉。当与成熟的长波长荧光探针组合后，可实现对目标分子的比例荧光检测，甚至可通过颜色实现肉眼直观检测。该聚合物平台的开发也使其后期应用于柔性纳米材料成为可能。
附图说明
图1是本发明中实施例2中MS-3在CDCL3中的核磁的碳氢谱图；
图2是本发明中实施例2中MS-5在CDCL3中的核磁的氢谱图；
图3是本发明中实施例3中MS-2在CDCL3中的核磁的氢谱图；
图4是本发明中实施例4中MS-1在CDCL3中的核磁的氢谱图；
图5是本发明中实施例5中MS1-Hg的紫外可见光谱滴定图；
图6是本发明中实施例5中MS1-Hg在537nm处的紫外吸光度变化趋势；。
图7是本发明中实施例例5中MS1-Hg的荧光光谱滴定图；
图8是本发明中实施例例5中MS1-Hg随Hg(NO3)2倍数增加时F564/F478的荧光强度比值变化；
图9是本发明中实施例例5中MS1-Hg在不同pH值下加Hg2+前后的荧光光谱测定。
具体实施方式
为了更清楚地说明本发明内容，用具体实施例说明如下，具体实施例不限定本发明内容范围。
实施例1
MS-4的合成：在罗丹明6g水合肼(200mg，0.46mmol)的二甲基甲酰胺(约3mL)溶液中，加入摩尔质量为1.5倍的异硫氰酸苯酯(94μl，0.69μmol)，逐渐升温至60℃，加热搅拌四小时，TLC确定反应结束后，将反应液用二氯甲烷萃取，有机相减压旋蒸，固体柱层析分离得粉色目标产物，收率50％。
实施例2
MS-3的合成：1克对羟基苯甲醛，等摩尔的烯丙基氯，三倍摩尔量的碳酸钾置于50毫升烧瓶，加入15毫升乙腈。反应液加热回流二小时，冷却过滤，滤液减压旋干得白色固体粉末(MS-3对烯丙基氧苯甲醛)，收率70％。产品未经提纯直接用于下一步反应。
香豆素水合肼的合成：香豆素酯(1g，3.46mmol)溶于15ml甲醇溶液中，慢慢滴入2ml水合肼，反应液常温搅拌30分钟，TLC确定反应结束后，析出的固体过滤得黄色固体粉末0.54g，收率56％。产品未经提纯直接用于下一步反应。
MS-5的合成：香豆素水合肼500毫克，1.5倍摩尔量的MS-3置于50毫升的烧瓶中，加入25毫升的乙醇，加热回流搅拌，反应四小时后，冷却过滤，得固体产品，收率30％。
实施例3
MS-2的合成：将5g甲基丙烯酸甲酯(0.0499mol)和3.3687g(0.0207mol)MS-3混合，并加入0.04g引发剂偶氮二异丁腈(0.0002mol)和20mL二甲基甲酰胺，用氮气鼓泡10分钟去除溶液中的氧气。反应液放置在一个恒温油浴中，在70℃下加热反应24小时，得到透明的聚合物。随后将反应混合物倒入甲醇(200ml)中，聚合物在甲醇中沉淀析出，超声震荡，过滤，甲醇多次洗涤除去固体部分未反应单体，将过滤所得聚合产物放在30℃真空干燥箱中干燥24小时至恒重。产量：4.1557g。
实施例4
MS-1的合成：将536mg MS-2和50.32mg(0.0207mol)的香豆素肼混合于5mL二甲基甲酰胺中，加入20μl冰醋酸，在70℃下加热反应24小时。将反应混合物倒入甲醇(200ml)中，聚合物在甲醇中沉淀析出，超声震荡，过滤，甲醇多次洗涤除去固体部分未反应单体，将过滤所得聚合产物MS-1在30℃真空干燥箱中干燥24小时至恒重。产量：481.6mg。
实施例5汞离子检测溶液MS1-Hg的制备
取MS-1和MS-4各制备成10-3mol/L的二氯甲烷溶液。分别取1微升和100微升放入10毫升的容量瓶，吹干溶剂，加入乙醇水(体积比8：2)混合溶液配制成检测溶液MS1-Hg。
实施例6紫外吸收光谱测定
本发明测定了检测溶液MS1-Hg的紫外吸收光谱，测试结果如图5所示。未加入Hg2+时，可以观察到在439纳米处有一个明显的吸收峰，这是属于香豆素荧光团的特征吸收峰，此时在538纳米处几乎没有吸收，这说明此时罗丹明荧光团处于闭环状态。随着Hg2+向溶液中逐渐加入，在538纳米处出现了一个新的吸收峰，该吸收峰随着汞离子的加入逐渐增加，当加入的Hg2+达到溶液中MS-4摩尔浓度的4倍时，538纳米处的吸收峰达到平衡(如图6所示)。而在439纳米处的吸收峰并没有随着Hg2+加入而有所改变。这说明汞离子加入后，罗丹明荧光团部分逐渐开环，但香豆素荧光团和罗丹明荧光团在基态下没有发生相互影响。
实施例7检测溶液MS1-Hg的荧光光谱测定
本发明测定了检测溶液MS1-Hg的荧光发射光谱，测试结果如图7所示。加入汞离子前，当在440纳米激发波长下，只能看到在478纳米处的香豆素的发射峰。随着汞离子的加入，478纳米处的荧光峰逐渐减弱，而564纳米处荧光发射峰逐渐增强，这是罗丹明6G开环后的荧光发射峰。当汞离子加入四倍后，564纳米和478纳米处的荧光强度比值趋于平稳(如图8所示)。这说明当汞离子加入后由于罗丹明衍生物开环，使香豆素荧光团的发射光谱和开环的罗丹明荧光团吸收光谱出现了交盖，从而出现了香豆素供体和罗丹明受体之间的荧光共振能量转移。因此我们可以观察到香豆素荧光团在478纳米处的下降，以及罗丹明荧光团在564纳米处的增强。当汞离子加入到一定量后，罗丹明完全开环，此时在加入汞离子荧光也不再有明显的变化。
实施例8不同pH值下加Hg2+前后的荧光光谱测定
本发明测定了检测溶液MS1-Hg的在不同pH下的荧光光谱，测试结果如图9所示。可以看到，未加入汞离子前的检测液在pH 4.5-10之间荧光是比较稳定的，在pH小于4才出现明显的荧光增强，这主要是由于罗丹明部分在酸性条件下发生了开环反应所导致的。加入汞离子后检测液在pH 4.5-10之间荧光也相对稳定。说明在正常的环境和生理条件下，本专利中的检测溶液MS1-Hg可以实现对汞离子的检测，而不受pH变化影响。
上述实施例只是用于对本发明的举例和说明，而非意在将本发明限制于所描述的实施例范围内。此外本领域技术人员可以理解的是，本发明不局限于上述实施例，根据本发明的教导还可以做出更多种的变型和修改，这些变型和修改均落在本发明所要求保护的范围内。
Claims (2)
1.一种可用于FRET荧光探针供体的聚合物通用荧光平台，其特征在于，该荧光平台的命名MS-1，具体的结构式如下：
所述荧光平台，其制备方法的制备路线如下：
MS-1的制作步骤如下：
S1. MS-3的合成：对羟基苯甲醛、烯丙基氯和碳酸钾在乙腈溶液中加热回流二小时得到化合物MS-3；
S2. MS-2的合成：甲基丙烯酸甲酯与MS-3在偶氮二异丁腈的引发下于二甲基甲酰胺中加热24小时聚合得到聚合物MS-2；
S3. MS-1的合成：MS-2在二甲基甲酰胺溶液中进一步与香豆素水合肼加热反应24小时反应得到聚合物荧光平台MS-1；
步骤S1中烯丙基氯的加入量与对羟基苯甲醛的摩尔量相同，碳酸钾的加入量为对羟基苯甲醛的三倍摩尔量；
步骤S1中乙腈的加入量以对羟基苯甲醛为基准，1克对羟基苯甲醛加入15毫升乙腈；
步骤S2中MS-3的加入量为甲基丙烯酸甲酯0.41倍摩尔量，偶氮二异丁腈的加入量为甲基丙烯酸甲酯质量的0.4%；
步骤S2中二甲基甲酰胺的加入量以甲基丙烯酸甲酯为基准，1克甲基丙烯酸甲酯加入4毫升二甲基甲酰胺；
步骤S3中香豆素水合肼的加入量为MS-2质量的9.4%。
2.一种权利要求1中聚合物通用荧光平台在荧光检测中的应用。
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