CN116375674B - 一种用于苦味酸检测的双态荧光探针及其制备方法 - Google Patents
一种用于苦味酸检测的双态荧光探针及其制备方法 Download PDFInfo
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Abstract
一种用于苦味酸检测的双态荧光探针及其制备方法。该荧光探针通过两步反应得到，在稀溶液状态下和固态下具有强荧光发射性质，能够在四氢呋喃‑水混合溶剂体系中实现苦味酸的高灵敏检测，荧光淬灭常数为2062M‑1，检测限低至1.81μM。本发明的荧光探针在食品、环境等领域中具有广泛应用的潜力。
Description
技术领域
本发明涉及荧光分子探针领域，尤其涉及一种用于苦味酸检测的双态荧光探针及其制备方法。
背景技术
苦味酸化学名称为2,4,6-三硝基苯酚，是一种具有强烈毒性和致癌性的有机化合物，对环境和人类健康的危害性极大。苦味酸的主要来源包括含苦味酸的染料、农药、医药、化工、印染等行业的废水、废气、废渣等。这些废弃物可能通过污染物的渗入、废水处理等方式进入到环境中，威胁着人们的健康。因此，对苦味酸的检测具有重要意义，开发快速、准确、灵敏的苦味酸检测方法非常必要。目前，常用的苦味酸检测方法包括高效液相色谱法、气相色谱法、荧光探针法等。
荧光探针法作为一种新兴的检测方法，具有快速、灵敏、高效、经济的优势，在苦味酸的检测中具有广泛的应用价值。荧光探针法采用荧光分子与苦味酸作用，形成荧光信号，通过检测荧光信号的强度和光谱特性实现苦味酸浓度的定量检测。荧光探针法与传统的检测方法相比，具有更高的灵敏度和准确性，且操作简单、成本低廉，因此在食品、环境等领域中有着广泛的应用前景。荧光探针法在苦味酸检测中的优势和应用前景非常广泛，为公众健康和环境保护提供了重要的支持。尽管荧光分子探针在苦味酸检测中的应用已经有所发展，但目前仍面临一些挑战。例如，寻找具有良好可溶性、高选择性和高灵敏度的探针仍然是一个关键的任务。由于苦味酸的化学结构与2,4,6-***，2,4-二硝基甲苯等化合物相似，因此开发具有高选择性的荧光探针具有一定难度。另外，现有荧光探针普遍采用单一发光型分子，如溶液发光型的聚集淬灭(ACQ)分子或固态光型的聚集诱导发光(AIE)分子，其应用范围受到限制。因此，开发新型双态荧光分子作为苦味酸探针可扩展其苦味酸检测的适用范围。
发明内容
本发明的目的在于解决现有技术中的上述问题，提供一种用于苦味酸检测的双态荧光探针及其制备方法，用于高灵敏、定量检测苦味酸。
为达到上述目的，本发明采用如下技术方案：
一种用于苦味酸检测的双态荧光探针，结构如下：
简写为FCPB，其在溶液和固体状态下均具有荧光性质。
一种用于苦味酸检测的双态荧光探针的制备方法，合成路线如下：
本发明制备方法包括以下步骤：
1)含氰基中间体CNPB的合成：将4-氰甲基苯硼酸和1-溴芘溶解于四氢呋喃溶液，加入双[二叔丁基-(4-二甲基氨基苯基)膦]二氯化钯(II)(Pd-132)和碳酸钾(K2CO3)，在氮气保护下，将上述溶液加热反应，反应结束待溶液冷却至室温后，将其倒入饱和食盐水，用有机溶剂萃取，随后洗脱，通过柱色谱提纯得到中间化合物CNPB；
2)将步骤1)制备的中间体CNPB和4-氟水杨醛溶解于乙酸和N,N-二甲基甲酰胺中，然后加入哌啶，在氮气(N2)保护下，加热反应，反应结束待溶液冷却至室温后，将其倒入冰水，用有机溶剂萃取，随后洗脱，通过柱色谱提纯得到探针分子FCPB。
步骤1)中，加热温度为60～100℃，反应时间为12～18h。
步骤2)中，加热温度为80～120℃，反应时间为10～16h。
步骤1)和步骤2)中，采用二氯甲烷萃取。
步骤1)和步骤2)中，以石油醚和二氯甲烷作为洗脱剂。
步骤1)各配料的摩尔比如下：4-氰甲基苯硼酸:1-溴芘:碳酸钾:双[二叔丁基-(4-二甲基氨基苯基)膦]二氯化钯(II)＝1:1～1.5:1～5:0.05～0.2。
步骤2)各配料的摩尔比如下：CNPB:4-氟水杨醛:哌啶＝1:1～1.5:10～100。
所述的FCPB的应用，用于检测苦味酸。
相对于现有技术，本发明技术方案取得的有益效果是：
(1)本发明的荧光分子为双态荧光分子，在溶液和固态状态下均具有良好的荧光发射性质，具有较好的适用性；
(2)本发明的双态荧光探针对苦味酸检测具有单一选择性，对其他常见芳香硝基化合物几乎不响应，且对苦味酸的检测具有高灵敏性；
(3)本发明的双态荧光探针的荧光淬灭曲线为线性拟合曲线，可用于苦味酸的定量检测；
(4)本发明的双态荧光探针的检测过程简单、快捷，无需复杂操作程序和昂贵设备；
(5)本发明的双态荧光探针合成路线简单、成本低廉，利于实际应用推广。
附图说明
图1为FCPB的核磁氢谱(1H NMR)；
图2为FCPB的核磁碳谱(13C NMR)；
图3为FCPB的质谱(MS)；
图4为FCPB在溶液和固体状态下的归一化荧光谱图；
图5为FCPB对不同芳香硝基化合物的荧光响应谱图；
图6为FCPB在476nm处的荧光强度在加入不同芳香硝基化合物及苦味酸前后的变化；
图7为FCPB对苦味酸的荧光滴定谱图；
图8为FCPB最大荧光强度对苦味酸浓度的线性拟合曲线图；
图9为FCPB的荧光光谱和苦味酸的紫外光谱；
图10为FCPB的HOMO、LUMO轨道以及苦味酸的LUMO轨道电子云分布。
具体实施方式
为了使本发明所要解决的技术问题、技术方案及有益效果更加清楚、明白，以下结合附图和实施例，对本发明做进一步详细说明。
实施例1
本实施例所述用于检测苦味酸的双态荧光探针FCPB的制备方法如下：
1)合成CNPB：将0.96g的4-氰甲基苯硼酸、1.41g的1-溴芘、24mL四氢呋喃、78mg的Pd-132和8mL的碳酸钾水溶液(2M)加入到50mL圆底烧瓶中。在氮气保护下升温至80℃反应8小时。反应结束后冷却至室温，并将反应液加入到100mL饱和食盐水中。用三次50mL二氯甲烷萃取有机相，将有机相合并后，用无水硫酸钠干燥3小时。过滤后，通过减压蒸馏得到粗产物。粗产物通过柱层析分离提纯(石油醚:二氯甲烷＝1:1)，得到1.14克白色固体CNPB，产率为72％。1H NMR(400MHz,CDCl3)δ:8.00～8.23(m，8H)，7.94(d，J＝7.8Hz，1H)，7.64(d，J＝8.0Hz，2H)，7.51(d，J＝8.0Hz，2H)，3.88(s，2H)。
2)合成FCPB：称取0.14g 4-氟水杨醛和0.35g CNPB，溶解于2mL乙酸和2mL N,N-二甲基甲酰胺中，加入3mL哌啶，氮气保护下，110℃反应12小时。待反应结束后，将反应液冷却后倒入100mL冰水中。用50mL二氯甲烷萃取3次，经过无水硫酸钠干燥、过滤和减压蒸馏得粗产物，最后通过层析分离提纯(石油醚:二氯甲烷＝1:1)，得到0.23g黄绿色固体FCPB，产率53％。1H NMR(400MHz,CDCl3)δ:7.85～8.26(m,9H),7.90(d,J＝8.0Hz,2H),7.85(s,1H),7.69(d,J＝8.0Hz,2H),6.88(d,J＝7.5Hz,1H),6.79(s,1H).13C NMR(101MHz,CDCl3):δ161.70,155.98,140.98,140.38,137.38,134.67,131.59,131.10,130.76,130.70,128.79,128.60,128.39,127.65,127.63,127.57,127.51,126.12,125.42,125.21,125.08,125.01,124.96,124.78,112.10,100.55,49.10,34.76,27.01,25.37,24.38.HR-MS(APCI-MS):m/z＝441.2457.calcd.for[C31H18FO2]+＝441.1291。
FCPB的核磁和质谱表征图谱如图1、图2和图3所示。
实施例2
双态荧光探针的光物理性质
溶液荧光光谱测试：用万分子一天平准确称量4.4mg FCPB，溶解于1mL无水四氢呋喃，制成浓度为0.01mM母液。用移液器准确取10μL母液，加入至1000mL四氢呋喃，稀释成浓度为10μM的测试溶液。将溶液倒入比色皿中，使用LS-55荧光光度计测试光谱，激发波长为393nm，扫描范围为400～600nm。
固体荧光光谱：称取50mg FCPB加入至0.5mm石英样品池，使用LS-55荧光光度计测试光谱，激发波长为393nm，扫描范围为400～600nm。
如图4所示，FCPB在四氢呋喃溶液和固体状态下的荧光发射波长分别为476nm和493nm。溶液和固体状态下的荧光量子产率分别为0.60和0.31，表明FCPB在稀溶液和固体状态下均具有良好的发光性质，是典型的双态发光型荧光分子。
实施例3
FCPB的苦味酸检测应用
双态荧光探针对苦味酸检测的选择性测试：以四氢呋喃/水(体积比1/1)作为混合溶剂，配制浓度为10μM的FCPB探针溶液。将包括苦味酸(PA)、二硝基甲苯(DNP)、硝基苯(NB)、邻硝基苯酚苯(o-NP)、对氯硝基苯(p-ClNB)、对氟硝基苯(p-FNB)、对硝基苯胺(p-NA)、对硝基苯酚(p-NP)、对硝基甲苯(p-NT)的九种芳香硝基化合物溶解于无水四氢呋喃，制成浓度为0.01M的母液。向9份体积为5mL的FCPB探针溶液中加入50μL上述芳香硝基化合物，震荡混合均匀，制备成测试溶液，另外制备一份体积为5mL的FCPB探针溶液，不加入任何芳香硝基化合物，作为参比。将测试溶液或参比溶液倒入荧光比色皿，使用LS-55荧光光度计测试光谱，激发波长为393nm，扫描范围为400～600nm。如图5所示，在九种芳香硝基化合物中，仅有苦味酸(PA)对FCPB的荧光造成了显著的淬灭，而其他八种芳香硝基化合物对荧光强度未产生明显的影响，说明FCPB对苦味酸的识别具有单一的选择性。
双态荧光探针对苦味酸检测的抗干扰性：以四氢呋喃/水(体积比1/1)作为混合溶剂，配制十份浓度为10μM的FCPB探针溶液，并标记1，2，3，4，5，6，7，8，9，10。将1号样品作为空白对照样品，2号样品作为阴性对照样品，剩余3-10号样品作为实验组样品。向3-10号样品中分别加入相当于FCPB浓度10当量的二硝基甲苯(DNP)、硝基苯(NB)、邻硝基苯酚苯(o-NP)、对氯硝基苯(p-ClNB)、对氟硝基苯(p-FNB)、对硝基苯胺(p-NA)、对硝基苯酚(p-NP)、对硝基甲苯(p-NT)八种芳香硝基化合物，使用LS-55荧光光度计测试光谱，激发波长为393nm，扫描范围为400～600nm。随后，向2-10号样品中加入相当于FCPB浓度10当量的苦味酸(PA)后，再在相同条件下测试荧光光谱。随后，将476nm处荧光强度摘出并作图，得到如图6所示结果，在加入苦味酸前，各组溶液的荧光与空白对照组及阴性参照组几乎相同，进一步说明其他芳香硝基化合物对FCPB的荧光强度几乎没有影响，而加入苦味酸后，实验组及阴性对照组的荧光发生显著淬灭，说明苦味酸造成了FCPB的荧光淬灭，且其他芳香硝基化合物的存在并不影响FCPB对苦味酸的检测。上述结果表明除了苦味酸外，其他方向硝基化合物不会对FCPB的荧光强度产生影响，并且这些芳香硝基化合物的存在也不会影响FCPB对苦味酸的检测。
双态荧光探针对苦味酸检测的动力学：向5mL浓度为10μM的FCPB的四氢呋喃/水(V/V1/1)溶液中逐次加入5μL苦味酸溶液(0.1M)并使用LS-55荧光光度计记录谱图。将476nm波长处荧光强度与苦味酸浓度变化作图并进行线性拟合，得到如图7所示结果，结果表明增加苦味酸的浓度导致荧光逐渐淬灭，当加入相当于FCPB浓度10倍当量的苦味酸后，其荧光淬灭程度达到79％。使用Stern-Volmer(S-V)方程进行分析：(I0/I＝1+KSV[Q])，其中，I0为化合物初始发射强度，I为加入苦味酸后的发射强度，KSV为淬灭常数，[Q]为苦味酸的浓度。对线性部分进行拟合后(0-3当量苦味酸范围)，计算得到淬灭常数为2062M-1。
双态荧光探针对苦味酸检测的检测限：检测限(limit of detection，LoD)是评估苦味酸荧光探针灵敏度的关键参数。向5mL浓度为10μM的FCPB的四氢呋喃/水(V/V 1/1)溶液中逐次加入5μL苦味酸溶液(0.1M)至最大3.0当量，绘制476nm波长处荧光强度与苦味酸浓度变化的点图，并进行线性拟合，得到如图8所示结果，FCPB对苦味酸的响应接近于线性变化，表明可以定量检测苦味酸，根据图8计算斜率K。准备10份上述FCPB溶液，测试荧光光谱，并取476nm处的荧光强度值，计算标准偏差σ。依据LoD计算公式：LoD＝3σ/K，计算检测限为1.81μM，表明本发明探针对苦味酸检测的灵敏度高。
实施例4
双态发光型荧光探针的苦味酸检测机理
分别称取FCPB和苦味酸溶于无水四氢呋喃，制成浓度为0.01M的母液后，用移液器取10μL母液加入1000μL无水四氢呋喃溶液中，获得浓度为10μM的测试溶液。将测试溶液转移至荧光比色皿，使用LS-55荧光光度计测试FCPB的荧光光谱，使用UV-759紫外分光光度计测试苦味酸的紫外吸收光谱，将两个谱图归一化后比较谱图重叠，得到如图9所示结果。可以看出，FCPB的荧光光谱和苦味酸的紫外吸收光谱有一定的重叠的，说明荧光共振能量转移(FRET)是可能的荧光淬灭机理。基于密度泛函理论，使用高斯09包和B3LYP/6-31+G(d)数组，计算FCPB的HOMO和LUMO轨道及苦味酸LUMO轨道的电子云分布及轨道能级，得到如图10所示结果。可以看出，FCPB的HOMO轨道能级高于苦味酸的LUMO轨道能级，说明光诱导电子转移(PET)不会在FCPB和苦味酸之间发生，光诱导电子(PET)过程不是苦味酸介导FCPB的荧光淬灭的原因。因此，FCPB的苦味酸检测机理可能为荧光共振能量转移。
以上所述仅是本发明的较佳实施例，并非对本发明作任何形式上的限制，任何熟悉本专业的技术人员，在不脱离本发明技术方案范围内，依据本发明的技术实质，对以上实施例所作的任何简单的修改、等同替换与改进等，均仍属于本发明技术方案的保护范围之内。
Claims (10)
1.一种用于苦味酸检测的双态荧光探针，其特征在于，结构如下：
简写为FCPB，其在溶液和固体状态下均具有荧光性质。
2.如权利要求1所述的一种用于苦味酸检测的双态荧光探针的制备方法，其特征在于，合成路线如下：
3.如权利要求2所述的制备方法，其特征在于，包括以下步骤：
1)含氰基中间体CNPB的合成：将4-氰甲基苯硼酸和1-溴芘溶解于四氢呋喃溶液，加入双[二叔丁基-(4-二甲基氨基苯基)膦]二氯化钯(II)和碳酸钾，在氮气保护下，将上述溶液加热反应，反应结束待溶液冷却至室温后，将其倒入饱和食盐水，用有机溶剂萃取，随后洗脱，通过柱色谱提纯得到中间化合物CNPB；
2)将步骤1)制备的中间体CNPB和4-氟水杨醛溶解于乙酸和N,N-二甲基甲酰胺中，然后加入哌啶，在氮气保护下，加热反应，反应结束待溶液冷却至室温后，将其倒入冰水，用有机溶剂萃取，随后洗脱，通过柱色谱提纯得到探针分子FCPB。
4.如权利要求3所述的制备方法，其特征在于：步骤1)中，加热温度为60～100℃，反应时间为12～18h。
5.如权利要求3所述的制备方法，其特征在于：步骤2)中，加热温度为80～120℃，反应时间为10～16h。
6.如权利要求3所述的制备方法，其特征在于：步骤1)和步骤2)中，采用二氯甲烷萃取。
7.如权利要求3所述的制备方法，其特征在于：步骤1)和步骤2)中，以石油醚和二氯甲烷作为洗脱剂。
8.如权利要求3所述的制备方法，其特征在于，步骤1)各配料的摩尔比如下：4-氰甲基苯硼酸:1-溴芘:碳酸钾:双[二叔丁基-(4-二甲基氨基苯基)膦]二氯化钯(II)＝1:1～1.5:1～5:0.05～0.2。
9.如权利要求3所述的制备方法，其特征在于，步骤2)各配料的摩尔比如下：CNPB:4-氟水杨醛:哌啶＝1:1～1.5:10～100。
10.权利要求1所述的一种用于苦味酸检测的双态荧光探针以及权利要求2～9任一项制备方法所制备的用于苦味酸检测的双态荧光探针的应用，其特征在于：用于检测苦味酸。
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