CN113264954A - 一种用于检测过氧化氢的荧光探针分子及其制备方法 - Google Patents
一种用于检测过氧化氢的荧光探针分子及其制备方法 Download PDFInfo
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Abstract
本发明公开一种用于检测过氧化氢的荧光探针分子及其制备方法，采用将化合物2‑R基荧光素、碳酸钾、4‑溴甲基苯硼酸频哪醇酯和乙腈混合，加热搅拌，冷凝回流反应，待反应冷却至室温后加入乙腈溶剂，得到粗产物；将得到的粗产物经正向硅胶色谱柱纯化，即得到白色固体检测过氧化氢的荧光探针分子2',7'‑二R‑3',6'‑双((4‑(4,4,5,5‑四甲基‑1,3,2‑二氧杂环戊烷‑2‑基)苄氧基)‑3H‑螺[异苯并呋喃‑1,9'‑氧杂蒽]，通过荧光信号强度变化来检测过氧化氢含量。由于过氧化氢和探针分子发生化学引起硼酸酯基团发生重排消除，而释放出高发光效率的2‑R基荧光素，提高了检测的特异性和灵敏性，本发明制备过程简单，成本低，兼具优异的特异性和灵敏性，能够用于现场快速检测过氧化氢，具备广阔的应用前景。
Description
技术领域
本发明涉及一种用于检测过氧化氢的荧光探针分子及其制备方法，属于有机化学领域和分析检测领域，通过荧光信号强度变化来检测过氧化氢。
背景技术
过氧化氢又称双氧水，是一种强氧化剂，广泛应用于工业漂白、电镀、外科消毒和军用火箭推进剂等领域。过氧化氢参与多种重要的人体生理过程[Journal of theAmerican Chemical Society,2012,134，1305-1315、Chemical Communications,2013,49，9929-9931和Science,2006,312，1882-1883.]。人体内过氧化氢浓度异常与许多疾病有关，如阿尔茨海默病、癌症、糖尿病、DNA损伤、心血管疾病和神经退行性等疾病[Nature,2004,430，686-689和Nature,2007,448，767-774.]。然而，过氧化氢对人体呼吸道有强腐蚀性，与眼睛直接接触后可导致失明。更重要的是，过氧化氢是一种典型的危化品氧化剂，也是一种非制式***物。由于其超强氧化性和助燃性，制作***简单，具有较大危害性。因此，针对过氧化氢的超灵敏、快速、特异性的精准识别检测具有重要现实意义。
迄今为止，针对过氧化氢的检测方法主要包括：比色法[Dyes and Pigments,2021,186,108954和Sensors and Actuators B:Chemical,2018,271，336-345]、电化学传感法[Analytica Chimica Acta,2020,1118,26-35]、离子迁移谱法[The Journal ofPhysical Chemistry B,2013,117,164-173]以及表面增强拉曼光谱法[AnalyticalChemistry,2016,88,7191-7197]等检测方法。然而，这些方法要么需要昂贵的仪器、繁琐的检测程序和半定量能力等缺点。作为比较，荧光光谱检测法具有响应快、灵敏度高、选择性好、价格低廉等优势而受到广泛关注[Biosensors and Bioelectronics,2016,79,237-243和Nature Chemistry,2012,4,973-984]。因此，基于过氧化氢的荧光检测是一种优选方法。
目前，对过氧化氢的荧光检测尚没有一种具备检测速度快、灵敏度高、抗干扰能力强、性能稳定、价格低廉的荧光探针分子。因此，迫切需要开发出一种具备上述优势且制备简单的荧光探针分子以满足对过氧化氢检测的需求，以解决过氧化氢现场、快速检测的不足的问题。制备性能优异的荧光探针检测过氧化氢是当前和今后食品安全、环境保护、公共卫生等领域中快速、现场检测的首选方法，同时也为人民生命安全提供有力的保障和技术支撑。
发明内容
本发明的目的在于，提供一种用于检测过氧化氢荧光探针分子及其制备方法，采用将化合物2-R基荧光素、碳酸钾、4-溴甲基苯硼酸频哪醇酯和乙腈混合，加热反应，待反应冷却至室温后加入乙腈溶剂，得到粗产物；将得到的粗产物经正向硅胶色谱柱纯化，即得到白色固体检测过氧化氢的荧光探针分子2',7'-二R-3',6'-双((4-(4,4,5,5-四甲基-1,3,2-二氧杂环戊烷-2-基)苄氧基)-3H-螺[异苯并呋喃-1,9'-氧杂蒽]。本发明所述的荧光探针分子制备过程简单，成本低，兼具优异的特异性和灵敏性，能够用于现场快速检测过氧化氢的用途，具备广阔的应用前景。
本发明所述的一种用于检测过氧化氢的荧光探针分子，该荧光探针的化学名称为2',7'-二R-3',6'-双((4-(4,4,5,5-四甲基-1,3,2-二氧杂环戊烷-2-基)苄氧基)-3H-螺[异苯并呋喃-1,9'-氧杂蒽]，其结构式为：
其中：R是氢或卤素。
所述用于检测过氧化氢的荧光探针分子的制备方法，按下列步骤进行：
a、按摩尔比为1:2:3:500将化合物2-R基荧光素、碳酸钾、4-溴甲基苯硼酸频哪醇酯和乙腈加入到100mL圆底烧瓶中，在温度90℃加热搅拌，冷凝回流反应20h后，停止反应，其中：R是氢或卤素；
b、待反应冷却至室温后，加入50mL乙腈，充分搅拌，过滤除去杂质，干燥得到粗产物；
c、将步骤b得到的粗产物经正向硅胶色谱柱纯化，洗脱剂为二氯甲烷，洗脱产物干燥，即得到白色固体检测过氧化氢的荧光探针分子
本发明所述的检测过氧化氢的荧光探针分子为2',7'-二R-3',6'-双((4-(4,4,5,5-四甲基-1,3,2-二氧杂环戊烷-2-基)苄氧基)-3H-螺[异苯并呋喃-1,9'-氧杂蒽]，具有以荧光素衍生物为荧光基团，4-甲氧基苯硼酸频哪醇酯为识别基团，具体的合成步骤为：
本发明所述的一种用于检测过氧化氢的荧光探针分子的检测原理为：在碱性条件下，过氧化氢氧化探针分子上的硼酸酯基团，导致发生重排消除反应，最终生成了2-R基荧光素、硼酸频哪醇酯、4-亚甲基环己-2,5-二烯-1-酮，从而将探针中的荧光团(2-R基荧光素)释放，实现了荧光点亮检测过氧化氢；反应前后检测的荧光强度变化与过氧化氢的浓度之间呈线性关系，从而可根据荧光强度变化实现定量检测过氧化氢。
本发明所述的一种用于检测过氧化氢的荧光探针分子能够实现对过氧化氢的快速、特异性检测；实现了过氧化氢检测的目的。该荧光探针分子检测过氧化氢的步骤：
(1)将碳酸钠(0.6g，5.7mmol)和碳酸氢钠(3.7g，4.4mmol)混合，溶解在乙腈溶剂(100mL)中，得到碱性缓冲溶液；
(2)将50mL步骤(1)得到的碱性缓冲溶液，溶解41.6mg的探针分子，得到1mM探针分子溶液；
(3)将过氧化氢标准溶液与步骤(2)得到的荧光探针反应后，测定荧光强度，荧光强度对应过氧化氢含量作校正曲线进行定量分析；
(4)将步骤(2)得到的探针分子溶液均匀分布在硅胶板上，得到硅胶板机传感器；
(5)依据步骤(3)得到的校正曲线计算得到待测样品中的过氧化氢含量。
本发明所述的一种用于检测过氧化氢的荧光探针分子，检测过氧化氢的检测限为13.6nmol/L。过氧化氢标准溶液在0-80μmol/L浓度下，检测分子的荧光强度与过氧化氢的浓度呈良好的线性关系，检测时间小于20s。
与现有过氧化氢检测技术相比，本发明的有益效果是：在碱性条件下本发明所述的荧光探针分子高灵敏、快速、特异性识别过氧化氢，生成高荧光效率的2-R基荧光素物，实现对过氧化氢的荧光检测。具有检测高灵敏性、特异性好、响应速度快等优势。本发明的检测过氧化氢的荧光探针分子在检测过程中响应快、灵敏度高，特异性强、成本低廉等优势使得其极易在现场及时检测领域得到实际应用推广。
附图说明
图1本发明实施例1中的荧光探针分子的一维氢核磁谱图，其中横坐标为化学位移，纵坐标为信号强度；
图2本发明实施例3中的荧光探针分子对过氧化氢的选择性检测识别图，其中横坐标为不同化合物等干扰物，纵坐标为荧光强度；
图3本发明实施例4中的荧光探针的荧光强度和过氧化氢浓度的线形关系图，其中横坐标为过氧化氢的浓度，纵坐标为荧光强度；
图4本发明实施例5中的硅胶板基探针分子的荧光强度和过氧化氢浓度图，图中的荧光图片为不同量的过氧化氢与探针分子反应后的荧光图片。
具体实施方式
以下是对本发明的具体实施方式进行说明。
实施例1
荧光探针分子2',7'-二氯-3',6'-双((4-(4,4,5,5-四甲基-1,3,2-二氧杂环戊烷-2-基)苄氧基)-3H-螺[异苯并呋喃-1,9'-氧杂蒽]的合成：
a、将二氯荧光素1.5mmol，0.6g、碳酸钾4mmol，0.56g、4-溴甲基苯硼酸频哪醇酯6mmol，1.8g和50mL乙腈加入到100mL圆底烧瓶中，在温度90℃加热搅拌溶解，冷凝回流过夜反应20h，待反应完全后，停止反应；
b、待反应冷却至室温，加入50mL乙腈，充分搅拌，过滤除去杂质，得到粗产物；
c、将步骤b得到的粗产物经正向硅胶色谱柱纯化，洗脱剂为二氯甲烷，洗脱产物干燥，即得到白色固体检测过氧化氢的荧光探针分子2',7'-二氯-3',6'-双((4-(4,4,5,5-四甲基-1,3,2-二氧杂环戊烷-2-基)苄氧基)-3H-螺[异苯并呋喃-1,9'-氧杂蒽]。
图1为探针分子2',7'-二氯-3',6'-双((4-(4,4,5,5-四甲基-1,3,2-二氧杂环戊烷-2-基)苄氧基)-3H-螺[异苯并呋喃-1,9'-氧杂蒽]的氢谱核磁数据：
1H NMR(400MHz,DMSO-d6)δ8.04(d,J＝7.5Hz,1H),7.86-7.60(m,6H),7.47(t,J＝10.3Hz,4H),7.35(d,J＝7.5Hz,1H),7.19(s,2H),6.83(s,2H),5.51-5.22(m,4H),1.30(s,25H)。
实施例2
荧光探针分子2',7'-二氢-3',6'-双((4-(4,4,5,5-四甲基-1,3,2-二氧杂环戊烷-2-基)苄氧基)-3H-螺[异苯并呋喃-1,9'-氧杂蒽]的合成：
a、将荧光素1.5mmol，0.5g、碳酸钾4mmol，0.56g、4-溴甲基苯硼酸频哪醇酯6mmol，1.8g和50mL乙腈加入到100mL圆底烧瓶中，在温度90℃加热搅拌溶解，冷凝回流过夜反应20h，待反应完全后，停止反应；
b、待反应冷却至室温，加入50mL乙腈后，充分搅拌，过滤除去杂质，得到粗产物；
c、将步骤b得到的粗产物经正向硅胶色谱柱纯化，洗脱剂为二氯甲烷，洗脱产物干燥后即得到白色固体检测过氧化氢的荧光探针分子；
氢谱核磁数据：1H NMR(400MHz,DMSO-d6)δ11.04(s,2H),7.98(d,J＝7.6Hz,1H),7.76(dt,J＝29.5,7.4Hz,2H),7.30(d,J＝7.6Hz,1H),6.88(s,2H),6.62(s,2H)。
实施例3
荧光探针分子对过氧化氢的选择性检测：
使用实施例1中制备的荧光探针分子评价该类探针分子对过氧化氢的特异性检测识别，图2是该荧光探针在加入各种常见干扰物或强氧化剂时，检测试纸的荧光强荧光发生变化，例如分别加入1mmol/L的干扰物为(Bi(NO3)3、CoCl2、Cs2CO3、FeCl3、FeSO4、HgCl2、K2Cr2O7、KCl、KI、KMnO4、MgCl2、Na2SO3、NaClO4、NaNO2、NH4NO3、NiCl2、Pb(CH3CO2)2、CH4N2S、CH4ON、CH4N2O或Zn(CH3CO2)2)探针激发波长为365纳米，发射波长为526纳米；当在荧光探针溶液中加入的是过氧化氢时，该荧光探针溶液的荧光强度剧烈增加，而加入各种常见干扰物种时，该荧光探针溶液的荧光强度几乎没有明显变化，因此，则说明该荧光探针分子对过氧化氢具有良好的选择性识别的功能。
实施例4
荧光探针分子强度的变化与过氧化氢的浓度之间的线性关系：
使用实施例1中的荧光探针分子作为评价该类探针分子的荧光强度与过氧化氢离子浓度的线性关系，图3是荧光探针分子的发射中心波长在526纳米处的荧光强度随着过氧化氢浓度的变化关系曲线，图3的结果表明：荧光探针分子溶液的荧光强度变化与过氧化氢的浓度变化在0～80umol/L范围内呈现出良好的线性关系，因此，该类探针分子具备优异的检测过氧化氢的能力，表现出良好的检测性能。
实施例5
图4是硅胶板基负载荧光探针分子对过氧化氢的实际检测性能和效果图：
将实施例1中的荧光探针分子均匀分散在硅胶板上，得到检测过氧化氢的检测传感器；通过滴加过氧化氢溶液，并在紫外灯照射下的观察传感器上探针分子的荧光强度变化，图4所示的测试结果表明：负载有荧光探针分子的硅胶板能够高效的检测过氧化氢，显示出明显的荧光强度变化，表现出良好的过氧化氢检测性能。
需要更进一步说明的是，以上的实施实例仅仅是对本发明目的优选实施方式进行描述的一种以二氯荧光素为荧光基团，苯硼酸频哪醇酯为识别基团的衍生物，对于本发明实施实例中尚未述及的同类荧光衍生物，均与所述实施实例中均能实现相同的目的，并且达到相同的检测效果和技术效果。
并非对本发明的范围进行限定，在不脱离本发明设计精神的前提下，本领域及相关技术人员，凡是对本发明的技术方案做出的各种改变和改进，所产生的功能为超出本发明的技术方案时，均应属于本发明的权利要求书中明确的保护范围。
Claims (2)
1.一种用于检测过氧化氢的荧光探针分子，其特征在于该荧光探针的化学名称为2',7'-二R-3',6'-双((4-(4,4,5,5-四甲基-1,3,2-二氧杂环戊烷-2-基)苄氧基)-3H-螺[异苯并呋喃-1,9'-氧杂蒽]，其结构式为：
其中：R是氢或卤素。
2.根据权利要求1所述的用于检测过氧化氢的荧光探针分子的制备方法，其特征在于按下列步骤进行：
a、按摩尔比为1:2:3:500将化合物2-R基荧光素、碳酸钾、4-溴甲基苯硼酸频哪醇酯和乙腈加入到圆底烧瓶中，在温度90℃加热搅拌，冷凝回流反应20 h后，停止反应，其中：R是氢或卤素；
b、待反应冷却至室温后，加入50 mL乙腈，充分搅拌，过滤除去杂质，干燥得到粗产物；
c、将步骤b得到的粗产物经正向硅胶色谱柱纯化，洗脱剂为二氯甲烷，将洗脱产物干燥，即得到白色固体检测过氧化氢的荧光探针分子。
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| CN114657780A (zh) * | 2022-03-21 | 2022-06-24 | 武汉纺织大学 | 用于过氧化氢荧光检测的纳米纤维膜的制备方法 |
| CN114657780B (zh) * | 2022-03-21 | 2023-11-28 | 武汉纺织大学 | 用于过氧化氢荧光检测的纳米纤维膜的制备方法 |
| KR102648134B1 (ko) * | 2022-09-13 | 2024-03-14 | 경북대학교 산학협력단 | 신규한 플루오레세인 유도체 및 이의 과산화수소 검출 용도 |
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