CN104262287A - 一种亚硫酸根比率荧光探针的制备及应用 - Google Patents
一种亚硫酸根比率荧光探针的制备及应用 Download PDFInfo
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Abstract
本发明公开了一种可以用于亚硫酸根离子荧光识别的新型化合物，具体涉及一种亚硫酸根比率荧光探针的制备方法及其应用，属于化学分析检测技术领域。其分子结构式如下：，该荧光探针用于环境或生物样品中亚硫酸根离子的荧光传感、分析，加入亚硫酸根后，醛基与亚硫酸根发生加成反应，生成羟基磺酸盐，对亚硫酸根离子识别的选择性好，抗干扰能力强，可以灵敏快速地从多种阴离子中区分出亚硫酸根离子，具有很好的应用前景。
Description
技术领域
本发明涉及的是化学分析检测技术领域，具体涉及一种亚硫酸根比率荧光探针的制备方法以及该荧光探针在检测亚硫酸根离子方面的应用。
背景技术
亚硫酸盐是一种重要的食品添加剂，可作为食品防腐剂和漂白剂，并且通过抑制非酶褐变和酶促褐变防止食品褐变。亚硫酸盐的使用需要在一定的安全范围内，人摄入少量的亚硫酸盐不会对身体造成伤害，然而摄入过多的亚硫酸盐可能造成胃肠障碍、引起剧烈腹痛、肾功能障碍能疾病。尤其是对亚硫酸盐过敏的人，即使摄入少量的亚硫酸盐也易引发哮喘、皮肤过敏、胃肠不适等。此外，工业废水中含有过多亚硫酸盐，会严重影响水中溶解氧的浓度，从而对生态环境造成危害。因此，发明方便快捷、高灵敏度检测亚硫酸盐含量的方法对食品安全和环境监测是十分重要的。传统检测亚硫酸盐的方法包括盐酸副玫瑰苯胺比色法、中和滴定法等。荧光探针具有灵敏度高、选择性好、原位检测等特点，非常适于检测亚硫酸根离子，然而近几年报导的大多数检测亚硫酸根离子的荧光探针是以荧光发射强度的增强或减弱作为响应信号，易受到诸如检测底物浓度、外部环境和仪器设备灵敏度因素的影响。比率荧光探针通过对两个不同波长处荧光强度作比值，并以其比值为检测信号，有效解决了上述荧光探针存在的问题。因此，发明新型亚硫酸根比率型荧光探针具有十分重大的意义。
发明内容
本发明目的之一是提供一种合成简单、反应条件温和、成本较低的荧光探针合成方法；目的之二是提供一种灵敏度高、选择性好，抗干扰能力强，能够实现比率荧光法检测亚硫酸根离子的荧光探针。
本发明使用比率荧光法检测亚硫酸根荧光探针，以苯并噻唑衍生物为荧光团，以醛基为亚硫酸根的识别基团。
本发明解决问题采取的技术方案为，一种比率荧光检测亚硫酸根荧光探针，其分子结构式如下， 。该荧光探针的合成路线如下，
具体制备方法包括以下步骤： 1) 将邻氨基苯硫酚和4-甲基水杨酸溶于甲苯中，氮气保护下加热至55℃，搅拌2.5 h；然后停止加热，自然冷却至35℃，向反应液中缓慢滴加三氯化磷，升温至85℃并搅拌5 h；停止反应，除去溶剂，经柱层析分离得到产物1。2) 将上步所得产物1和三乙胺溶于二氯甲烷中，再向其中缓慢滴加乙酰氯，室温搅拌2 h；加入蒸馏水淬灭反应，用二氯甲烷萃取，饱和食盐水洗涤，无水硫酸钠干燥，旋蒸除去溶剂，经柱层析分离得到产物2。3) 将上步得到产物2，N-溴代丁二酰亚胺，过氧化苯甲酰溶于无水四氯化碳中，回流24 h；停止反应，将反应液倒入冰水中，氯仿萃取，无水硫酸钠干燥，旋蒸除去溶剂，经柱层析分离得到产物3。4) 将上步得到产物3和环六亚甲基四胺溶于氯仿中，回流搅拌8 h，旋蒸除去氯仿，加入50%的乙酸溶液，回流搅拌2 h；加水淬灭反应，再用氢氧化钠溶液调节pH至中性, 用氯仿萃取，无水硫酸钠干燥，旋蒸除去溶剂；然后将所得固体溶于二氯甲烷中，加入氢氧化钾，搅拌回流1 h；停止加热，反应液冷却至室温，用稀盐酸溶液调节pH至中性，二氯甲烷萃取，无水硫酸钠干燥，旋蒸除去溶剂，经柱层析分离得到产物4，即探针分子。在本说明书的实施例中更详细地说明了该探针的合成和检测方法。
本发明的荧光分子探针使用方法如下，将探针分子溶解在含有30%乙腈、pH为7.4的HEPES缓冲溶液中，室温下进行测试。当加亚硫酸根时，由于醛基可以与亚硫酸根发生加成反应，生成羟基磺酸盐，从而引起荧光光谱的变化。探针分子与亚硫酸根作用原理如下，。
本发明的亚硫酸根比率荧光探针的具体特征如下：探针分子在315 nm 和357 nm处有两个吸收峰, 荧光发射峰在539 nm处，随着亚硫酸根的加入，探针分子在315 nm 和357 nm处的吸收峰逐渐下降，在290 nm和332 nm产生新吸收峰。而荧光光谱在539 nm处的发射峰逐渐降低直至消失，而在460 nm处出现了新的发射峰，并且逐渐增强，荧光颜色也由原来的黄色荧光变为绿色荧光。460 nm和539 nm两波长所对应的荧光强度比值从0.036升高到 3.11，荧光强度比值是原来的86倍。
本发明所述的探针分子合成简单，成本较低，对亚硫酸根的选择性好、抗干扰能力强、响应速度快使得该比率荧光探针在生物化学，环境科学等领域具有实际的应用价值。
附图说明
图1为本发明的荧光探针 (5.0×10-6 mol/L) 在缓冲溶液 (V乙腈/VHEPES = 3/7，pH = 7.4) 中，与亚硫酸根反应前后的紫外吸收光谱变化情况，横坐标为波长，纵坐标为吸光度。
图2为本发明的荧光探针 (5.0×10-6 mol/L) 在缓冲溶液 (V乙腈/VHEPES = 3/7，pH = 7.4)中，与不同浓度亚硫酸根作用后的荧光光谱变化情况，横坐标为波长，纵坐标为荧光强度。
图3为本发明的荧光探针 (5.0×10-6 mol/L) 在缓冲溶液（V乙腈/VHEPES = 3/7，pH = 7.4)中，与低浓度浓度亚硫酸根作用反应后荧光强度比值(I460 nm/I539 nm)与亚硫酸根浓度的线性关系，横坐标为亚硫酸根的浓度，纵坐标为荧光强度比值(I460 nm/I539 nm)。
图4为本发明的荧光探针 (5.0×10-6 mol/L) 在缓冲溶液 (V乙腈/VHEPES = 3/7，pH = 7.4)中，与阴离子 (F-, Cl-, Br-, I-, NO3 -, NO2 -, AcO-, N3 -, SO4 2-, S2O3 2-, ClO-, SCN-, CN-, H2O2, PO43-, S2-, CO3 2-) 和氨基酸 (Cys, Gsh, Hcy) 作用的后的荧光强度比值 (I460 nm/I539 nm) 柱状图。
图5为亚硫酸根与阴离子(F-, Cl-, Br-, I-, NO3 -, NO2 -, AcO-, N3 -, SO4 2-, S2O3 2-, ClO-, SCN-, CN-, H2O2, PO43-, S2-, CO3 2-)和氨基酸(Cys, Gsh, Hcy)共存时，与本发明的荧光探针(5.0×10-6 mol/L)在缓冲溶液（V乙腈/VHEPES=3/7，pH=7.4)中作用后的荧光强度比值(I460 nm/I539 nm)柱状图。
图6为本发明的荧光探针 (5.0×10-6 mol/L) 在不同pH值缓冲溶液（V乙腈/VHEPES = 3/7) 中，与亚硫酸根作用前后的荧光强度比值 (I460 nm/I539 nm)，横坐标为pH，纵坐标为荧光强度比值 (I460 nm/I539 nm)。
图7为本发明的荧光探针 (5.0×10-6 mol/L) 在缓冲溶液 (V乙腈/VHEPES = 3/7，pH =7.4)中，与亚硫酸根作用过程中荧光强度比值随时间的变化，横坐标为时间，纵坐标为荧光强度比值(I460 nm/I539 nm)。
具体实施方式
实施例1：化合物1的合成
将邻氨基苯硫酚 (8.2 g, 66 mmol) 和4-甲基水杨酸 (10.0 g, 66 mmol) 溶于70 mL甲苯中，氮气保护下加热至55 ℃，搅拌2.5 h；然后停止加热，自然降温至35℃，向反应液中缓慢滴加6.2 mL三氯化磷，升温至85℃并搅拌5 h；停止反应，旋蒸除去溶剂，经柱层析分离得到产物1。产量：6.4 g。产量：40.3％。化合物1表征如下：1H NMR (CDCl3): δ = 12.46 (s, 1H), 7.97-7.95 (d, 1H), 7.89-7.87 (d, 1H), 7.58-7.56 (d, 1H), 7.51-7.47 (t, 1H), 7.41-7.37 (t, 1H), 6.92 (s, 1H), 6.78-6.76 (d, 1H), 2.37 (s, 3H).
实施例2：化合物2的合成
上步所得产物1 (5.0 g, 20.7 mmol) 和三乙胺 (2.5 g, 24.9 mmol) 溶于60mL二氯甲烷中，再向其中缓慢滴加乙酰氯 (1.95 g, 24.9 mmol)，室温搅拌2 h；停止反应，加入40 mL水淬灭反应，用二氯甲烷萃取，饱和食盐水洗涤，无水硫酸钠干燥，旋蒸除去溶剂，经柱层析分离得到产物2。产量：4.7 g。产量：80％。化合物2表征如下：1H NMR (400 MHz, CDCl3) δ 8.23 (d, J = 8.1 Hz, 1H), 8.11 (d, J = 8.1 Hz, 1H), 7.94 (d, J = 7.5 Hz, 1H), 7.53 (t, J = 7.7 Hz, 1H), 7.43 (m, 1H), 7.24 (d, J = 8.1, 1H), 7.09 (s, 1H), 2.51 (s, 3H), 2.46 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 169.31, 162.75, 153.05, 148.12, 142.45, 135.21, 130.07, 127.43, 126.26, 125.20, 124.17, 123.38, 123.23, 121.31, 21.74, 21.39.
实施例3：化合物3的合成
将上步得到的产物2 (3.5 g, 12.4 mmol)，N-溴代丁二酰亚胺(2.4 g, 13.5 mmol)，过氧化苯甲酰(0.06 g, 0.025 mmol)溶于50 mL干燥四氯化碳中，回流24 h；停止反应，将反应液倒入冰水中，氯仿萃取，无水硫酸钠干燥，旋蒸除去溶剂，真空干燥后经柱层析分离得到产物3。产量：2.3 g。产率：51.3％。化合物3表征如下：1H NMR (400 MHz, CDCl3) δ 8.35 (d, J = 8.1 Hz, 1H), 8.12 (d, J = 7.9 Hz, 1H), 7.96 (d, J = 7.4 Hz, 1H), 7.58 – 7.52 (m, 1H), 7.48 – 7.42 (m, 2H), 7.33 (s, 1H), 4.54 (s, 2H), 2.52 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 168.97, 161.79, 152.91, 148.23, 141.31, 135.35, 130.68, 127.00, 126.46, 126.03, 125.56, 124.31, 123.47, 121.39, 31.73, 21.71.
实施例4：探针分子的合成
将上步得到的产物3 (1.5 g, 4.1 mmol) 和环六亚甲基四胺 (1.3g, 9.1 mmol) 溶于40mL氯仿中，回流搅拌8 h，旋蒸除去氯仿，加入20 mL 50%的乙酸溶液，回流搅拌2 h，加水淬灭反应，再用氢氧化钠溶液调节体系pH至中性, 用氯仿萃取，无水硫酸钠干燥，旋蒸除去溶剂；然后将所得的固体溶于20 mL二氯甲烷中，加入0.3 g氢氧化钾，搅拌回流1 h，停止加热，反应液冷却至室温，用稀盐酸溶液调节pH至中性，二氯甲烷萃取，无水硫酸钠干燥，旋蒸除去溶剂，经柱层析分离得到最终产物4，即探针分子。产量：0.3170 g。产率：30％。最终产物4表征如下：1H NMR (400 MHz, CDCl3) δ 10.04 (s, 1H), 8.07 (d, J = 7.8 Hz, 1H), 7.97 (d, J = 8.0 Hz, 1H), 7.87 (d, J = 8.0 Hz, 1H), 7.63 – 7.55 (m, 2H), 7.50 (t, J = 7.5 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 191.41, 167.93, 158.29, 151.66, 139.04, 132.98, 128.99, 127.12, 126.31, 122.66, 121.67, 121.28, 119.64, 119.50.
实施例5：本发明亚硫酸根比率荧光探针的应用
将探针溶于缓冲溶液 (V乙腈/VHEPES = 3/7，pH = 7.4) 中配制成5.0×10-6 mol/L的溶液，向溶液中加入F-, Cl-, Br-, I-, NO3 -, NO2 -, AcO-, N3 -, SO4 2-, S2O3 2-, ClO-, SCN-, CN-, H2O2, Cys, GSH, Hcy, PO43-, S2-, CO3 2-没有引起荧光的变化，加入SO3 2-引起了荧光变化，该荧光探针对亚硫酸根表现出高灵敏度、高选择性的识别。当SO3 2-与干扰物质F-, Cl-, Br-, I-, NO3 -, NO2 -, AcO-, N3 -, SO4 2-, S2O3 2-, ClO-, SCN-, CN-, H2O2, Cys, GSH, Hcy, PO43-, S2-, CO3 2-共存时，探针不受干扰因素的影响，表现出来很好的抗干扰能力。该探针分子与亚硫酸根响应速度快，30秒内即可观察到荧光的变化。探针分子在pH为3至10的范围内都可以对亚硫酸根选择性识别，表现出了较宽的应用范围。
Claims (3)
1.一种亚硫酸根离子比率荧光探针，其结构为：
2.如权利要求1所述的亚硫酸根离子荧光探针的制备方法，其特征在于按以下步骤进行制备：
(a)将邻氨基苯硫酚和4-甲基水杨酸溶于甲苯中，氮气保护下加热至55℃，搅拌2.5h；然后停止加热，自然冷却至35℃，向反应液中缓慢滴加三氯化磷，升温至85℃并搅拌5h；停止反应，除去溶剂，经柱层析分离得到产物。
(b)将上步所得产物和三乙胺溶于二氯甲烷中，再向其中缓慢滴加乙酰氯，室温搅拌2h；加入蒸馏水淬灭反应，用二氯甲烷萃取，饱和食盐水洗涤，无水硫酸钠干燥，旋蒸除去溶剂，经柱层析分离得到产物。
(c)将上步得到产物，N-溴代丁二酰亚胺，过氧化苯甲酰溶于无水四氯化碳中，回流24h；停止反应，将反应液倒入冰水中，氯仿萃取，无水硫酸钠干燥，旋蒸除去溶剂，经柱层析分离得到产物。
(d)将上步得到产物和环六亚甲基四胺溶于氯仿中，回流搅拌8h，旋蒸除去氯仿，加入50％的乙酸溶液，回流搅拌2h；加水淬灭反应，再用氢氧化钠溶液调节pH至中性,用氯仿萃取，无水硫酸钠干燥，旋蒸除去溶剂；然后将所得固体溶于二氯甲烷中，加入氢氧化钾，搅拌回流1h；停止加热，反应液冷却至室温，用稀盐酸溶液调节pH至中性，二氯甲烷萃取，无水硫酸钠干燥，旋蒸除去溶剂，经柱层析分离得到产物。
3.根据权利要求1所述的亚硫酸根离子荧光探针的用途，其特征在于该亚硫酸根离子荧光探针用于环境或生物样品中的亚硫酸根离子的荧光检测和分析。
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