CN116496232A - 一种次氯酸荧光探针及其制备方法和应用 - Google Patents
一种次氯酸荧光探针及其制备方法和应用 Download PDFInfo
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- CN116496232A CN116496232A CN202310301154.3A CN202310301154A CN116496232A CN 116496232 A CN116496232 A CN 116496232A CN 202310301154 A CN202310301154 A CN 202310301154A CN 116496232 A CN116496232 A CN 116496232A
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
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- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
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- C09K2211/1025—Heterocyclic compounds characterised by ligands
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- Nitrogen Condensed Heterocyclic Rings (AREA)
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- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
本发明公开了一种次氯酸荧光探针，分子式为C23H19NO3S，结构式如式Ⅰ所示。所述次氯酸荧光探针通过将10‑乙基‑3‑甲酰基吩噻嗪与3,4‑二羟基苯乙酮溶于溶剂中，加入哌啶，回流反应得粗产物，分离纯化所得。本发明合成工艺简单易行，制备得到的荧光探针对次氯酸选择性好、灵敏度高，检测限可达53.4nM，响应速度快，性能稳定，生物相容性好，能够在生理条件下对次氯酸进行实时检测。
Description
技术领域
本发明属于荧光探针技术领域，具体涉及一种次氯酸荧光探针及其制备方法和在检测水中和细胞中次氯酸根的应用。
背景技术
活性氧(ROS)是含氧的化学反应性物质的总称，包括超氧自由基、羟基自由基、过氧化氢、单线态氧(1O2)、α-氧和次氯酸/次氯酸盐等，在生命系统中发挥着重要作用。次氯酸(HClO)是一种弱酸性的活性氧，在抵抗病原体入侵和维持细胞氧化还原平衡等生理过程中起着关键作用。由于其强氧化性，HClO常被作为漂白剂或消毒剂广泛用于家庭清洁、饮用水和游泳池中。生物体内的内源性次氯酸是在白细胞包括巨噬细胞、单核细胞和中性粒细胞中，由过氧化氢和氯离子在髓过氧化物酶(MPO)的催化下反应生成的。在生理条件下，HClO处于平衡状态，部分解离成次氯酸根阴离子。尽管HClO有助于保护机体健康，但过量的HClO会引起氧化应激反应，导致一系列生理疾病，如炎症性疾病、动脉粥样硬化、心血管疾病、癌症和肾脏疾病等。鉴于HClO在生物学上的重要性，监测生命系统中次氯酸浓度的变化，研究活细胞中次氯酸的动态分布，在当今细胞生物学和医疗诊断等领域具有重要意义。
可用于检测次氯酸/次氯酸盐的方法有很多，如碘量滴定法、比色法、化学发光法、库仑法、极谱法和辐解法等。然而，这些方法往往比较繁琐，有些还需要昂贵的实验仪器和专业的技术人员。相比于上述方法，荧光探针检测法所需的仪器相对简单，操作方便，选择性和灵敏度高，检测范围广，响应时间快速，而且检测过程对样品没有破坏，对细胞危害也很小，荧光检测结合显微镜可以提供实时结果，因而受到研究者的广泛青睐，成为检测次氯酸的有力工具。
目前报道的用于检测次氯酸的荧光探针，按照荧光基团的不同可分为：BODIPY、罗丹明、荧光素、花菁、对甲氧基苯酚、香豆素、吩噻嗪和金属配合物等荧光探针。但是这些荧光探针仍然具有一些缺陷，如合成步骤复杂、水溶性差、对次氯酸的选择性差、背景荧光过高、灵敏度低、响应速度慢等，这些缺陷很大程度上影响了探针的应用。
发明内容
针对现有技术的不足，本发明的目的在于提供一种次氯酸荧光探针及其制备方法，本发明提供的荧光探针合成简单、性能稳定、对次氯酸根离子选择性好、灵敏度高、响应速度快、生物相容性好。
为实现上述目的，本发明采用如下技术方案：
本发明提供的一种次氯酸荧光探针，分子式为C23H19NO3S，结构式如式Ⅰ所示，
所述次氯酸荧光探针的制备方法，包括如下步骤：
将10-乙基-3-甲酰基吩噻嗪与3,4-二羟基苯乙酮溶于溶剂中，加入哌啶，回流反应得粗产物，分离纯化即得所述次氯酸荧光探针。
优选的，所述溶剂为甲醇。
优选的，所述10-乙基-3-甲酰基吩噻嗪与3,4-二羟基苯乙酮、哌啶的摩尔比为1:(1～1.5):(0.1～0.5)。
优选的，所述回流反应的温度为60～70℃，反应时间为36～60h。
优选的所述分离纯化为通过硅胶柱洗脱纯化，洗脱剂为体积比1:10的甲醇和乙酸乙酯。
本发明还提供上述次氯酸荧光探针在检测次氯酸根离子中的应用。
优选的，所述次氯酸荧光探针在检测次氯酸根离子中的应用，包括如下步骤：
将所述次氯酸荧光探针溶于亲水性有机溶剂中，得到荧光探针储备液；
将所述荧光探针储备液与PBS缓冲液混合，得到荧光探针检测液；
在所述荧光探针检测液中加入待测样品，进行荧光检测。
优选的，所述亲水性有机溶剂为无水乙醇、四氢呋喃、甲醇、N,N-二甲基甲酰胺或二甲基亚砜中的一种。
本发明还提供上述次氯酸荧光探针在制备检测次氯酸根离子产品中的应用。
与现有技术相比，本发明的有益效果在于：
本发明合成工艺简单易行，制备得到的荧光探针对次氯酸选择性好、灵敏度高，检测限可达53.4nM，响应速度快，性能稳定，生物相容性好，能够在生理条件下对次氯酸进行实时检测。
附图说明
图1为荧光探针PTZ-DAP随NaClO浓度变化的荧光光谱图；
图2为荧光探针PTZ-DAP随NaClO浓度变化的荧光强度拟合曲线图；
图3为荧光探针PTZ-DAP在不同p H值下与次氯酸钠反应前后荧光强度变化图；
图4为荧光探针PTZ-DAP与次氯酸反应随时间变化的荧光强度变化图；
图5为荧光探针PTZ-DAP与不同物质在470nm处的荧光强度图；
图6为RAW.264.7细胞与荧光探针PTZ-DAP共培养12h时的细胞活力变化图；
图7为荧光探针PTZ-DAP检测RAW264.7细胞中外源性HClO的荧光显微镜成像图；
图8为荧光探针PTZ-DAP检测RAW264.7细胞中内源性HClO的荧光显微镜成像图。
具体实施方式
以下结合具体实施例对本发明作进一步的详细说明，以使本领域的技术人员更加清楚地理解本发明。所举实例只用于解释本发明，并非用于限定本发明的范围。在本发明实施例中，若无特殊说明，所有原料组分均为本领域技术人员熟知的市售产品；若未具体指明，所用的技术手段均为本领域技术人员所熟知的常规手段。
实施例1
荧光探针PTZ-DAP的合成，合成路线如下：
(1)10-乙基吩噻嗪的合成：将吩噻嗪(9.96g，50mmol)和NaOH(16g，400mmol)加入二甲基亚砜(100ml)中，遮光搅拌1h；向溶液中滴加溴乙烷(5.72g，52.5mmol)，在室温下搅拌反应20h；反应完成后，将混合物倒入水(400ml)中并用二氯甲烷萃取；有机相用无水硫酸镁干燥，过滤，减压浓缩，粗产物通过硅胶柱洗脱纯化，洗脱剂为体积比1:5的二氯甲烷和石油醚，得到白色固体(9.16g，产率80.6％)。
(2)10-乙基-3-甲酰基吩噻嗪的合成：将10-乙基吩噻嗪(4.546g，20mmol)溶于1,2-二氯乙烷(40ml)；在室温和氮气气氛下，将N-甲基甲酰胺(2.958ml，24mmol)和氯氧化磷(2.796ml，30mmol)混合，搅拌30分钟后，将混合液滴加到10-乙基吩噻嗪溶液中，在氮气气氛85℃下搅拌反应6h；反应完成后，冷却至室温，缓缓倒入冰水中，然后用二氯甲烷萃取；有机相用无水硫酸钠干燥，过滤，减压浓缩，粗产物通过硅胶柱洗脱纯化，洗脱剂为体积比1:5的石油醚和乙酸乙酯，得到黄色固体(2.75g，产率53.9％)。
(3)PTZ-DAP的合成：将10-乙基-3-甲酰基吩噻嗪(2.04g，8mmol)与3,4-二羟基苯乙酮(1.52g，10mmol)溶于甲醇(30ml)中，加入哌啶(0.1ml，0.4mmol)，在65℃下回流反应48h；粗产物通过硅胶柱洗脱纯化，洗脱剂为体积比1:10的甲醇和乙酸乙酯，得到黄色油状液体(0.42g，产率13.5％)。
1H NMR(600MHz,CDCl3):δ10.04(s,2H),8.48(d,J＝1.6Hz,1H),8.18(dd,J＝8.8,1.6Hz,2H),7.73(t，J＝7.4Hz,2H),7.58(t,J＝9.5Hz,5H),7.40(t,J＝7.5Hz,2H),3.52-3.46(m,2H),1.42(t,J＝7.0Hz,3H).
上述的检测结果分析证实了合成的化合物为化学式Ⅰ所示的结构。
取实施例1制备的PTZ-DAP，分别加入无水乙醇(EtOH)、四氢呋喃(THF)、甲醇(MeOH)、N,N-二甲基甲酰胺(DMF)和二甲基亚砜(DMSO)中，配制成探针浓度为10mM的储备液，置于冰箱备用。
实施例2
荧光探针PTZ-DAP响应次氯酸根离子的荧光光谱检测。
将DMF与pH值为7.4的PBS缓冲液按9:1的体积比混合均匀，将含DMF的储备液稀释成探针浓度为10μM的检测液，取0.5mL检测液加入0.1mL不同浓度(0μM～720μM)的次氯酸钠(NaClO)溶液，使用荧光分光光度计检测荧光强度。
图1是荧光探针PTZ-DAP随NaClO浓度变化的荧光光谱，可以看出，随着NaClO浓度的增加，荧光强度显著增加。以NaClO浓度为横坐标，以为荧光强度为纵坐标作图，得到荧光探针PTZ-DAP随NaClO浓度变化的荧光强度拟合曲线图2。根据公式Detection limit＝3σ/k计算最低检测限，其中σ是未加次氯酸钠溶液的标准偏差，k是荧光强度与次氯酸钠溶液浓度的线性关系的斜率。计算得到荧光探针PTZ-DAP对次氯酸根离子的检测限为53.4nM。
实施例3
溶液pH对荧光探针PTZ-DAP检测次氯酸根离子的影响。
使用1mol/L氢氧化钠水溶液或1mol/L的盐酸将pH值为7.4的PBS缓冲液调节为pH值3～10的缓冲液；用DMF将储备液稀释成探针浓度为1mM的检测液。荧光检测分为两组：一组取10μL检测液，1mL不同pH值的缓冲液，用水定容至2mL；另一组取10μL检测液，1mL不同pH值的缓冲液，再加入200μL100μM的次氯酸钠溶液,用水定容至2mL。然后使用荧光分光光度计进行荧光光谱检测，结果如图3所示。
可以看出，荧光探针PTZ-DAP与次氯酸根反应前，探针分子随着pH的改变荧光基本没有变化，背景荧光较低，与次氯酸反应之后，荧光显著增强，而且随着pH的改变荧光基本没有变化，一直保持较强的荧光，在pH值为3～10的范围内都具有很好的稳定性。
实施例4
荧光探针PTZ-DAP与次氯酸反应的动力学测试。
将DMF与pH值为7.4的PBS缓冲液按9:1的体积比混合均匀，将储备液稀释成探针浓度为10μM的检测液，再加入次氯酸钠溶液使得次氯酸根离子的终浓度为10μM。在激发波长340nm条件下，使用荧光分光光度计进行荧光光谱检测，记录其在470nm处荧光强度，结果如图4所示。
可以看出，随着次氯酸钠的加入，荧光迅速增强，5秒钟之内即可达到平衡并且在1h内荧光强度基本保持不变。结果表明，荧光探针PTZ-DAP对次氯酸钠响应快速，对次氯酸钠可进行实时的检测。
实施例5
荧光探针PTZ-DAP对次氯酸根离子的选择性测试。
将DMF与pH值为7.4的PBS缓冲液按9:1的体积比混合均匀，将储备液稀释成探针浓度为10μM的检测液，再分别加入终浓度100μM的各种金属离子溶液、氨基酸溶液、活性氧、活性氮溶液和10μM的次氯酸钠，使用荧光分光光度计分别进行荧光光谱检测。结果如图5所示。1～30分别为：(1)空白组；(2)H2O2；(3)O2 ·-；(4)1O2；(5)·OH；(6)NO；(7)TBO·；(8)TBHP；(9)ONOO-；(10)脯氨酸；(11)甘氨酸；(12)谷氨酸；(13)天冬氨酸；(14)半胱氨酸；(15)亮氨酸；(16)谷胱甘肽；(17)赖氨酸；(18)甲硫氨酸；(19)氯化钡；(20)硫酸钛；(21)氯化钙；(22)硫酸镁；(23)硫酸铜；(24)氯化钾；(25)氯化钠；(26)亚硝酸钠；(27)硝酸锌；(28)硫化钠；(29)氯化铈；(30)次氯酸钠。
可以看出，只有次氯酸钠的加入可以使荧光探针PTZ-DAP在470nm处的荧光信号显著增强，其它各种活性氧、氨基酸、阴离子和阳离子均不会引起荧光探针PTZ-DAP荧光信号的明显变化，这说明本发明所提供的荧光探针PTZ-DAP能够实现对次氯酸根离子的选择性检测。
实施例7
荧光探针PTZ-DAP对细胞毒性测试。
细胞毒性实验采用MTT法。将处于对数期生长的大鼠巨噬细胞RAW.264.7细胞接种到96孔板，每孔加1ml细胞液使得每孔含有104个细胞，放入37℃含有5％CO2的培养箱内用DMEM培养基孵育使细胞贴壁。用DMEM将含DMSO的储备液稀释成不同浓度(0μM，100μM，200μM，300μM，400μM，500μM)的检测液；每孔加入10μL，对照组加入10μL细胞培养用1×PBS缓冲液，每个浓度做八个平行样。孵育12h后添加10μL0.5mg/ml的MTT，放入培养箱中继续孵育4h；吸出上清液，每孔添加150μL DMSO，置于摇床上震荡20分钟，取出用酶标仪测定每孔在490nm处的吸光度。
细胞存活率计算公式为：
Cell viability(％)＝(OD490sample-OD490blank)/(OD490control-OD490blank)×100％
其中OD490sample表示用不同浓度的探针PTZ-DAP处理细胞的孔的吸光值；OD490blank表示只有DMEM的孔的吸光值；OD490control表示未经探针PTZ-DAP处理细胞的孔的吸光值。
结果如图6所示，可以看出，用0-50μM不同浓度的探针PTZ-DAP孵育细胞24h后，即使探针PTZ-DAP浓度达到50μM，细胞存活效果仍然令人满意，细胞的存活率在95％以上，这表明探针PTZ-DAP对细胞具有低毒性，并且可以很好地应用于细胞而没有严重的细胞损伤，适用于生物成像实验。
实施例8
荧光探针PTZ-DAP对次氯酸的细胞成像。
(1)细胞外源性次氯酸钠成像
实验之前用DMSO将储备液稀释成探针浓度为15μM的检测液。实验分为三组：第一组为对照组，先把探针孵育到细胞上，半个小时后用1×PBS缓冲液洗涤三次，再加入PBS缓冲液继续孵育半个小时；第二组先把探针孵育到细胞上，半个小时后用1×PBS缓冲液洗涤三次，再加入10μM次氯酸钠溶液继续孵育半个小时；第三组把探针孵育到细胞上，半个小时后用1×PBS缓冲液洗涤三次，再加入30μM次氯酸钠溶液继续孵育半个小时。吸出次氯酸钠溶液，用PBS缓冲液洗涤三次后用荧光显微镜观察荧光变化。
结果如图7所示，a、c、e分别为三组的明场图像，b、d、f分别为对应的蓝色通道。可以看出，仅用探针处理RAW264.7细胞30分钟后，在蓝色通道几乎观察不到荧光，而在用1eq的次氯酸钠处理30分钟后，在细胞中产生微弱的蓝色荧光，表明探针与次氯酸钠发生了特异性结合作用；随着次氯酸钠的浓度变大，与3eq的次氯酸钠共同孵育时，细胞内的蓝色荧光信号明显增强，表明探针对细胞内次氯酸钠反应。结果表明，荧光探针PTZ-DAP表现出良好的细胞膜渗透性，具有在RAW264.7细胞中检测到外源性HClO的能力。
(2)细胞内源性次氯酸钠成像
实验之前用DMSO将储备液稀释成探针浓度为10μM的检测液。实验分为三组：第一组为对照组，先把探针孵育到细胞上，半个小时后用1×PBS缓冲液洗涤三次，再加入PBS缓冲液继续孵育半个小时后进行成像；第二组细胞先用1μg/ml LPS孵育半个小时，后用1×PBS缓冲液洗涤三次，再孵育探针半小时进行成像；第三组细胞先用1μg/ml LPS和5μg/mlPMA孵育半个小时，后用1×PBS缓冲液洗涤三次，再加入250μM 4-氨基苯甲酰肼孵育一个小时后用1×PBS缓冲液洗涤三次，最后加入探针PTZ-DAP孵育细胞半个小时，再用1×PBS缓冲液洗涤三次后用荧光显微镜成像。
结果如图8所示，a、c、e分别为三组的明场图像，b、d、f分别为对应的蓝色通道。可以看出，在对照组中，活的RAW264.7巨噬细胞在培养基中与探针PTZ-DAP一起在37℃下孵育30分钟，未观察到明显荧光。而在实验组中，巨噬细胞与LPS和PMA孵育30分钟后，再与探针PTZ-DAP共孵育30分钟，蓝色通道可以观察到明显荧光。当细胞先与LPS和PMA孵育30分钟，再加入可以降低细胞内次氯酸水平的MPO抑制剂ABAH培育30分钟后，与探针PTZ-DAP共孵育30分钟，蓝色通道可以明显观察到荧光减弱。结果表明，荧光探针PTZ-DAP能够对活的RAW264.7巨噬细胞中的内源性HClO进行成像。
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (10)
1.一种次氯酸荧光探针，其特征在于，分子式为C23H19NO3S，结构式如式Ⅰ所示，
2.权利要求1所述的次氯酸荧光探针的制备方法，其特征在于，包括如下步骤：
将10-乙基-3-甲酰基吩噻嗪与3,4-二羟基苯乙酮溶于溶剂中，加入哌啶，回流反应得粗产物，分离纯化即得所述次氯酸荧光探针。
3.根据权利要求2所述的次氯酸荧光探针的制备方法，其特征在于，所述溶剂为甲醇。
4.根据权利要求2所述的次氯酸荧光探针的制备方法，其特征在于，所述10-乙基-3-甲酰基吩噻嗪与3,4-二羟基苯乙酮、哌啶的摩尔比为1:(1～1.5):(0.1～0.5)。
5.根据权利要求2所述的次氯酸荧光探针的制备方法，其特征在于，所述回流反应的温度为60～70℃，反应时间为36～60h。
6.根据权利要求2所述的次氯酸荧光探针的制备方法，其特征在于，所述分离纯化为通过硅胶柱洗脱纯化，洗脱剂为体积比1:10的甲醇和乙酸乙酯。
7.权利要求1所述的次氯酸荧光探针在检测次氯酸根离子中的应用。
8.根据权利要求7所述的次氯酸荧光探针在检测次氯酸根离子中的应用，其特征在于，包括如下步骤：
将所述次氯酸荧光探针溶于亲水性有机溶剂中，得到荧光探针储备液；
将所述荧光探针储备液与PBS缓冲液混合，得到荧光探针检测液；
在所述荧光探针检测液中加入待测样品，进行荧光检测。
9.根据权利要求8所述的次氯酸荧光探针在检测次氯酸根离子中的应用，其特征在于，所述亲水性有机溶剂为无水乙醇、四氢呋喃、甲醇、N,N-二甲基甲酰胺或二甲基亚砜中的一种。
10.权利要求1所述的次氯酸荧光探针在制备检测次氯酸根离子产品中的应用。
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