CN108690040A - 一种超灵敏高选择性检测次氯酸的比色荧光探针 - Google Patents
一种超灵敏高选择性检测次氯酸的比色荧光探针 Download PDFInfo
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Abstract
本发明涉及一种超灵敏高选择性检测次氯酸的比色荧光探针。具体地,本发明的探针为一类荧光素类化合物,其可作为次氯酸荧光探针用于次氯酸的检测。这类探针可实现如下的技术效果中的至少一个:高灵敏高选择性地识别次氯酸并进行准确检测;可以快速对次氯酸实现响应;可以实现对次氯酸的即时检测;性质稳定,可以长期保存使用;有利于在实际样本中对(体内/体外)次氯酸进行检测。
Description
技术领域
本发明涉及荧光素类化合物作为次氯酸荧光探针,其可在纯水体系下对细胞内源性次氯酸进行快速超灵敏高选择性检测,或者其可测定实际水样中次氯酸的浓度。
背景技术
在人体内,次氯酸(HClO)可以以髓过氧化物酶(MPO)催化反应的过氧化氢和氯离子的调节方式产生,其与先天宿主防御和对杀死各种病原体非常重要。在体外,由于泳池水次氯酸浓度过高导致的游泳池中毒事件现依旧频频发生。生物体内产生或蓄积过量的次氯酸,会引起多种疾病,包括心血管疾病,动脉粥样硬化,骨关节炎,类风湿性关节炎和肺损伤,严重还会导致癌症。因此,检测实际水体和生物体系中次氯酸的浓度已成为一个重要的课题,现亟待发展快速、灵敏、高选择性检测次氯酸的手段。
当前,常见的次氯酸检测手段主要是碘还原滴定法、分光光度法、化学发光分析法、库伦法等。但这些分析手段在实际应用中既昂贵又繁琐,且常常需要特殊的昂贵实验仪器和高技能专业操作人员。因此,高效、廉价、简捷的次氯酸检测手段成为重要的研究课题。
在各种离子/分子的检测方法中,比色荧光探针检测法由于操作简单,可在现场使用,选择性和灵敏度高,检测范围广,响应时间快速,能够进行原位检测和实时监测,检测过程对样品没有破坏而极为引人注目。但是目前报道的荧光探针仍存在一些问题,包括灵敏度低、选择性差以及合成复杂等。总之,发展具有高灵敏度、高选择性、合成步骤简单的次氯酸比色荧光探针是本领域技术人员急需解决的问题。
发明内容
本领域急需一种制备简单的快速高灵敏高选择性识别次氯酸的比色荧光探针,从而能够有效检测体内或体外次氯酸。为此,本发明合成了一类新颖的次氯酸的荧光探针,其合成简单、超灵敏、稳定性高、和/或选择性高,和/或能够快速特异性识别次氯酸。
具体而言,本发明提供了一种次氯酸比色荧光探针,其为荧光素类化合物,其结构如下:
优选的,本发明的荧光探针是:
在本发明的次氯酸荧光探针的制备方法中,第一步反应温度是70℃油浴;反应时间是24h,和/或间苯二酚和4-乙氨基酮酸的摩尔比约为1∶1至1∶1.2,优选的为1∶1;第二步反应温度为常温,和/或第一步的产物与二甲氨基硫代甲酰氯的摩尔比为约1∶4至1∶12,优选为1∶10或1∶12。
本发明还提供了用于检测样本(例如游泳池水样本)中次氯酸浓度的检测制剂,其包含本发明的探针。优选地,本发明的检测制剂还包含产品的使用说明书。
本发明的次氯酸荧光探针可与次氯酸进行特异性作用,产生荧光光谱的变化,从而实现对次氯酸的定量检测。
具体而言,本发明的次氯酸荧光探针分别与过氧化氢、过氧化亚硝酸盐、氯酸盐以及其他物质进行作用均不能导致荧光光谱的明显改变,从而实现对次氯酸的特异性识别。
可选择地,本发明的次氯酸荧光探针的稳定性好,进而能够长期保存使用。
进一步的,本发明的次氯酸荧光探针是快速高选择性次氯酸比色探针,且合成简单,成本低廉,有利于商业化的推广应用。
附图说明
图1是探针(5μM)对HClO(10μM)响应时间的测试结果。
图2和图3是不同浓度HClO(50-450nM)对探针(5μM)荧光光谱的影响及定量线性关系。
图4是不同分析物对探针(5μM)荧光光谱的影响。其中:1.空白;2.钾离子K+;3.钙离子Ca2+;4.镁离子Mg2+;5.钠离子Na+;6.锌离子Zn2+;7.铝离子Al3+;8.硝酸根离子NO3 -;9.亚硝酸根离子NO2 -;10.铜离子Cu2+;11.铁离子Fe3+;12.亚铁离子Fe2+;13.半胱氨酸Cys;14.过氧化氢H2O2;15.过氧化叔丁醇TBHP;16.羟基自由基·OH;17.过氧叔丁醇自由基·OtBu;18.一氧化氮NO;19.单线态氧1O2;20.超氧阴离子自由基O2 -;21.氯离子Cl-;22.谷胱甘肽GSH;23.过氧化亚硝酸盐ONOO-;24.次氯酸HClO(10μM)。
具体实施方式:
本发明提供了上述快速高灵敏高选择性次氯酸荧光探针的合成路线、方法及其光谱性能。
本发明的次氯酸荧光探针是一类荧光素类化合物,其具有以下结构通式:
上式中:R1,R2,R3,R4,R5,R6,R7,R8,R9和R10为氢原子,直链或支链烷基,直链或支链烷氧基,磺酸基,酯基,羧基;R1,R2,R3,R4,R5,R6,R7,R8,R9和R10可以相同或不同。
该类次氯酸比色荧光荧光探针的合成路线和方法如下:
具体地,本发明的荧光探针可以通过如下方法制备,第一步为间苯二酚和4-乙氨基酮酸溶于三氟乙酸溶液加热回流24h,旋蒸之后加入乙酸乙酯回流15分钟,抽滤得到红色固体。将一定摩尔比(例如1∶4-1∶12)的第一步的产物与二甲氨基硫代甲酰氯溶于二氯甲烷并加入DIPEA进行常温搅拌一段时间(例如20h)使用二氯甲烷体系进行柱色谱分离,得到橙色固体435mg,产率为87%。
因此,本发明还提供了荧光素类化合物在制备用于检测次氯酸的比色荧光探针中的用途。
本发明还提供了二甲硫代甲酸酯类化合物在制备用于检测次氯酸的荧光探针中的用途。
本发明的快速高灵敏高选择性识别次氯酸荧光探针的显著特征是能够快速高选择性特异性地识别次氯酸,能够准确对次氯酸进行定量分析。
下面将通过借助以下实施例来更详细地说明本发明。以下实施例仅是说明性的,应该明白,本发明并不受下述实施例的限制。
实施例1
(方案1)第一步为间苯二酚和4-乙氨基酮酸溶于三氟乙酸溶液加热回流24h,旋蒸之后加入乙酸乙酯回流15分钟,抽滤得到红色固体。将的第一步的产物387mg(1mmol)与二甲氨基硫代甲酰氯430.16mg(4mmol)溶于二氯甲烷并加入DIPEA进行常温搅拌20h使用二氯甲烷体系进行柱色谱分离,得到橙色固体370mg,产率为74%。
(方案2)第一步为间苯二酚和4-乙氨基酮酸溶于三氟乙酸溶液加热回流24h,旋蒸之后加入乙酸乙酯回流15分钟,抽滤得到红色固体。将的第一步的产物387mg(1mmol)与二甲氨基硫代甲酰氯645.24mg(6mmol)溶于二氯甲烷并加入DIPEA进行常温搅拌20h使用二氯甲烷体系进行柱色谱分离,得到橙色固体402mg,产率为80%。
(方案3)第一步为间苯二酚和4-乙氨基酮酸溶于三氟乙酸溶液加热回流24h,旋蒸之后加入乙酸乙酯回流15分钟,抽滤得到红色固体。将的第一步的产物387mg(1mmol)与二甲氨基硫代甲酰氯860.32mg(8mmol)溶于二氯甲烷并加入DIPEA进行常温搅拌20h使用二氯甲烷体系进行柱色谱分离,得到橙色固体430mg,产率为86%。
(方案4)第一步为间苯二酚和4-乙氨基酮酸溶于三氟乙酸溶液加热回流24h,旋蒸之后加入乙酸乙酯回流15分钟,抽滤得到红色固体。将的第一步的产物387mg(1mmol)与二甲氨基硫代甲酰氯1075.4mg(10mmol)溶于二氯甲烷并加入DIPEA进行常温搅拌20h使用二氯甲烷体系进行柱色谱分离,得到橙色固体435mg,产率为87%。
(方案5)第一步为间苯二酚和4-乙氨基酮酸溶于三氟乙酸溶液加热回流24h,旋蒸之后加入乙酸乙酯回流15分钟,抽滤得到红色固体。将的第一步的产物387mg(1mmol)与二甲氨基硫代甲酰氯1290.49mg(12mmol)溶于二氯甲烷并加入DIPEA进行常温搅拌20h使用二氯甲烷体系进行柱色谱分离,得到橙色固体420mg,产率为84%。
本发明的发明人进行了如下测试:
实施例2
探针(5μM)对HClO(10μM)响应时间的测试结果。上述测定是在5mM PBS,pH 7.4的水溶液中进行的,所使用的探针是实施例1中所制备的探针,且所有光谱测试都是在25℃下测得的。结果参见图1。
从图1可以看出,加入10μM次氯酸后,荧光强度骤然增强,并且在5s内能够响应完成。
实施例3
不同浓度次氯酸(0-10μM)对探针(5μM)荧光光谱的影响。上述测定是在5mM PBS,pH 7.4的水溶液中进行的,所使用的探针是实施例1中所制备的探针,且所有光谱测试都是在25℃下作用1min后测得的。结果参见图2。
从图3可以看出,伴随着探针溶液中次氯酸浓度的增加,荧光光谱逐渐增强,且在50-450nM的次氯酸浓度范围内,次氯酸的浓度和荧光强度显现线性关系,且检出限低至4.5nM。因此,本发明的探针能较精确地确定待测样本中次氯酸的含量。
实施例4
(a)不同分析物对探针(5μM)荧光光谱的影响。分析物包括:1.空白;2.钾离子K+;3.钙离子Ca2+;4.镁离子Mg2+;5.钠离子Na+;6.锌离子Zn2+;7.铝离子Al3+;8.硝酸根离子NO3 -;9.亚硝酸根离子NO2 -;10.铜离子Gu2+;11.铁离子Fe3+;12.亚铁离子Fe2+;13.半胱氨酸Cys;14.过氧化氢H2O2;15.过氧化叔丁醇TBHP;16.羟基自由基·OH;17.过氧叔丁醇自由基·OtBu;18.一氧化氮NO;19.单线态氧1O2;20.超氧阴离子自由基O2 -;21.氯离子Cl-;22.谷胱甘肽GSH;23.过氧化亚硝酸盐ONOO-;24.次氯酸HClO(10μM)。它们的浓度(除特殊标注)均为1000μM。所有测试条件是5mM PBS,pH=7.4的水溶液中进行的,所使用的探针是实施例1中所制备的探针,且所有光谱测试都是在25℃下作用1min后测得的。结果参见图4。具体地,移取50μL的探针储备液(1mM)放进10mL比色管中,然后加入5mL超纯水,再移取20μL上述分析物储备液加入比色管内,然后移取1mL的PBS溶液(pH 7.4,10mM),加入比色管内,最后用超纯水定容至10mL。摇匀,静置5min,即可测定。结果如图4所示。
从图4可以看出,探针对次氯酸具有很高的选择性,能够专一性地和次氯酸进行反应,反应前后,荧光光谱有明显变化,而其他离子与探针作用后荧光强度并不发生明显变化。而且其他离子不会明显干扰探针对次氯酸的定性与定量检测。
虽然用上述实施方式描述了本发明,应当理解的是,在不背离本发明的精神的前提下,本发明可进行进一步的修饰和变动,且这些修饰和变动均属于本发明的保护范围之内。
Claims (7)
1.用于检测样本中次氯酸含量的化合物,其具有以下结构:
上式中:R1,R2,R3,R4,R5,R6,R7,R8,R9和R10为氢原子,直链或支链烷基,直链或支链烷氧基,磺酸基,酯基,羧基;R1,R2,R3,R4,R5,R6,R7,R8,R9和R10可以相同或不同。
2.根据权利要求1所述的化合物,其为如下结构的化合物:
3.根据权利要求1所述的化合物,其中所述的样本是游泳池水样本。
4.根据权利要求1所述的化合物,其中所述的样本是生物样本。
5.根据权利要求1所述的化合物,其中所述检测是定性检测或定量检测。
6.根据权利要求1所述的化合物,其中所述检测是在缓冲体系下进行的。
7.根据权利要求1所述的化合物,其中所述缓冲体系的pH为7.4。
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