CN104017570A - 识别微丝菌用荧光探针的制备方法 - Google Patents
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Abstract
本发明提供一种识别微丝菌用荧光探针的制备方法,该方法先采用卤代长碳链化合物对咔唑吡啶苯乙烯类菁染料的4-甲基吡啶部分进行修饰,而后再与3-甲酰基-N-乙基咔唑反应合成具有长疏水链的荧光探针。本发明效果为,该探针制备过程中将咔唑吡啶苯乙烯类菁染料的制备和修饰同步化,制备方法简单,制得的荧光探针背景干扰小,荧光强度高,荧光性质稳定。利用微丝菌具有疏水表面这一特性,本申请中所制备的具有长疏水链的荧光探针可与其结合,从而达到荧光识别微丝菌的目的。
Description
技术领域
本发明属于用于污水处理领域中微生物检测用荧光探针的制备方法,具体涉及一种卤代长碳链化合物修饰咔唑吡啶苯乙烯类菁染料得到识别微丝菌用荧光探针的制备方法。
背景技术
自活性污泥法诞生一个多世纪以来,污泥膨胀问题就一直是困扰污水处理厂正常运行的一个世界性难题。活性污泥膨胀由丝状菌过度生长引起的丝状膨胀和非丝状膨胀,其中前者占主导,据报道约有90%的污泥膨胀问题是由活性污泥中丝状菌的大量生长而引起的。人们对丝状膨胀及其关键菌群的研究集中在2个方面:一是引发丝状膨胀的关键菌群本身的研究,包括其分离鉴定、生理生态特性、以及污泥体系中相关丝状菌群的识别与定量表征等等;其二是从工艺操作角度探讨工艺操作条件对丝状菌群的影响,寻求控制污泥膨胀的对策。
微丝菌是污泥起泡和膨胀过程中最常见的一种丝状菌,特别是涉及营养物去除的污水处理系统中,因而对这类丝状菌进行鉴别研究,对污泥膨胀现象的预警和控制具有重要意义。传统鉴别方法主要是根据微丝菌的形态学特征和染色反应状态来进行。但由于微丝菌较难分离和纯培养,人们对其代谢和生理生态特性了解较少。近年来,随着PCR、核酸测序等相关分子生物学技术的迅速发展,采用荧光原位杂交(fluorescence in situhybridization,FISH)技术对微丝菌进行鉴别,避免了传统的方法进行鉴定和计数的局限性,且测定过程中细胞形态不被破坏,能够真实反映自然环境下微生物的情况及分布等特点。但是,FISH只能对已知核酸序列的微生物进行标识,且FISH方法存在细胞穿透率较低、低rRNA含量以及存在微生物自身荧光的影响和荧光褪色等缺点。
J.L.Nielsen等人采用荧光微球法对微丝菌进行检测,发现微丝菌表面具有一定的疏水性。利用它的这一特性,本申请中采用长碳链化合物对荧光染料进行修饰,构建一种具有长疏水链的荧光探针,使其与微丝菌结合,从而达到荧光识别微丝菌的目的。
发明内容
本发明目的是提供一种识别微丝菌用荧光探针的制备方法,利用卤代长碳链化合物对荧光染料进行修饰,使制得的荧光探针具有长疏水链,能与具有疏水表面的微丝菌结合,从而达到识别微丝菌的目的。
为实现上述目的,本发明采用的技术方案是提供一种识别微丝菌用荧光探针的制备方法,该方法是采取将咔唑吡啶苯乙烯类菁染料的合成和修饰同步化的合成路线,先引入卤代长碳链化合物对其4-甲基吡啶部分进行修饰,而后再与3-甲酰基-N-乙基咔唑部分反应,制得具有长疏水链的荧光探针,包括以下步骤:
(1)长链烷基-4-甲基吡啶卤化盐的合成
在圆底烧瓶中分别加入0.8-1.5份4-甲基吡啶,0.7-1.3份卤代长碳链化合物及28份甲苯,磁力搅拌充分溶解进行反应,反应温度为120℃,待反应完成后,冷却至室温,去除上层甲苯得到油状物,并用正己烷洗涤,最后将洗涤完的油状物在旋转蒸发仪中进行减压蒸馏,得到长链烷基-4-甲基吡啶卤化盐;
(2)3-甲酰基-N-乙基咔唑的合成
在圆底烧瓶中加入三氯氧磷20份,在冰水浴冷却搅拌条件下,向圆底烧瓶中依次滴加DMF19-22份,65份含3-3.5份N-乙基咔唑的1,2-二氯乙烷溶液,滴毕,回流反应8h,反应液冷却至室温,并倒入冰水中,搅拌,用二氯甲烷萃取,在旋转蒸发仪中进行减压蒸馏后,经柱层析分离得3-甲酰基-N-乙基咔唑;
(3)荧光探针的合成
将步骤(1)中制备的长链烷基-4-甲基吡啶卤化盐溶于70份无水乙醇中,并加入0.9-1.3份步骤(2)中制备的3-甲酰基-N-乙基咔唑,滴加3-5滴哌啶,回流反应12h,得到红色溶液,反应液浓缩后,加入乙醚析出沉淀,抽滤,制得滤饼,将滤饼在甲醇中重新结晶,得具有长疏水链的荧光探针。
本发明的效果为:
(1)所制备的荧光探针,是在咔唑吡啶苯乙烯类菁染料的合成路线中,先引入卤代长碳链化合物对其4-甲基吡啶部分进行修饰,而后再与3-甲酰基-N-乙基咔唑部分反应,从而制备得到卤代长碳链修饰的荧光探针,这一制备方法实现了咔唑吡啶苯乙烯类菁染料的合成和修饰的同步化,且该制备方法简单,制得的荧光探针背景干扰小,荧光强度高,荧光性质稳定。
(2)卤代长碳链化合物的修饰使得制备的荧光探针具有长疏水链,能与具有疏水表面的微丝菌结合,从而达到荧光识别微丝菌的目的。
附图说明
图1为本发明荧光探针与微丝菌荧光下作用关系的镜检图;
图2为本发明荧光探针与微丝菌自然光下作用关系的镜检图。
具体实施方式
结合实例对本发明的识别微丝菌用荧光探针的制备方法加以说明。
本发明的识别微丝菌用荧光探针的制备方法,实现了荧光染料的制备和修饰同步化,是在咔唑吡啶苯乙烯类菁染料的合成路线中,先引入卤代长碳链化合物对其4-甲基吡啶部分进行修饰,而后再与3-甲酰基-N-乙基咔唑部分反应,制得具有长疏水链的荧光探针,这一过程主要包括以下步骤:
(1)长链烷基-4-甲基吡啶卤化盐的合成
在圆底烧瓶中分别加入0.8-1.5份4-甲基吡啶,0.7-1.3份卤代长碳链化合物及28份甲苯,磁力搅拌充分溶解进行反应,反应温度为120℃,待反应完成后,冷却至室温,去除上层甲苯得到油状物,并用正己烷洗涤,最后将洗涤完的油状物在旋转蒸发仪中进行减压蒸馏,得到长链烷基-4-甲基吡啶卤化盐;
(2)3-甲酰基-N-乙基咔唑的合成
在圆底烧瓶中加入三氯氧磷20份,在冰水浴冷却搅拌条件下,向圆底烧瓶中依次滴加DMF19-22份,65份含3-3.5份N-乙基咔唑的1,2-二氯乙烷溶液,滴毕,回流反应8h,反应液冷却至室温,并倒入冰水中,搅拌,用二氯甲烷萃取,在旋转蒸发仪中进行减压蒸馏后,经柱层析分离得3-甲酰基-N-乙基咔唑;
(3)荧光探针的合成
将步骤(1)中制备的长链烷基-4-甲基吡啶卤化盐溶于70份无水乙醇中,并加入0.9-1.3份步骤(2)中制备的3-甲酰基-N-乙基咔唑,滴加3-5滴哌啶,回流反应12h,得到红色溶液,反应液浓缩后,加入乙醚析出沉淀,抽滤,制得滤饼,将滤饼在甲醇中重新结晶,得具有长疏水链的荧光探针。
所制得的具有长疏水链的荧光探针与微丝菌的作用过程如下:取制得的荧光探针配置成探针溶液,而后用含微丝菌的细菌溶液对探针溶液进行稀释,将稀释后的探针溶液滴加至载玻片上,采用荧光倒置显微镜分别在荧光和自然光条件下进行对比观察,分别如图1和图2所示。对比可以看出,图1中微丝菌菌丝的亮度明显强于图2,说明本申请所制得的具有长疏水链的荧光探针可很好地识别微丝菌。
所述卤代长碳链化合物为1-溴十二烷,1-溴十四烷,1-溴十六烷,1-溴十八烷,1-碘十二烷,1-碘十六烷或1-碘十八烷。
实施例:
实施例1
在圆底烧瓶中分别加入0.4650g4-甲基吡啶,1.5268g1-溴十六烷及15mL甲苯,磁力搅拌充分溶解,120℃回流,反应完成后,冷却至室温,去除上层甲苯,用正己烷洗涤剩余黄色油状物三次,将洗涤后的产物在旋转蒸发仪中减压蒸馏去除溶剂得到1-十六烷基-4-甲基吡啶溴化盐,产率91.5%。在圆底烧瓶中加入9.5mL三氯氧磷,冰水浴冷却搅拌下,滴加7.7mL DMF,滴毕,于室温搅拌30min,缓慢滴加16ml含有3.15g N-乙基咔唑的1,2-二氯乙烷溶液,滴毕,回流反应8h。反应完成后冷却至室温后,将反应液倒入冰水中搅拌1h,用二氯甲烷萃取冰水溶液,再用P2O5干燥含有产物的二氯甲烷溶液,之后减压蒸馏去除二氯甲烷,产物经柱层析分离得3-甲酰基-N-乙基咔唑。在得到的1-十六烷基-4-甲基吡啶溴化盐的反应瓶中,加入1.1155g3-甲酰基-N-乙基咔唑,无水乙醇20.00mL,滴加3滴哌啶,回流反应12h得红色溶液,反应液浓缩后,加乙醚析出固体,抽滤,滤饼于甲醇中重结晶,得到所需的荧光探针,产率49.6%。对荧光探针进行光谱检测,测得荧光探针最大激发波长为435nm,发射波长为574nm。
称取制备的1-溴十六烷修饰的荧光探针30.1mg,将其溶解于50mL乙醇中,配制成浓度为1mmol/L的探针溶液,采用细菌溶液对其进行稀释从而配置成浓度为0.010mmol/L的探针溶液,采用荧光倒置显微镜分别在自然光和荧光条件下进行对比观察,可以明显看出,在荧光条件下进行检测,可观察到光亮的微丝菌菌丝,且明显区别于菌胶团,如图1所示。而在自然光条件下进行检测,微丝菌菌丝不显示亮度且不易区别于菌胶团,如图2所示。对比说明,本申请制备的荧光探针可与微丝菌结合,从而达到识别微丝菌的目的。
实施例2
在圆底烧瓶中分别加入0.6985g4-甲基吡啶,1.8023g1-溴十四烷及15mL甲苯,磁力搅拌充分溶解,120℃回流,反应完成后,冷却至室温,去除上层甲苯,用正己烷洗涤剩余黄色油状物三次,将洗涤后的产物在旋转蒸发仪中减压蒸馏去除溶剂得到1-十四烷基-4-甲基吡啶溴化盐,产率92.7%。在圆底烧瓶中加入9.5mL三氯氧磷,冰水浴冷却搅拌下,滴加8.9mLDMF,滴毕,于室温搅拌30min,缓慢滴加25ml含有3.445g N-乙基咔唑的1,2-二氯乙烷溶液,滴毕,回流反应8h。反应完成后冷却至室温后,将反应液倒入冰水中搅拌1h,用二氯甲烷萃取冰水溶液,再用P2O5干燥含有产物的二氯甲烷溶液,之后减压蒸馏去除二氯甲烷,产物经柱层析分离得3-甲酰基-N-乙基咔唑。在得到的1-十四烷基-4-甲基吡啶溴化盐的反应瓶中,加入1.45g3-甲酰基-N-乙基咔唑,无水乙醇20.00mL,滴加5滴哌啶,回流反应12h得红色溶液,反应液浓缩后,加乙醚析出沉淀,抽滤,滤饼于甲醇中重结晶,得到所需的荧光探针,产率31%。对荧光探针进行光谱检测,测得荧光探针最大激发波长为438nm,发射波长为572nm。如实施例1中一样,将所制得的荧光探针配置成浓度为0.015mmol/L的探针溶液,采用倒置显微镜进行观察,在荧光条件下可观察到明亮的微丝菌菌丝,所制备探针可识别微丝菌。
实施例3
在圆底烧瓶中分别加入0.3725g4-甲基吡啶,0.8723g1-溴十二烷及15mL甲苯,磁力搅拌充分溶解,120℃回流,反应完成后,冷却至室温,去除上层甲苯,用正己烷洗涤剩余黄色油状物三次,将洗涤后的产物在旋转蒸发仪中减压蒸馏去除溶剂得到1-十二烷基-4-甲基吡啶溴化盐,产率94%。在圆底烧瓶中加入9.5mL三氯氧磷,冰水浴冷却搅拌下,滴加7.32mL DMF,滴毕,于室温搅拌30min,缓慢滴加25mL含有2.953g N-乙基咔唑的1,2-二氯乙烷溶液,滴毕,回流反应8h。反应完成后冷却至室温后,将反应液倒入冰水中搅拌1h,用二氯甲烷萃取冰水溶液,再用P2O5干燥含有产物的二氯甲烷溶液,之后减压蒸馏去除二氯甲烷,产物经柱层析分离得3-甲酰基-N-乙基咔唑。在得到的1-十六烷基-4-甲基吡啶溴化盐的反应瓶中,加入1.004g3-甲酰基-N-乙基咔唑,无水乙醇20.00mL,滴加3滴哌啶,回流反应12h得红色溶液,反应液浓缩后,加乙醚析出沉淀,抽滤,滤饼于甲醇中重结晶,得到所需的荧光探针,产率30.7%。对荧光探针进行光谱检测,测得荧光探针最大激发波长为440nm,发射波长为566nm。如实施例1中一样,将所制得的荧光探针配置成浓度为0.020mmol/L的探针溶液,采用倒置显微镜进行观察,在荧光条件下可观察到明亮的微丝菌菌丝,所制备探针可识别微丝菌。
实施例4
将上述实施例1中的0.5268g1-溴十六烷换成1.667g1-溴十八烷,得到1-十八烷基-4-甲基吡啶溴化盐,产率89.9%。其它操作均按实施例3中步骤,不作调整,最终得到十八烷基链修饰的咔唑吡啶苯乙烯类菁染料荧光探针。对荧光探针进行光谱检测,测得荧光探针最大激发波长为435nm,发射波长为577nm。如实施例1中一样,将所制得的荧光探针配置成浓度为0.006mmol/L的探针溶液,采用倒置显微镜进行观察,在荧光条件下可观察到明亮的微丝菌菌丝,所制备探针可识别微丝菌。
实施例5
将上述实施例1中的0.5268g1-溴十六烷换成1.4812g1-碘十二烷,得到1-十二烷基-4-甲基吡啶碘化盐。其它操作均按实施例3中步骤,不作调整,最终得到十二烷基链修饰的咔唑吡啶苯乙烯类菁染料荧光探针。对荧光探针进行光谱检测,测得荧光探针最大激发波长为441nm,发射波长为565nm。如实施例1中一样,将所制得的荧光探针配置成浓度为0.020mmol/L的探针溶液,采用倒置显微镜进行观察,在荧光条件下可观察到明亮的微丝菌菌丝,所制备探针可识别微丝菌。
实施例6
将上述实施例1中的0.5268g1-溴十六烷换成1.7617g1-碘十六烷,得到1-十六烷基-4-甲基吡啶碘化盐。其它操作均按实施例3中步骤,不作调整,最终得到十六烷基链修饰的咔唑吡啶苯乙烯类菁染料荧光探针。对荧光探针进行光谱检测,测得荧光探针最大激发波长为436nm,发射波长为574nm。如实施例1中一样,将所制得的荧光探针配置成浓度为0.010mmol/L的探针溶液,采用倒置显微镜进行观察,在荧光条件下可观察到明亮的微丝菌菌丝,所制备探针可识别微丝菌。
实施例7
将上述实施例1中的0.5268g1-溴十六烷换成1.902g1-碘十八烷,得到1-十八烷基-4-甲基吡啶碘化盐。其它操作均按实施例3中步骤,不作调整,最终得到十八烷基链修饰的咔唑吡啶苯乙烯类菁染料荧光探针。对荧光探针进行光谱检测,测得荧光探针最大激发波长为443nm,发射波长为578nm。如实施例1中一样,将所制得的荧光探针配置成浓度为0.006mmol/L的探针溶液,采用倒置显微镜进行观察,在荧光条件下可观察到明亮的微丝菌菌丝,所制备探针可识别微丝菌。
Claims (2)
1.一种识别微丝菌用荧光探针的制备方法,该方法是采取将咔唑吡啶苯乙烯类菁染料的合成和修饰同步化的合成路线,先引入卤代长碳链化合物对其4-甲基吡啶部分进行修饰,而后再与3-甲酰基-N-乙基咔唑部分反应,制得具有长疏水链的荧光探针,包括以下步骤:
(1)长链烷基-4-甲基吡啶卤化盐的合成
在圆底烧瓶中分别加入0.8-1.5份4-甲基吡啶,0.7-1.3份卤代长碳链化合物及28份甲苯,磁力搅拌充分溶解进行反应,反应温度为120℃,待反应完成后,冷却至室温,去除上层甲苯得到油状物,并用正己烷洗涤,最后将洗涤完的油状物在旋转蒸发仪中进行减压蒸馏,得到长链烷基-4-甲基吡啶卤化盐;
(2)3-甲酰基-N-乙基咔唑的合成
在圆底烧瓶中加入三氯氧磷20份,在冰水浴冷却搅拌条件下,向圆底烧瓶中依次滴加DMF19-22份,65份含3-3.5份N-乙基咔唑的1,2-二氯乙烷溶液,滴毕,回流反应8h,反应液冷却至室温,并倒入冰水中,搅拌,用二氯甲烷萃取,在旋转蒸发仪中进行减压蒸馏后,经柱层析分离得3-甲酰基-N-乙基咔唑;
(3)荧光探针的合成
将步骤(1)中制备的长链烷基-4-甲基吡啶卤化盐溶于70份无水乙醇中,并加入0.9-1.3份步骤(2)中制备的3-甲酰基-N-乙基咔唑,滴加3-5滴哌啶,回流反应12h,得到红色溶液,反应液浓缩后,加入乙醚析出沉淀,抽滤,制得滤饼,将滤饼在甲醇中重新结晶,得具有长疏水链的荧光探针。
2.根据权利要求1所述识别微丝菌用荧光探针的制备方法,其特征是:所述卤代长碳链化合物为1-溴十二烷或1-溴十四烷或1-溴十六烷或1-溴十八烷或1-碘十二烷或1-碘十六烷或1-碘十八烷。
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---|---|---|---|---|
CN104479668A (zh) * | 2014-10-15 | 2015-04-01 | 华中科技大学 | 一种荧光染料探针 |
CN104479668B (zh) * | 2014-10-15 | 2016-08-31 | 华中科技大学 | 一种荧光染料探针 |
CN104845614A (zh) * | 2015-04-30 | 2015-08-19 | 天津城建大学 | 一种dna标记用荧光探针及其合成方法和用途 |
CN104845614B (zh) * | 2015-04-30 | 2016-07-13 | 天津城建大学 | 一种dna标记用荧光探针及其合成方法和用途 |
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WO2015180230A1 (zh) | 2015-12-03 |
CN104017570B (zh) | 2016-01-20 |
AU2014393669B2 (en) | 2017-05-18 |
AU2014393669A1 (en) | 2015-12-10 |
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