CN109490298B - 一种可视化偶联反应试剂盒 - Google Patents
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Abstract
一种可视化偶联反应试剂盒,用于羧基和氨基的共价偶联,其特征在于:所述试剂盒由活化液、反应液、活化剂、稳定剂、指示剂和磁盘组成;其中所述活化液为2‑(N‑吗啡啉)乙磺酸缓冲液;其中所述反应液为磷酸盐缓冲液;其中所述活化剂为1‑(3‑二甲氨基丙基)‑3‑乙基碳二亚胺盐酸盐(EDC);其中所述稳定剂为N‑羟基硫代琥珀酰亚胺(sulfo‑NHS);其中所述指示剂为表面羧基化修饰的Fe3O4颗粒。本发明应用功能化磁性纳米颗粒为羧基活化的可视化指示剂,精准把握添加氨基反应物的时间,有效解决EDC/sulfo‑NHS偶联反应的偶联效率低和重复性差的问题,可广泛应用于科研和生产,具有广阔的市场前景。
Description
技术领域
本发明涉及一种化学反应试剂盒,具体说是涉及一种用于羧基和氨基EDC/sulfo-NHS偶联反应的试剂盒。
背景技术
偶联反应是由两个分子通过化学反应而得到一个分子的过程,不仅可以用于有机合成,在生物、医药和检测分析等领域也得到广泛的应用。其中,羧基(-COOH)和氨基(-NH2)的偶联是一种重要的偶联反应,特别是在生物和医药领域具有广泛的应用。
EDC/sulfo-NHS偶联反应因具有反应速度快、副反应少、操作简单和生物活性保持率高的优点,是羧基和氨基偶联中应用最广的偶联法,特别是用于酶、抗体等蛋白质与其他分子的偶联,其反应式如下:
反应物1(-COOH)+ EDC→ 活性酯中间体
活性酯中间体+ 反应物2(-NH2)→反应物1和反应物2的偶联物
EDC/sulfo-NHS偶联反应的第一步是反应物1羧基的活化,反应物1的羧基在EDC存在的条件下被活化生成非常容易水解的活性酯中间体,sulfo-NHS的存在可以适当延缓该中间体的水解速度;EDC/sulfo-NHS偶联反应的第二步在反应物1羧基活化后加入具有氨基的反应物2,反应物2和活性酯中间体反应,生成酰胺键将反应物1和反应物2共价偶联。可见,EDC/sulfo-NHS偶联反应因活性酯中间体的极不稳定而导致偶联效率低和重复性差,不仅影响反应原料利用率低造成资源浪费,还影响偶联产物的稳定性,阻碍其应用。如何提高EDC/sulfo-NHS偶联反应的效率及其重复性,是长期困扰着相关领域研发和生产人员的问题。
准确掌握EDC/sulfo-NHS偶联反应中反应物羧基的活化过程,精准把握反应物2的添加时间节点,使反应物2和活性酯中间体的反应时间最优化,是提高EDC/sulfo-NHS偶联反应的偶联效率和重复性的关键。为此,需要一种既可以实时反映反应物1的羧基活化程度,又对偶联反应没有干扰的指示剂,用于准确把握EDC/sulfo-NHS偶联反应的时间节点。
本发明设计一种功能化磁性纳米颗粒作为指示剂,用于反映反应物1的羧基EDC/sulfo-NHS活化程度,通过指示剂在反应物1羧基活化过程中产生的可视变化,准确掌握EDC/sulfo-NHS偶联反应中反应物羧基的活化过程,精准把握反应物2的添加时间,有效解决EDC/sulfo-NHS偶联反应遇到的偶联效率低和重复性差的问题。
发明内容
本发明的目的正是基于功能化磁性纳米颗粒设计一种用于羧基和氨基EDC/sulfo-NHS偶联反应的试剂盒。
本发明的目的可通过下述技术措施来实现:
本发明是一种用于羧基和氨基EDC/sulfo-NHS偶联反应的试剂盒,由活化液、反应液、活化剂、稳定剂、指示剂和磁盘组成;其中所述活化液为pH 4.0-6.0的MES缓冲液;其中所述反应液为pH 7.0-9.0的磷酸盐缓冲液;其中所述活化剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC);其中所述稳定剂为N-羟基硫代琥珀酰亚胺(sulfo-NHS);其中所述指示剂为直径在100-500 nm之间的表面羧基化修饰的四氧化三铁功能化磁性纳米颗粒。
本发明的用于EDC/sulfo-NHS偶联反应的试剂盒的使用方法如下:
a. 将含氨基的反应物2加入反应液中,搅拌均匀后备用;
b. 将含羧基的反应物1和指示剂加入活化液中,充分搅拌使指示剂处于稳定的悬浮状态后,再依次加入稳定剂和活化剂,并充分搅拌均匀,然后在室温条件下静置;
c. 当活化液出现指示剂聚沉现象时,把反应液置于磁盘上让指示剂和活化液彻底分离,然后把活化液转移到在a步骤中准备好的反应液中,在室温条件下反应2小时,即可完成反应物1羧基和反应物2氨基的共价偶联,生成反应物1和反应物2的偶联物。
本发明具有以下有益效果:
本发明用于羧基和氨基EDC/sulfo-NHS偶联反应,应用功能化磁性纳米颗粒为指示剂,通过指示剂在反应物1羧基EDC/sulfo-NHS活化过程中的可视变化,准确掌握反应物1羧基的活化过程,精准把握反应物2的添加时间节点,有效解决EDC/sulfo-NHS偶联反应遇到的偶联效率低和重复性差的问题,不但可以节省偶联原料,还能改善和拓宽该偶联反应在各个领域中的应用。由于本试剂盒简单高效、操作方便及成本较低,可广泛应用于科研实验室及生产车间,因此本发明具有宽阔的市场前景。
具体实施方式
本发明以下将结合实施例作进一步描述,但并不限制本发明。
实施例1
一种由活化液、反应液、活化剂、稳定剂、指示剂和磁盘组成的用于EDC/sulfo-NHS偶联反应试剂盒,其特征如下:
(1) 活化液为1.0 mL 0.01 mol/L的MES缓冲液(pH 5.0);
(2) 反应液为2.0 mL 0.01 mol/L磷酸盐缓冲液(pH 7.0);
(3) 活化剂为50克1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC);
(4) 稳定剂为50克N-羟基硫代琥珀酰亚胺(sulfo-NHS);
(5) 指示剂为1 mL 50 mg/mL的直径200 nm的表面羧基化修饰的四氧化三铁颗粒。
本EDC/sulfo-NHS偶联反应试剂盒的使用方法如下:
a. 将含氨基的反应物2加入反应液中,搅拌均匀后备用;
b. 将含羧基的反应物1和指示剂加入活化液中,充分搅拌使指示剂处于稳定的悬浮状态后,再依次加入稳定剂和活化剂,并充分搅拌均匀,然后在室温条件下静置;
c. 当活化液出现指示剂聚沉现象时,把反应液置于磁盘上让指示剂和活化液彻底分离,然后把活化液转移到在a步骤中准备好的反应液中,在室温条件下反应2小时,即可完成反应物1羧基和反应物2氨基的共价偶联,生成反应物1和反应物2的偶联物。
实施例2
一种由活化液、反应液、活化剂、稳定剂、指示剂和磁盘组成的EDC/sulfo-NHS偶联反应试剂盒,其特征如下:
(1) 活化液为10 mL 0.1 mol/L的MES缓冲液(pH 6.0);
(2) 反应液为20 mL 0.1 mol/L磷酸盐缓冲液(pH 8.0);
(3) 活化剂为300克1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC);
(4) 稳定剂为400克N-羟基硫代琥珀酰亚胺(sulfo-NHS);
(5) 指示剂为5 mL 50 mg/mL的直径400 nm的表面羧基化修饰的四氧化三铁颗粒。
本EDC/sulfo-NHS偶联反应试剂盒的使用方法如下:
a. 将含氨基的反应物2加入反应液中,搅拌均匀后备用;
b. 将含羧基的反应物1和指示剂加入活化液中,充分搅拌使指示剂处于稳定的悬浮状态后,再依次加入稳定剂和活化剂,并充分搅拌均匀,然后在室温条件下静置;
c. 当活化液出现指示剂聚沉现象时,把反应液置于磁盘上让指示剂和活化液彻底分离,然后把活化液转移到在a步骤中准备好的反应液中,在室温条件下反应2小时,即可完成反应物1羧基和反应物2氨基的共价偶联,生成反应物1和反应物2的偶联物。
Claims (1)
1.一种可视化偶联反应试剂盒,可实现偶联反应物1的羧基和偶联反应物2的氨基的EDC/sulfo-NHS共价偶联,其特征在于:所述偶联反应试剂盒由活化液、反应液、活化剂、稳定剂、指示剂和磁盘组成;其中所述活化液为pH 4.0-6.0的MES缓冲液;其中所述反应液为pH 7.0-9.0的磷酸盐缓冲液;其中所述活化剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC);其中所述稳定剂为N-羟基硫代琥珀酰亚胺(sulfo-NHS);其中所述指示剂为直径在100-500nm之间的表面羧基化修饰的四氧化三铁功能化磁性纳米颗粒;所述可视化偶联反应试剂盒的使用方法如下:
a.将含氨基的反应物2加入反应液中,搅拌均匀后备用;
b.将含羧基的反应物1和指示剂加入活化液中,充分搅拌使指示剂处于稳定的悬浮状态后,再依次加入稳定剂和活化剂,并充分搅拌均匀,然后在室温条件下静置;
c.当活化液出现指示剂聚沉现象时,把反应液置于磁盘上让指示剂和活化液彻底分离,然后把活化液转移到在a步骤中准备好的反应液中,在室温条件下反应2小时,即可完成反应物1羧基和反应物2氨基的共价偶联,生成反应物1和反应物2的偶联物。
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