CN104597233B - 用于增强化学发光的增强液及制备化学发光液的方法 - Google Patents

用于增强化学发光的增强液及制备化学发光液的方法 Download PDF

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CN104597233B
CN104597233B CN201410617881.1A CN201410617881A CN104597233B CN 104597233 B CN104597233 B CN 104597233B CN 201410617881 A CN201410617881 A CN 201410617881A CN 104597233 B CN104597233 B CN 104597233B
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任琪
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Abstract

本发明提供一种用于增强化学发光的增强液及制备化学发光液的方法,包括以下几个步骤:步骤A:将葡萄糖甲胺溶解于水中调节pH至8至9,再将权利要求1所述的N‑烷基二甲基甘露糖胺季铵盐,吖啶橙,BSA,甘氨酸,MgCl2,ZnCl2,分别加入至二乙醇胺溶液中,搅拌溶解,定容,得到增强液;步骤B:用增强液将化学发光底物稀释得到化学发光液。本发明采用以上技术方案,其优点在于,该增强液用于免疫检测领域,提高了检测灵敏度高和线性范围,降低了成本,且无污染。解决了以往试剂依赖进口,价格昂贵,且不能达到最佳发光性能等问题。

Description

用于增强化学发光的增强液及制备化学发光液的方法
技术领域
本发明涉及一种用于增强化学发光的增强液及制备化学发光液的方法。
背景技术
化学发光作为国外近年快速发展且广泛使用的免疫分析技术,它具有高灵敏度,检测线性范围宽和无污染等特点。目前化学发光免疫分析中主要有鲁米诺,异鲁米诺,吖啶和环二氧乙烷衍生物(如CSPD,CDP-SATR,AMPPD等)等系统。吖啶和异鲁米诺直接标记示踪属于直接化学发光。鲁米诺和环二氧乙烷衍生物(如CSPD,CDP-SATR,AMPPD等)属于酶促类化学发光。CSPD,CDP-SATR,AMPPD及其增强液多为进口产品价格昂贵。
美国专利5145772公开了聚乙烯苄基二甲基苄基氯化铵形成胶束对环二氧乙烷化合物的发光增强效果。美国专利US5547836公开了聚乙烯苄基三甲基氯化铵可对环二氧乙烷化合物发光增强。中国专利CN1719254A公开了一CSPD为底物,十六烷基三甲基氯化铵,BSA,十八烷基荧光素,肉蔻酰甘油磷酸二钠为增强液的发光液,但其成本高,制备复杂,不利于广泛应用
发明内容
本发明提出一种的用于新型环二氧乙烷化合物化学发光底物A的增强液,包含至少一种化学发光法底物:N-烷基二甲基甘露糖胺季铵盐,R为8-40烷基或8-40烯基,Y为负离子,其结构式如下:
本发明采用以上技术方案,其优点在于,该增强液用于免疫检测领域,提高了检测灵敏度高和线性范围,降低了成本低,且无污染。解决了以往试剂依赖进口,价格昂贵,且不能达到最佳发光性能等问题。
优选的,所述N-烷基二甲基甘露糖胺季铵盐采用N-十二烷基二甲基甘露糖胺氯化铵,N-十四烷基二甲基甘露糖胺氯化铵,N-十六烷基二甲基甘露糖胺氯化铵,N-十八烷基二甲基甘露糖胺氯化铵,N-十二烷基二甲基甘露糖胺溴化铵,N-十四烷基二甲基甘露糖胺溴化铵,N-十六烷基二甲基甘露糖胺溴化铵和N-十八烷基二甲基甘露糖胺溴化铵中的至少一种。
优选的,还包括:1-50mg/L吖啶橙、1-100g/L BSA、1-10g/L甘氨酸、0.1-0.5mol/L葡萄糖甲胺、0.1-10mmol/L氯化镁或者乙酸镁、0.1-10mmol/L氯化锌。
优选的,葡萄糖甲胺pH=8.5。
本发明还提供一种采用该增强液制备化学发光液的方法,包括以下几个步骤:
步骤A:将葡萄糖甲胺溶解于水中调节pH至8至9,再将N-烷基二甲基甘露糖胺季铵盐、吖啶橙、BSA、甘氨酸、MgCl2、ZnCl2分别加入至二乙醇胺溶液中,搅拌溶解,定容,得至增强液;
步骤B:用增强液将化学发光底物稀释得到化学发光液。
优选的,化学发光底物采用环二氧乙烷化合物化学发光底物。
优选的,化学发光底物采用9-[3-(5-氯-2-螺旋金刚烷)-1,2二氧环乙烷]-3-氯-7-磷氧酰基-10-丙酸基-9,10二氢化吖啶二钠盐,其结构式为:
本发明的有益效果是:
1:与市售增强液相比提高了新型环二氧乙烷化合物化学发光法底物的发光液的稳定性
2:与市售增强液相比提高了新型环二氧乙烷化合物化学发光法底物的发光液的性能
3:相比市售CDP-STAR+Emerald-IITM发光液发光性信号著提高。
4:相比市售CDP-STAR+Emerald-IITM发光液检测时间明显缩短。
5:相对进口化学发光液和增强剂更为经济,新型化学发光底物化合物产品灵敏度高,稳定性好,价格低。
附图说明
图1是实施例1中,将10ul 2*10-16mol的碱性磷酸酶作为样品加入微孔板中,然 后加入100ul化学发光液1,在化学发光仪(BIOTEK-Synergy2)上记录发光信号,检测其发光动力学曲线。
图2是实施例1中,将10ul 2*10-16mol的碱性磷酸酶作为样品加入微孔板中,然后加入100ul化学发光液2,在化学发光仪(BIOTEK-Synergy2)上记录发光信号,检测其发光动力学曲线。
图3是实施例1中,将10ul 2*10-16mol的碱性磷酸酶作为样品加入微孔板中,然后加入100ul化学发光液3,在化学发光仪(BIOTEK-Synergy2)上记录发光信号,检测其发光动力学曲线。
图4是实施例1中,将10ul 2*10-16mol的碱性磷酸酶作为样品加入微孔板中,然后加入100ul化学发光液4,在化学发光仪(BIOTEK-Synergy2)上记录发光信号,检测其发光动力学曲线。
图5是实施例2中,将10ul 2*10-16mol的碱性磷酸酶作为样品加入微孔板中,然后分别加入100ul化学发光液1-A,化学发光液1-B,化学发光液5-A和化学发光液5-B,在化学发光仪(BIOTEK-Synergy2)上记录发光信号,检测其发光动力学曲线。
图6是实施例2中,将10ul 2*10-16mol的碱性磷酸酶作为样品加入微孔板中,然后分别加入100ul化学发光液2-A,化学发光液2-B在化学发光仪(BIOTEK-Synergy2)上记录发光信号,检测其发光动力学曲线。
图7是实施例2中,将10ul 2*10-16mol的碱性磷酸酶作为样品加入微孔板中,然后分别加入100ul化学发光液1,化学发光液6,在化学发光仪(BIOTEK-Synergy2)上记录发光信号,检测其发光动力学曲线。
具体实施方式
下面结合附图,对本发明的较优的实施例作进一步的详细说明:
实施例1:
步骤1:将葡萄糖甲胺按19.5g浓度溶解于900mL水中调节pH至8.5,N-十二烷基二甲基甘露糖胺氯化铵1g,吖啶橙6mg,BSA 10g,甘氨酸30mg,MgCl21mmol,ZnCl20.2mmol,分别加入至二乙醇胺溶液中,搅拌溶解,定容至1L,得增强液(1);
步骤2:将增强液(1)将9-[3-(5-氯-2-螺旋金刚烷)-1,2二氧环乙烷]-3-氯-7-磷氧酰基-10-丙酸基-9,10二氢化吖啶二钠盐稀释至450mg/L得到化学发光液1。
步骤3:将CDP-STAR增强液Emerald-IITM(Tropix)稀释9-[3-(5-氯-2-螺旋金刚烷)-1,2二氧环乙烷]-3-氯-7-磷氧酰基-10-丙酸基-9,10二氢化吖啶二钠盐稀释至450mg/L得到化学发光液2
步骤4:用增强液(1)将CDP-STAR稀释至400mg/L得到化学发光液3
使用CDP-STAR增强液Emerald-IITM(Tropix)将CDP-STAR稀释至400mg/L得到化学发光液4
测试步骤:
1.将10ul 2*10-16mol的碱性磷酸酶作为样品加入微孔板中,然后加入100ul化学发光液1,在化学发光仪(BIOTEK-Synergy2)上记录发光信号,检测其发光动力学曲线,如图1所示;
2.将10ul 2*10-16mol的碱性磷酸酶作为样品加入微孔板中,然后加入100ul化学发光液2,在化学发光仪(BIOTEK-Synergy2)上记录发光信号,检测其发光动力学曲线,如图2所示;
3.将10ul 2*10-16mol的碱性磷酸酶作为样品加入微孔板中,然后加入100ul化学发光液3,在化学发光仪(BIOTEK-Synergy2)上记录发光信号,检测其发光动力学曲线,如图3所示;
4.将10ul 2*10-16mol的碱性磷酸酶作为样品加入微孔板中,然后加入100ul化学发光液4,在化学发光仪(BIOTEK-Synergy2)上记录发光信号,检测其发光动力学曲线,如图4所示;
由图1和图2可知,增强液和Emerald-IITM(Tropix)用于新型环二氧乙烷化合物化学发光法底物A时Emerald-IITM所配置发光液2发光效率明显低于增强液所配置发光液1
由图3和图4可知,我们发现增强液和Emerald-IITM(Tropix)用于环二氧乙烷化合物CDP-STAR时Emerald-IITM所配置发光液4发光效率明显高于增强液所配置发光液3
实施例2
步骤1:将葡萄糖甲胺按19.5g浓度溶解于900mL水中调节PH至8.5,N-十二烷基二甲基甘露糖胺氯化铵1g,吖啶橙6mg,BSA 10g,MgCl21mmol,ZnCl20.2mmol,分别加入至二乙醇胺溶液中,搅拌溶解,定容至1L增强液(2)
步骤2:使用增强液(2)将9-[3-(5-氯-2-螺旋金刚烷)-1,2二氧环乙烷]-3-氯-7-磷氧酰基-10-丙酸基-9,10二氢化吖啶二钠盐(A)稀释至450mg/L得到化学发光液5。
步骤3:将化学发光液1分成两份:化学发光液1-A和发光液1-B,将化学发光液1-B放置于370C,发光液1-A室温保存6天
步骤4:将化学发光液2分成两份:化学发光液2-A和发光液2-B,将化学发光液2-B放置于370C,发光液2-A室温保存6天
步骤5:将化学发光液5分成两份:化学发光液5-A和化学发光液5-B,将发光液5-B放置于370C,发光液5-A室温保存6天
测试方法:
步骤A:将10ul 2*10-16mol的碱性磷酸酶作为样品加入微孔板中,然后分别加入100ul化学发光液1-A,化学发光液1-B,化学发光液5-A和化学发光液5-B,在化学发光仪(BIOTEK-Synergy2)上记录发光信号,检测其发光动力学曲线。如图5
将10ul 2*10-16mol的碱性磷酸酶作为样品加入微孔板中,然后分别加入100ul化学发光液2-A,化学发光液2-B在化学发光仪(BIOTEK-Synergy2)上记录发光信号,检测其发光动力学曲线。如图6
由图5可知,发光液1室温和37℃分别存放6天后发光信号基本不变,而化学发光液5室温和37℃分别存放6天后37℃存放条件下的化学发光液5-B发光信号明显衰减。我们发现甘氨酸能够协同提高发光液的稳定性。
由图6可知,发光增强液Emerald-IITM(Tropix)所配发光液2在37℃存放6天后发光信号较室温存放下明显下降。
步骤3:将葡萄糖甲胺按19.5g浓度溶解于900mL水中调节PH至8.5,N-十二烷基二甲基甘露糖胺氯化铵1g,吖啶橙6mg,BSA 10g,甘氨酸30mg,MgCl21mmol,分别加入至二乙醇胺溶液中,搅拌溶解,定容至1L,得增强液(3)
使用增强液(3)将9-[3-(5-氯-2-螺旋金刚烷)-1,2二氧环乙烷]-3-氯-7-磷氧酰基-10-丙酸基-9,10二氢化吖啶二钠盐(A)稀释至450mg/L得到化学发光液6
将10ul 2*10-16mol的碱性磷酸酶作为样品加入微孔板中,然后分别加入100ul化学发光液1,化学发光液6,在化学发光仪(BIOTEK-Synergy2)上记录发光信号,检测其发光动力学曲线。
如图7所示化学发光液1和化学发光液6相比前者发光信号明显高于后者,本发明发现ZnCl2有着提高发光信号的作用。
以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明,不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干简单推演或替换,都应当视为属于本发明的保护范围。

Claims (6)

1.一种用于增强化学发光的增强液,其特征在于,包含至少一种N-烷基二甲基甘露糖胺季铵盐,R为8-40烷基或8-40烯基,Y为负离子,其结构式如下:
还包括:1-50mg/L吖啶橙、1-100g/L BSA、1-10g/L甘氨酸、0.1-0.5mol/L葡萄糖甲胺、0.1-10mmol/L氯化镁或者乙酸镁、0.1-10mmol/L氯化锌。
2.如权利要求1所述的增强液,其特征在于,所述N-烷基二甲基甘露糖胺季铵盐采用N-十二烷基二甲基甘露糖胺氯化铵,N-十四烷基二甲基甘露糖胺氯化铵,N-十六烷基二甲基甘露糖胺氯化铵,N-十八烷基二甲基甘露糖胺氯化铵,N-十二烷基二甲基甘露糖胺溴化铵,N-十四烷基二甲基甘露糖胺溴化铵,N-十六烷基二甲基甘露糖胺溴化铵和N-十八烷基二甲基甘露糖胺溴化铵中的至少一种。
3.如权利要求2所述的增强液,其特征在于,葡萄糖甲胺pH=8.5。
4.一种采用如权利要求2所述的增强液制备化学发光液的方法,其特征在于,包括以下几个步骤:
步骤A:将葡萄糖甲胺溶解于水中调节pH至8-9,再将N-烷基二甲基甘露糖胺季铵盐,吖啶橙,BSA,甘氨酸,MgCl2,ZnCl2,分别加入至二乙醇胺溶液中,搅拌溶解,定容,得到增强液;
步骤B:用增强液将化学发光底物稀释得到化学发光液。
5.如权利要求4所述的方法,其特征在于,所述步骤B中,化学发光底物采用环二氧乙烷化合物化学发光底物。
6.如权利要求4所述的方法,其特征在于,所述步骤B中,化学发光底物采用9-[3-(5-氯-2-螺旋金刚烷)-1,2二氧环乙烷]-3-氯-7-磷氧酰基-10-丙酸基-9,10二氢化吖啶二钠盐,其结构式为:
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