CN105784699A - 一种稳定性更好、灵敏度更高的钾离子试剂及检测方法 - Google Patents
一种稳定性更好、灵敏度更高的钾离子试剂及检测方法 Download PDFInfo
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- NPYPAHLBTDXSSS-UHFFFAOYSA-N Potassium ion Chemical compound [K+] NPYPAHLBTDXSSS-UHFFFAOYSA-N 0.000 title claims abstract description 32
- 229910001414 potassium ion Inorganic materials 0.000 title claims abstract description 32
- 238000001514 detection method Methods 0.000 title claims abstract description 27
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 5
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Abstract
本发明涉及血清钾离子检测技术领域，特别涉及一种钾离子检测试剂，试剂R1中含有咪唑缓冲液，MIT,BSA,蔗糖，海藻糖，GLDH，LDH，PK，聚乙二醇，MgCl2，十二烷基三甲基氯化铵，月桂基咪唑啉甜菜碱，防腐剂；试剂R2中含有Tris缓冲液，PEP·BSA，蔗糖·海藻糖，EDTA，NADH，防腐剂。采用咪唑缓冲液，添加稳定剂BSA,蔗糖，海藻糖，聚乙二醇，大大增强了试剂的稳定性；采用新型表面活性剂十二烷基三甲基氯化铵，月桂基咪唑啉甜菜碱，不仅显著改善测定的性能，而且增强了试剂的稳定性和分析灵敏度。
Description
技术领域
本发明涉及钾离子检测技术领域，特别涉及一种稳定性更好，灵敏度更高的钾离子检测试剂，还涉及使用此检测试剂的检测方法。
背景技术
人体内的钾主要来源于食物，食物中含有的钾90%以上短时间内就在肠道内被吸收，进入血液的钾90%在4h内经由肾脏排出体外。钾离子大部分存在于细胞内，是维持细胞正常生理活动的主要阳离子，在保持机体的正常渗透压和内环境酸碱平衡、参与机体糖及蛋白质代谢、保证神经肌肉的正常功能等方面具有重要作用。钾离子代谢紊乱会引起高钾血症和低钾血症，如果得不到及时治疗，会导致全身各器官系统特别是心血管系统、神经系统的生理功能和机体的物质代谢发生相应的障碍，严重时可导致死亡。血清钾的测定是一项临床常规和急诊检验项目，钾离子浓度的升高与降低与临床多种疾病相关，测定血清钾在临床诊断上具有重要的意义，因此如何选择一个简便、快捷、有效、准确的检测方法，是分析方法研究中的重要内容。
目前，钾离子的检测方法主要有火焰光度法、离子选择电极法（ISE）、干化学法和分光光度法法。
火焰光度法的优点是血清钾用血量少，操作简便，结果准确可靠，成本低廉。其缺点在于火焰光度法在检测过程中，灵敏度易受多种因素影响。此外，火焰光度法最大的缺点在于所使用的是丙烷等燃气，给实验室带来安全隐患。除此之外，火焰光度法测定钾不易实现自动化。离子选择性电极法是以测量电池的电位为基础的定量分析方法。离子选择性电极法可定量分析标本中钾离子浓度，具有高度选择性和敏感性。该方法具有标本用量少、检测快速、稳定、准确可靠的优点。但实验结果表明，电极法需要不断定标、电极需半年更换一次，维护费用较高。和其他生化项目一起检测时，还需对样本进行分样处理，增大了工作量，分析后的数据要重新汇总后再录入报告单中，易造成人为误差。而在大型生化仪上配置的电解质单元模块购机成本大，而且使用仪器的维护成本和配套试剂的费用很高，造成医院成本增加。干化学法分析仪以其快速、操作简便、干扰因素小、结果准确、精密度高、维护简单等优点越来越广泛的应用于临床。近年来，干化学分析及其检测仪器的开发和应用得到迅速发展。美国KodakEktachemDT-60干式生化分析仪（美国柯达公司产品）及配套用多层膜胶片，受到广大检验工作者的认可和好评。该仪器采电极干扰少，操作简单快速，易于掌握，结果准确可靠。多层膜电极法测定血清钾离子，线性良好，回收试验附合要求，测定结果的准确度和精密度与离子选择性电极法技术相当。干化学法具有无需定标、操作简单、保养容易、稳定性好、故障低、污染少等优点，适用于临床急诊和移动检测（救护车、野外）的需要，但检测成本费用较高，还不能广泛应用到临床常规检测中。分光光度法分为两类：一类是大环发色团法，另一类是酶法。大环发色团法的萃取步骤繁多，使用时有很大的局限性。在冠醚络合金属离子的理论基础上，Berry等首先建立了以酶促反应为主体的K+测定法。酶法测定K+使大批量全自动测定成为可能，同时该法具有操作简单、线性范围宽、污染率低，与离子选择电极相关性好，具有准确、快速、重复性好、适于大量标本的检测等优点，一般只需要在生化分析仪上即可完成血清钾检测，完全能满足临床需要，具有较好的临床应用价值。但其试剂的稳定性不理想，酶法本身易受温度，基质的酸碱度。
鉴于此，本发明在酶法的基础上，优化反应体系，采用咪唑-HCL缓冲液，添加多种稳定剂，显著改善了试剂的稳定性和灵敏度。是一种更加稳定、灵敏度更好的钾离子检测试剂。
发明内容
本发明的目的是提供一种用于检测钾离子的试剂及使用该试剂检测血清钾含量的方法。该试剂盒采用酶法，可以有效检测血清中钾离子的含量，灵敏度高，稳定性好等优点。
基本原理：
通过钾依赖性丙酮酸激酶催化底物磷酸烯醇式丙酮酸（PEP）的酶动力学反应检测钾，其产物丙酮酸盐在乳酸脱氢酶（LDH）作用下与NADH反应生成NAD+，其在340nm的吸光值下降与钾浓度呈比例。
K+
PEP+ADP酮酸盐+ATP
丙酮酸激酶
LDH
丙酮酸盐+NADH+H+ 乳酸+NAD+
本发明是通过以下步骤得到的：
一种钾离子检测试剂，包括试剂R1和试剂R2，所述试剂R1和试剂R2的组成如下：
试剂R1中含有：
咪唑-HCL缓冲液······································100mmol/L，
MIT·················································3g/L，
BSA·················································1g/L，
ɑ-KG················································5g/L，
MgCl2···············································15mmol/L，
ADP·················································5mmol/L，
蔗糖················································5g/L，
海藻糖··············································2g/L，
聚乙二醇············································5g/L，
黄原胶··············································2g/L
山梨醇··············································4g/L
甘油················································120g/L
麦芽糖··············································5g/L
GLDH················································5KU/L，
LDH·················································5KU/L，
PK··················································3KU/L，
曲拉通X-100········································2mL/L，
十二烷基三甲基氯化铵································0.5g/L，
月桂基咪唑啉甜菜碱·································3ml/L，
防腐剂··············································0.5g/L；
试剂R2的组分为：
Tris-HCL缓冲液······································100mmol/L，
PEP·················································2KU/L，
BSA·················································1g/L，
NADH················································3mmol/L
蔗糖················································5g/L，
海藻糖··············································2g/L，
EDTA················································2g/L，
防腐剂··············································0.5g/L。
所述的钾离子检测试剂，试剂R1中缓冲液为25℃，pH为7.2的咪唑-盐酸缓冲液。
所述的钾离子检测试剂，试剂R2中缓冲液为25℃，pH为9.3的Tris-盐酸缓冲液。
所述的钾离子检测试剂，所述防腐剂为NaN3。
所述的钾离子检测试剂来检测钾离子的检测方法，使用全自动生化分析仪利用固定时间法进行测定，检测主波长为340nm。
所述的检测方法，R1试剂和R2试剂的比例为3:1。
本发明的有益效果：
1）采用新的缓冲体系和稳定剂，显著改善了试剂的稳定性；
2）采用新型表面活性剂十二烷基三甲基氯化铵和月桂基咪唑啉甜菜碱，不仅显著改善测定的性能，而且增强了试剂的稳定性和灵敏度；
3）试剂的准确度和稳定性良好，价格便宜，使用方便，完全可以满足临床需要。
附图说明
图1为两种试剂的相关性曲线图，
图2为两种试剂效期稳定性曲线图，
图3为实施例1试剂检测方法，
图4为实施例2中分析灵敏度实验结果，
图5为实施例1试剂与市场常见并得到认可的钾离子测定试剂盒对比检测结果。
具体实施方式
下面结合具体实施例对本发明进行进一步说明：
实施例1
钾离子的检测试剂，包试剂R1和试剂R2：
1）其R1的组成为：
咪唑-HCL缓冲液········································100mmol/L，
MIT···················································3g/L，
BSA···················································1g/L，
ɑ-KG··················································5g/L，
MgCl2·················································15mmol/L，
ADP···················································5mmol/L，
蔗糖··················································5g/L，
海藻糖················································2g/L，
聚乙二醇··············································5g/L，
黄原胶················································2g/L
山梨醇················································4g/L
甘油··················································120g/L
麦芽糖················································5g/L
GLDH··················································5KU/L，
LDH···················································5KU/L，
PK····················································3KU/L，
曲拉通X-100··········································2mL/L，
十二烷基三甲基氯化铵··································0.5g/L，
月桂基咪唑啉甜菜碱···································3ml/L，
防腐剂················································0.5g/L；
2）试剂R2的组分为：
Tris-HCL缓冲液········································100mmol/L，
PEP···················································2KU/L，
BSA···················································1g/L，
NADH··················································3mmol/L，
蔗糖··················································5g/L，
海藻糖················································2g/L，
EDTA··················································2g/L，
防腐剂················································0.5g/L，
3）本实施例试剂的使用方法：
本实施例描述的钾离子检测试剂，在使用时采用具有双试剂功能的全自动生化分析仪，如日立7180全自动分析仪等，利用固定时间法进行测定。将R1和R2按照3:1的比例放置到对应的试剂位上，在样品盘的对应位置放置好蒸馏水、标准品和样本，操作如图3。
实施例2
分析灵敏度试验：
用本发明钾离子试剂盒测试已知浓度在4.22mmol/L的样本，记录吸光度差值。利用市场常见的国家食品药品监督管理局认可的某公司的钾离子试剂盒进行对照检测。检测结果如图4所示。
通过检测数据可知，对照组检测试剂盒的吸光度差值的绝对值比实施例1的高，说明在试剂中加入黄原胶、月桂基咪唑啉甜菜碱等稳定剂，大大的提高了试剂的分析灵敏度。
实施例3
相关性实验：利用实施例1配方配制试剂，与市场常见的国家食品药品监督管理局认可的某公司的钾离子试剂盒进行对照检测，同时检测了20个临床血清样本，检测结果如图5所示。并获得了两种试剂的相关性曲线（如图1所示），通过检测结果显示，两个试剂盒的相关系数为0.9992，说明了两者有极大的相关性。
实施例4
试剂的效期稳定性对比试验：对实施例1中的试剂，均匀分装13组，每组的试剂量为R1为15mL，R2为5mL；并且取13组市场常见的国家食品药品监督管理局认可的某公司的钾离子试剂盒作对照。放置到2-8℃冰箱中，每月的同一天取出一组试剂检测钾离子质控品（靶值为4.22mmol/L），检测结果如图2所示，实施例1试剂在2-8℃储存条件下比市场常见的钾离子测定试剂盒更加稳定。
通过验证，本试剂与同类检测试剂对比相关性好，临床检测样本结果一致，能够达到市场对产品的应用要求，并且灵敏度好，是一种更加稳定、良好的钾离子检测试剂。
Claims (6)
1.一种钾离子检测试剂，其特征在于包括试剂R1和试剂R2，所述试剂R1和试剂R2的组成如下：
试剂R1中含有
钾离子的检测试剂，包试剂R1和试剂R2：
1）其R1的组成为：
咪唑-HCL缓冲液········································100mmol/L，
MIT···················································3g/L，
BSA···················································1g/L，
ɑ-KG··················································5g/L，
MgCl2·················································15mmol/L，
ADP···················································5mmol/L，
蔗糖··················································5g/L，
海藻糖················································2g/L，
聚乙二醇··············································5g/L，
黄原胶················································2g/L，
山梨醇················································4g/L，
甘油··················································120g/L，
麦芽糖················································5g/L，
GLDH··················································5KU/L，
LDH···················································5KU/L，
PK····················································3KU/L，
曲拉通X-100··········································2mL/L，
十二烷基三甲基氯化铵··································0.5g/L，
月桂基咪唑啉甜菜碱···································3ml/L，
防腐剂················································0.5g/L；
2）试剂R2的组分为：
Tris-HCL缓冲液········································100mmol/L，
PEP···················································2KU/L，
BSA···················································1g/L，
NADH··················································3mmol/L
蔗糖··················································5g/L，
海藻糖················································2g/L，
EDTA··················································2g/L，
防腐剂················································0.5g/L。
2.根据权利要求1所述的钾离子检测试剂，其特征在于试剂R1中缓冲液为25℃，pH为7.2的咪唑-盐酸缓冲液。
3.根据权利要求1所述的钾离子检测试剂，其特征在于试剂R2中缓冲液为25℃，pH为9.3的Tris-盐酸缓冲液。
4.根据权利要求1所述的钾离子检测试剂，其特征在于所述防腐剂为NaN3。
5.一种使用权利要求1-4中任一项所述的钾离子检测试剂来检测血清钾的检测方法，其特征在于使用全自动生化分析仪利用固定时间法进行测定，检测主波长为340nm。
6.根据权利要求5所述的检测方法，其特征在于R1试剂和R2试剂的比例为3:1。
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