CN111304282A - 一种尿液酪氨酸的检测试剂盒 - Google Patents
一种尿液酪氨酸的检测试剂盒 Download PDFInfo
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Abstract
一种尿液酪氨酸的检测试剂盒，属于生物化学检测技术领域。本发明涉及的试剂盒由以下试剂组成：试剂A:PBS缓冲液；试剂B：酪氨酸羟化酶，四氢生物蝶呤；试剂C：复合酶；试剂D：高碘酸钠；试剂E:酪氨酸标准。本发明利用生物复合酶催化酪氨酸发生特异性显色反应，目前实验证明，除了维生素C外，还没有其它常见尿液中其它物质的干扰，结果准确可靠。另外试剂符合环保要求，对环境没有污染；对人体基本无毒性，使用相对安全。
Description
技术领域
本发明涉及生物化学检测领域，具体涉及一种尿液酪氨酸的检测试剂盒。
背景技术
酪氨酸是构成蛋白质的氨基酸，呈嗜水性，酪氨酸在人及动物体内由苯丙氨酸羟化而产生，是人体重要的半必需氨基酸，它是人体中合成多种蛋白类激素的重要原料，是机体生物合成儿茶酚胺的前身物质。酪氨酸既是生糖又是生酮氨基酸。因酪氨酸代谢异常导致的疾病包括白化病、阿尔茨海默症、苯丙酮酸尿症、白癜风和帕金森等。在正常个体中其含量相对稳定，而在肿瘤患者体液中，酪氨酸含量与健康人相比会有比较大的差异，近年来，越来越多的代谢组学研究表明，体液代谢产物可作为早期发现或检测多种肿瘤进程的标志物，尿酪氨酸浓度大于2.5mmol/L为异常，判断为阳性。医学研究发现，鼻咽癌、卵巢癌、宫颈癌、大肠癌患者的尿酪氨酸阳性率约80～85％，肝癌、胃癌患者的尿酪氨酸阳性率约70％。以尿液中的酪氨酸作为恶性肿瘤标志物，可应用于早期癌症筛查，且取材方便，无创伤，适合大规模推广。体液代谢产物同样可以作为早期发现和监测多种肿瘤进程的标志物，肿瘤病人尿液中酚类代谢物排出量增加。在酪氨酸的研究中，以血清中酪氨酸及其代谢产物的研究相对较多，而对人体尿液中酪氨酸研究相对较少。血清肿瘤标志物存在操作繁琐，成本高，有创性，早期检出率不高等缺点。因此，探索酪氨酸含量的分析新技术并应有于尿液样品测定，对癌症的辅助诊断、治疗和监测具有十分重要的临床意义。
近年来对氨基酸代谢尤其是酪氨酸的体液检测研究有许多技术成果，如专利CN201510962013.1公开了一种用于检测人体尿液中酪氨酸类肿瘤标志物的试剂。其主要成分是硝酸汞、硫酸汞、硝酸镍、硫酸钴等溶液组成。还有专利CN201510537446.2也公开了一种对羟基苯丙氨酸酪氨酸尿液检测试剂。还有一些其它多种技术，但是其检测的反应原理基本上是依托于Millon反应原理或硝化反应原理，结果会产砖红色螯合物，以此对酪氨酸进行一个定性的检测。虽然这些方法在应用中有一定的价值和效果，但仍有如下不足之处：
(1)准确度受到一定限制，上述技术方法容易受尿液颜色、尿中蛋白、还原性物质、酸碱度以及无机盐影响或干扰。
(2)安全性差且不环保，汞是有毒的化合物，可通过呼吸道、皮肤或消化道等不同途径侵入人体，集聚于肝、肾、大脑等部位，且具有累积性，造成神经性中毒和深部组织病变；废弃的汞盐会污染环境，并能通过食物链在生物体内富集，进而进入人体，汞盐若处理不当，对人体以及环境都有极大的危害。
(3)不方便，由于试剂的腐蚀性和挥发性，所以目前产品常用安瓿瓶盛装试剂，但对消费者而言开启困难而且容易产生外伤事故。
发明内容
针对上述检测试剂的不准确、不安全、不环保、不方便的不足，本发明提供一种尿液酪氨酸的检测试剂盒，具有操作简捷，性能稳定，特异性强的优点。
本发明解决技术问题采用的试剂盒由以下试剂组成：
试剂A:0.1mol/L PBS缓冲液，PH6.6-7.0；试剂B：酪氨酸羟化酶800U/mL-1700U/mL，四氢生物蝶呤0.2-1mg/mL；试剂C：复合酶，组成如下：多酚氧化酶900-1500LU/L，抗坏血酸氧化酶浓度为300U/mL-700U/mL，酶稳定剂0.01mg/mL-0.12mg/mL；试剂D：质量浓度为0.05-0.22％的高碘酸钠；试剂E:酪氨酸标准液0.10μmol/mL。
进一步的，试剂A为缓冲液，配制方式为称取磷酸二氢钾13.5g,磷酸氢二钠14.3g，取容器加蒸馏水到1L，调PH到6.6-7.0得到试剂A。
原理：本发明基于酪氨酸在复合酶并有试剂中辅因子的参与作用下，反应产生一种酪氨酸的衍生物，后者经多酚氧化酶和高碘酸盐的反应，可以形成一种特征性的颜色变化，用480nm波长通过比色测量，即可特异性的得到尿中酪氨酸的含量。
有益效果：
与现有的尿酪氨酸/对羟基苯丙氨酸检测试剂相比，本发明具有以下优点：
(1)利用生物复合酶催化酪氨酸发生特异性显色反应，目前实验证明，除了维生素C外，还没有其它常见尿液中其它物质的干扰，结果准确可靠。
(2)试剂符合环保要求，对环境没有污染；对人体基本无毒性，使用相对安全。
(3)检测快速无创方便，使用范围广泛，成本低廉，适合大众化推广应用。
附图说明
图1为本发明实施例6酪氨酸浓度曲线图。
具体实施方式
下面结合实施例主要是针对尿液中酪氨酸的检测对本发明作进一步的说明，下列实施例中如未注明具体条件的实验方法，通常按照本领域的常规手段进行。本实施例只用于对发明进行进一步的说明，不能理解为对本发明保护范围的限制，对其它尿液以外的体液等方面，本领域的技术人员根据上述发明的内容做出一些非本质的改进和调整也属于本发明的保护的范围。
实施例1
本实施例酪氨酸检测试剂盒配制如下：
试剂A:0.1mol/L PBS缓冲液，PH6.8；试剂B：酪氨酸羟化酶1500U/mL，四氢生物蝶呤0.4mg/mL；试剂C：多酚氧化酶1200LU/L，抗坏血酸氧化酶浓度为530U/mL，酶稳定剂0.10mg/mL；试剂D：质量分数为0.17％的高碘酸钠；试剂E:酪氨酸标准液0.10μmol/mL。其中试剂A的配制方式为称取磷酸二氢钾13.5g,磷酸氢二钠14.3g,取容器加蒸馏水到1L，调PH到6.8。
实施例2
本实施例酪氨酸检测试剂盒配制如下：
试剂A:0.1mol/L PBS缓冲液，PH6.6；试剂B：酪氨酸羟化酶800U/mL，四氢生物蝶呤1mg/mL；试剂C：多酚氧化酶900LU/L，抗坏血酸氧化酶浓度为700U/mL，酶稳定剂0.01mg/mL；试剂D：质量分数为0.22％的高碘酸钠；试剂E:酪氨酸标准液0.10μmol/mL。其中试剂A的配制方式为称取磷酸二氢钾13.5g,磷酸氢二钠14.3g,取容器加蒸馏水到1L，调PH到6.6。
实施例3
本实施例酪氨酸检测试剂盒配制如下：
试剂A:0.1mol/L PBS缓冲液，PH7.0；试剂B：酪氨酸羟化酶1700U/mL，四氢生物蝶呤0.2mg/mL；试剂C：多酚氧化酶900LU/L，抗坏血酸氧化酶浓度为300U/mL，酶稳定剂0.12mg/mL；试剂D：质量分数为0.05％的高碘酸钠；试剂E:酪氨酸标准液0.10μmol/mL。其中试剂A的配制方式为称取磷酸二氢钾13.5g,磷酸氢二钠14.3g,取容器加蒸馏水到1L，调PH到7.0。
实施例4
本实施例酪氨酸检测试剂盒配制如下：
试剂A:0.1mol/L PBS缓冲液，PH6.7；试剂B：酪氨酸羟化酶800U/mL，四氢生物蝶呤0.2mg/mL；试剂C：多酚氧化酶900LU/L，抗坏血酸氧化酶浓度为300U/mL，酶稳定剂0.01mg/mL；试剂D：质量分数为0.05％的高碘酸钠；试剂E:酪氨酸标准液0.10μmol/mL。其中试剂A的配制方式为称取磷酸二氢钾13.5g,磷酸氢二钠14.3g,取容器加蒸馏水到1L，调PH到6.7。
实施例5
本实施例酪氨酸检测试剂盒配制如下：
试剂A:0.1mol/L PBS缓冲液，PH6.9；试剂B：酪氨酸羟化酶1700U/mL，四氢生物蝶呤1mg/mL；试剂C：多酚氧化酶1500LU/L，抗坏血酸氧化酶浓度为700U/mL，酶稳定剂0.12mg/mL；试剂D：质量分数为0.22％的高碘酸钠；试剂E:酪氨酸标准液0.10μmol/mL。其中试剂A的配制方式为称取磷酸二氢钾13.5g,磷酸氢二钠14.3g,取容器加蒸馏水到1L，调PH到6.9。
实施例6
标准曲线的制作：所述酪氨酸标准液的配制，称取0.4528g酪氨酸，用水定容于250ml,既得10mmol/L贮藏液，再由此配成浓度为0.2-1mmol/L的系列标准溶液；用纯水配制浓度分别为0.2mmol/L、0.4mmol/L、0.6mmol/L、0.8mmol/L、1mmol/L的标准溶液。
标准曲线如表1所示：
表1标准曲线表
实施例1-5的试剂盒应用检测酪氨酸，制得酪氨酸浓度曲线图如图1所示。本实施例的检测下限在标准曲线的基础上，经过试验检测为0.181。
实施例7
将实施例1-5的试剂盒的进行稳定性实验：将对羟基测定试剂盒置于密封袋中，放置37℃烘箱中，每隔30天观察各试剂外观及进行显色效果测试。测试方法：配制用纯水配制浓度分别为0.2mmol/L、0.4mmol/L、0.6mmol/L、0.8mmol/L、1mmol/L的标准溶液。按照操作方法依次进行显色，显色完全后观察显色效果。
测试结果如表2所示：
表2试剂盒稳定性测试表格
实施例8
样品加标回收实验:收集不同的样品，采用该方法进行对羟基的测试，与国标法进行比对进行回收实验，回收率在97.0-112.0％之间，测试结果如表3所示。
表3实样品检测与加标回收率实验
上述实验说明，本发明的检测方法稳定性好，检测结果准确可靠，适用于尿液的快速检测。
实施例9
本实施例酪氨酸检测试剂盒配制如下：
试剂A:0.1mol/L PBS缓冲液，PH6.9；试剂B：酪氨酸羟化酶1100U/mL，四氢生物蝶呤0.6mg/mL；试剂C：多酚氧化酶1300LU/L，抗坏血酸氧化酶浓度为600U/mL，酶稳定剂0.11mg/mL；试剂D：质量分数为0.2％的高碘酸钠；试剂E:酪氨酸标准液0.10μmol/mL。其中试剂A的配制方式为称取磷酸二氢钾13.5g,磷酸氢二钠14.3g,取容器加蒸馏水到1L，调PH到6.9。
使用本实施例的试剂盒进行样品及标准的重复性检测：
随机取自愿者晨尿5份；另配制标准品3份浓度分别为：1标准0.2mmol/L；2标准0.4mmol/L；3标准0.8mmol/L。检测结果如表4所示：
表4样品及标准的重复性检测表
从检测的样品和标准品可以看出，本试剂检测的重复性良好，标准品的值和标准曲线比对复合。
注意事项：多种氨基酸、蛋白质、糖类、离子及维生素等干扰试验中发现，维生素C浓度达到2.8mmol/L以上时，酪氨酸氧化反应会有干扰造成假阴性，在实际检测过程中需要注意。因此，检测试纸中加入了排除此干扰的试剂成分，可以有效的提高抗VC干扰的能力。
以上所述仅为本发明的优选实施例，并非对本发明做出任何形式上和实质上的限制。本领域的技术人员，在不脱离本发明技术方案的范围内，当可利用以上所揭示的技术内容而做出的更改、修饰与演变的等同变化均为本发明的等效实施例；同时，凡依据本发明的实质技术对以上实施例所作的任何等同变化的更改、修饰与演变等均在本发明的由权利要求界定的范围。
Claims (4)
1.一种尿液酪氨酸的检测试剂盒，其特征在于，该试剂盒由以下试剂组成：
试剂A:0.1mol/LPBS缓冲液，PH6.6-7.0；试剂B：酪氨酸羟化酶800U/mL-1700U/mL；四氢生物蝶呤0.2-1mg/mL；试剂C：复合酶；试剂D：质量浓度为0.05-0.22％的高碘酸钠；试剂E:酪氨酸标准液0.10μmol/mL。
2.根据权利要求1所述的一种尿液酪氨酸的检测试剂盒，其特征在于，试剂C复合酶的组成如下：多酚氧化酶900-1500LU/L，抗坏血酸氧化酶浓度为300U/mL-700U/mL，酶稳定剂0.01mg/mL-0.12mg/mL。
3.根据权利要求1所述的一种尿液酪氨酸的检测试剂盒，其特征在于，试剂A为缓冲液，配制方式为称取磷酸二氢钾13.5g,磷酸氢二钠14.3g，取容器加蒸馏水到1L，调PH到6.6-7.0得到试剂A。
4.根据权利要求1所述的一种尿液酪氨酸的检测试剂盒，其特征在于，该试剂盒由以下试剂组成：试剂A:0.1mol/LPBS缓冲液，PH6.8；试剂B：酪氨酸羟化酶1500U/mL，四氢生物蝶呤0.4mg/mL；试剂C：多酚氧化酶1200LU/L，抗坏血酸氧化酶浓度为530U/mL，酶稳定剂0.10mg/mL；试剂D：质量分数为0.17％的高碘酸钠；试剂E:酪氨酸标准液0.10μmol/mL。
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| CN117110256A (zh) * | 2023-05-29 | 2023-11-24 | 兰州大学第一医院 | 一种基于N-GQDs荧光淬灭原理的尿液酪氨酸检测试剂及检测方法 |
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| CN113684243A (zh) * | 2021-08-17 | 2021-11-23 | 大连大学 | 一种尿液缬氨酸的检测试剂盒及检测方法 |
| CN117110256A (zh) * | 2023-05-29 | 2023-11-24 | 兰州大学第一医院 | 一种基于N-GQDs荧光淬灭原理的尿液酪氨酸检测试剂及检测方法 |
| CN117110256B (zh) * | 2023-05-29 | 2024-04-19 | 兰州大学第一医院 | 一种基于N-GQDs荧光淬灭原理的尿液酪氨酸检测试剂及检测方法 |
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