CN108872220B - 一种无汞对羟基苯丙氨酸检测试剂及制备方法和应用 - Google Patents
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Abstract
本发明属于检测试剂技术领域，具体涉及一种无汞对羟基苯丙氨酸检测试剂及制备方法和应用。所述试剂缓冲液以及分散在缓冲液中的酪氨酸酶和4‑氨基安替比林。所述制备方法是将上述组分计量混合。可应用于制备尿液中对羟基苯丙氨酸的试剂盒。本发明提供的技术方案采用酪氨酸酶对对羟基苯丙氨酸进行定性和定量分析，并成功将其改进为可以产品的尿液检测试剂，检测试剂组分环境友好，其中不再含有汞镍等重金属离子，使用后也不会造成环境污染，另外，温和的分散体系，使得制备和使用过程非常安全。
Description
技术领域
本发明属于检测试剂技术领域，具体涉及一种无汞对羟基苯丙氨酸检测试剂及制备方法和应用。
背景技术
肿瘤的早期诊断和早期治疗是提高肿瘤治愈率的关键。现临床常用的确诊手段有胸透、B超、CT、核磁共振等，常常是伴随着穿刺、抽血等加重病人痛苦甚至有可能发生交叉感染的手段，而且价格昂贵，更重要的是通过这些手段能检测出的肿瘤一般都处于中晚期，治愈率大大降低。
癌细胞的核苷酸代谢异常会生成一种单羟酚类代谢物，其中对羟基苯丙氨酸的含量远高于正常人，而这种物质能够通过尿液排放。因此检测对羟基苯丙氨酸的含量，可以推断人体是否患有癌症，能够及早的发现肿瘤，挽救生命，无需额外的开支，免去病人的恐惧和痛苦。
目前对羟基苯丙氨酸尿液检测试剂都是采用汞离子、亚汞离子显色，比如中国专利CN103323452A、CN104535565A、CN106706614A和CN107490689A中的技术方案均是基于这一原理。该方法有以下不足：1)试剂采用氨基酸和金属离子配位原理，尿液中的尿酸等在高浓度情况下产生极大干扰，造成假阴性结果；2)检测方法中都含有汞离子，汞的毒性不仅使制备过程存在一定安全隐患，另外，检测废液中汞镍等重金属离子也会对环境产生危害，因此生产和回收都需特殊的处理，增加使用成本；3)硫酸硝酸等强酸的使用在制备过程和使用过程也同样存在很多安全问题。
发明内容
本发明提供了一种无汞对羟基苯丙氨酸检测试剂及制备方法和应用，用以解决目前对羟基苯丙氨酸检测试剂中均含汞和强酸的问题。
为了解决上述技术问题，本发明的技术方案是：所述无汞对羟基苯丙氨酸检测试剂，其包括缓冲液以及分散在缓冲液中的酪氨酸酶
可选地，还包括4-氨基安替比林。
酪氨酸酶，也叫多酚氧化酶、儿茶酚氧化酶等，能够将无色的多酚类物质氧化为有颜色的茶红素、茶黄素等物质。但茶红素茶黄素颜色较浅，颜色梯度不明显，如果作为尿液检索试剂则灵敏度不足，而4-氨基安替比林则可以加深显色效果，从而使酪氨酸酶作为对羟基苯丙氨酸成为可能。
可选地，还包括分散在缓冲液中的蔗糖、白蛋白和Triton X-100(聚乙二醇辛基苯基醚)。酪氨酸酶作为显色剂还存在一个贮存稳定性的问题，通过在缓冲液中添加适量的蔗糖、白蛋白和Triton X-100，则可以配置的试剂可以长期保存，解决了其产品化的问题。
可选地，所述缓冲液为磷酸盐水溶液，pH值为5.0～8.0。
可选地，所述酪氨酸酶的浓度为50～2000U/mL。
可选地，所述4-氨基安替比林的浓度为0.5-10mg/mL。
可选地，所述蔗糖的浓度为0.02-0.1g/mL、白蛋白的浓度为0.005-0.05g/mL和Triton X-100的浓度为0.005-0.02g/mL。
本发明还提供了上述无汞对羟基苯丙氨酸检测试剂的制备方法，其包括如下步骤：配置缓冲液，将酪氨酸酶干粉和4-氨基安替比林溶解在所述缓冲液中，配置完成。
本发明还提供了一种尿液中对羟基苯丙氨酸的试剂盒，其包括盛装有上述无汞对羟基苯丙氨酸检测试剂的安瓿瓶。
可选地，所述安瓿瓶中无汞对羟基苯丙氨酸检测试剂的量为0.1-0.5mL。
本发明提供的技术方案采用酪氨酸酶对对羟基苯丙氨酸进行定性和定量分析，并成功将其改进为可以产品的尿液检测试剂，检测试剂组分环境友好，其中不再含有汞镍等重金属离子，使用后也不会造成环境污染，另外，温和的分散体系，使得制备和使用过程非常安全。
附图说明
图1是实施例4分光光度法定量测试实验中的标准曲线。
具体实施方式
为了便于理解，下面结合实施例阐述所述无汞对羟基苯丙氨酸检测试剂，应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。
所有原材料均为分析纯级别的普通化学制剂，制备过程也都在常温、常压下进行。
实施例1
1)配置pH＝6.5的磷酸盐缓冲液；
2)称取10KU酪氨酸酶干粉，溶解于10ml pH＝6.5的磷酸盐缓冲液中，配置完成；
3)将所述无汞对羟基苯丙氨酸检测试剂按每瓶0.1ml分装于安瓿瓶中。
实施例1的检测试剂包括了缓冲液和酪氨酸酶，其中酪氨酸酶的浓度为1000U/mL。
实施例2
1)配置pH＝7.4磷酸盐缓冲液；
2)称取5KU酪氨酸酶干粉，溶解于10ml pH＝7.4的磷酸盐缓冲液中；
3)然后加入0.1g牛血清白蛋白、0.2g蔗糖和0.1g Triton X-100，配置完成；
4)将所述无汞对羟基苯丙氨酸检测试剂按每瓶0.1ml分装于安瓿瓶中。
实施例2的检测试剂包括了缓冲液、酪氨酸酶、蔗糖、白蛋白和Triton X-100，其中酪氨酸酶的浓度为500U/mL，蔗糖浓度为0.02g/mL、白蛋白的浓度为0.01g/mL和Triton X-100的浓度为0.01g/mL。
实施例3
1)配置pH＝7.4磷酸盐缓冲液；
2)称取8KU酪氨酸酶干粉，溶解于10ml pH＝7.4的磷酸盐缓冲液中；
3)然后加入0.01g 4-氨基安替比林；
4)然后加入0.1g牛血清白蛋白、0.2g蔗糖和0.1g Triton X-100，配置完成；
5)将所述无汞对羟基苯丙氨酸检测试剂按每瓶0.1ml分装于安瓿瓶中。
实施例3的检测试剂包括了缓冲液、酪氨酸酶、4-氨基安替比林、蔗糖、白蛋白和Triton X-100，其中酪氨酸酶的浓度为800U/mL，4-氨基安替比林的浓度为1mg/mL，蔗糖浓度为0.02g/mL、白蛋白的浓度为0.01g/mL和Triton X-100的浓度为0.01g/mL。
实施例4效果验证试验
4.1定性测试实验
配置四个不同浓度的对羟基苯丙氨酸水溶液，从低到高依次为0mg/L，60mg/L，120mg/L，250mg/L和450mg/L，分别添加到上述实施例配置的检测试剂中，观察变色情况，结果如表1所示。
表1
通过表1可以发现，本发明提供的检测试剂加入含有对羟基苯丙氨酸的测试样品后，出现了明显的颜色变化，含量越高，变色程度越高。
4.2分光光度法定量测试实验
实施例3中添加了4-氨基安替比林，反应后的溶液除了采用肉眼观测定量分析外，可以使用分光光度法测试，测试结果如图1所示。根据不同浓度的酪氨酸溶液，可以制作标准曲线。得到待测样本的吸光度值后可以根据标准曲线得到定量的对羟基苯丙氨酸浓度。
4.3稳定性实验
将实施例1-3的试剂在50℃烘箱中保存一周，然后分别进行测试，实施例1在低浓度时不能明显显色，实施例2和3在能够正常显色。
4.4抗干扰实验
配置250mg/L的对羟基苯丙氨酸水溶液溶液，其中含有尿酸600μmol/L,分别采用本专利中实施例1，2，3以及专利CN104535565A中的试剂测试，其中1,2,3能够正常显色，专利CN104535565A的试剂不能显色。
最后应说明的是：以上实施例仅用以说明本发明的技术方案，而非对其限制。尽管参照前述实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换，而这些修改或替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
Claims (4)
1.一种无汞对羟基苯丙氨酸检测试剂，其特征在于，包括缓冲液以及分散在缓冲液中的酪氨酸酶，所述缓冲液中还分散有4-氨基安替比林、蔗糖、白蛋白和Triton X-100，所述4-氨基安替比林的浓度为0.5-10mg/mL，所述缓冲液为磷酸盐水溶液，pH值为5.0～8.0，所述酪氨酸酶的浓度为50～2000U/mL，所述蔗糖的浓度为0.02-0.1g/mL、白蛋白的浓度为0.005-0.05g/mL和Triton X-100的浓度为0.005-0.02g/mL。
2.权利要求1所述无汞对羟基苯丙氨酸检测试剂的制备方法，其特征在于，包括如下步骤：配置缓冲液，将其他组分溶解在所述缓冲液中，配置完成。
3.一种尿液中对羟基苯丙氨酸的试剂盒，其特征在于，包括盛装有权利要求1所述无汞对羟基苯丙氨酸检测试剂的安瓿瓶。
4.根据权利要求3所述尿液中对羟基苯丙氨酸的试剂盒，其特征在于，所述安瓿瓶中无汞对羟基苯丙氨酸检测试剂的量为0.1-0.5mL。
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| PCT/CN2018/110242 WO2020010739A1 (zh) | 2018-07-11 | 2018-10-15 | 一种无汞对羟基苯丙氨酸检测试剂及制备方法和应用 |
| US15/734,139 US20210215667A1 (en) | 2018-07-11 | 2018-10-15 | Mercury-free p-hydroxyl phenylalanine detection reagent and preparation method and application thereof |
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| CA2014119C (en) * | 1989-04-07 | 2000-11-21 | Diane L. Aunet | Methods and devices for the separation of plasma or serum from whole blood, collection of plasma or serum, and reagent delivery system |
| CN107014806B (zh) * | 2017-03-29 | 2019-05-31 | 汉宇集团股份有限公司 | 一种尿液酪氨酸的检测试纸 |
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