CN109971478A - 铽离子掺杂的纳米颗粒用于荧光双波长检测多巴胺的方法 - Google Patents
铽离子掺杂的纳米颗粒用于荧光双波长检测多巴胺的方法 Download PDFInfo
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Abstract
本发明公开了一种镧系离子掺杂的纳米颗粒荧光双波长光谱检测多巴胺的检测方法，其中镧系掺杂的纳米颗粒以钆(Gd3+)的稀土氟化物NaGdF4作为基质材料，掺杂发光镧系离子铽(Tb3+)，通过热共沉淀法合成。该方法制备简单，毒性低，化学光学稳定性高，制备的纳米颗粒可通过272nm和297nm两种波长激发产生荧光，在多巴胺浓度升高的情况下，以272nm波长激发则产生荧光淬灭效应，荧光强度降低，而以297nm波长激发则荧光增强，且荧光强度变化与浓度符合Langmuir吸附行为，检测限低至7.7nM，实现多巴胺的双波长荧光检测，且通过双波长激发检测可以互相验证，提升检测的准确性。
Description
技术领域
本发明涉及分析检测方法，具体涉及一种利用铽离子掺杂的纳米颗粒荧光双波长检测多巴胺的方法。
背景技术
多巴胺是一种哺乳动物中枢神经系统的神经递质，属于儿茶酚胺类物质，中文别名2-(3，4-二羟基苯基)乙胺。多巴胺主要产生于神经系统和肾上腺髓质，在行为和认知等许多大脑功能中起着重要作用。在脑内多巴胺的分泌可以调控大脑的情欲，兴奋以及上瘾行为，扮演着信息使者的角色，其水平的异常和一些精神系统变性疾病有关，如帕金森症，奥兹海默症，抑郁症等。基于此，快速准确的检测多巴胺的浓度具有重要意义。
目前常用的多巴胺检测方法有电化学法，色谱法以及光学法。多巴胺具有电化学活性，但是在电化学检测中，多巴胺的氧化电位和抗坏血酸以及尿酸等体液中常见物质的氧化电位相近，因此会受到其干扰。高效液相色谱法作为一种重要的分离手段，在多巴胺分析研究中得以应用，但是高效液相色谱价格相对比较昂贵，检测成本高。光学法则包括紫外-可见吸收、电致化学发光、分子荧光等方法，其中荧光法检测灵敏度较高，但常规的有机荧光探针稳定性较低，容易产生光漂白，在一定程度上影响了荧光法检测的准确性。
镧系离子经激光产生的荧光具有发射波长宽度窄，荧光寿命长，无光漂白效应，稳定性好等性质，因此基于镧系离子发光的荧光探针可替代传统的有机荧光探针用于多巴胺及其他生物检测。2017年Qianming Wang使用稀土铽(Tb3+)螯合物通过荧光增强实现对多巴胺的检测，检测限为0.82μM(Microchimica Acta，2017，184，2275-2280)，但稀土铽(Tb3+)螯合物的制备较为复杂，光化学稳定性相对较差，且检测限较高。与镧系螯合物相比，镧系掺杂纳米颗粒具有制备简单，尺寸可调节，化学稳定性高、可修饰性好、潜在生物毒性低等优点。
发明内容
本发明的目的在于提供一种准确，快速，灵敏度高的多巴胺光学检测的方法。
为了实现上述目的，本发明提供一种镧系离子掺杂的纳米颗粒荧光双波长光谱检测多巴胺的检测方法，其中镧系掺杂的纳米颗粒以钆(Gd3+)的稀土氟化物NaGdF4作为基质材料，掺杂发光镧系离子铽(Tb3+)，通过热共沉淀法合成。该纳米颗粒可通过272nm和297nm两种波长激发产生荧光，在多巴胺浓度升高的情况下，以272nm波长激发则产生荧光淬灭效应，荧光强度降低，而以297nm波长激发则荧光增强，实现多巴胺的双波长荧光检测，且通过双波长激发可以互相验证，提升检测的准确性。
本发明的实施方法包括：将醋酸钆(0.352mmol)，醋酸铽(0.048mmol)和油酸(4mL)，十八烯(6mL)混合150℃保持1小时，降到室温加入3.3mL氟化氨(0.4M)和2mL氢氧化钠(0.5M)，室温下搅拌两个小时，升温到100℃抽真空，充入氮气条件下升温到280℃反应2小时，离心分离，乙醇清洗并将产物分散在环己烷中。加入1M盐酸溶液充分混合，离心分离收集产物，产物为NaGdF4：Tb，将其分散在去离子水中保存。将NaGdF4：Tb纳米颗粒分散在缓冲溶液环境中，在272nm和297nm激发波长下，将多巴胺浓度除以荧光强度差值与浓度作出符合Langmuir行为的标准曲线，以三倍噪音强度作为荧光强度差值带入标准方程，求出检测限，以供未知样品测量荧光强度推算浓度。
上述纳米颗粒制备过程中，醋酸钆、醋酸铽纯度高于99.9％。
上述纳米颗粒制备过程中，醋酸钆、醋酸铽中钆和铽的摩尔比为88∶12。
上述荧光检测过程中，缓冲溶液pH为8.5。
上述荧光检测过程中，检测荧光强度的波长为488nm，543nm，584nm，620nm，优选为543nm。
上述纳米颗粒制备过程中，NaGdF4：Tb颗粒优选可继续通过热共沉淀法增加一层NaGdF4壳，形成NaGdF4：Tb@NaGdF4。
与现有技术相比，本发明采铽离子掺杂的纳米颗粒，双波长荧光检测，优化了检测探针，简化了检测步骤，缩短检测时间。本发明的其他特征和优点将在随后的具体实施方式部分予以详细说明。
附图说明
为了清楚的阐述本发明实施例中的技术方案，以下将对实施例中所需要使用的附图作简单的说明。
图1为实施例一中合成的NaGdF4：Tb的透射电镜图，以及尺寸分布图。
图2为实施例一中合成的NaGdF4：Tb的X射线衍射图。
图3为实施例二中合成的NaGdF4：Tb@NaGdF4的透射电镜图，以及尺寸分布图。
图4为实施例二中合成的NaGdF4：Tb@NaGdF4的X射线衍射图。
图5为实施例四中NaGdF4：Tb纳米颗粒在272nm激发波长处检测多巴胺的荧光光谱图。
图6为实施例四中NaGdF4：Tb纳米颗粒在297nm激发波长处检测多巴胺的荧光光谱图。
图7为实施例六中NaGdF4：Tb@NaGdF4纳米颗粒在272nm激发波长处检测多巴胺的荧光光谱图。
图8为实施例五中双波长荧光光谱检测方法铽离子掺杂纳米颗粒检测多巴胺的选择性图。
具体实施方式
以下对本发明的具体实施方式以及在检测多巴胺方面的应用进行具体说明。应当理解的是，此处所描述的具体实施方式仅用于说明和解释本发明，并不用于限制本发明。
实施例一：热共沉淀法合成了铽离子掺杂的纳米颗粒NaGdF4：Tb。将醋酸钆(0.352mmol)，醋酸铽(0.048mmol)和油酸(4mL)，十八烯(6mL)混合150℃保持1小时，降到室温加入3.3mL氟化氨(0.4M)和2mL氢氧化钠(0.5M)，室温下搅拌两个小时，升温到100℃抽真空，充入氮气条件下升温到280℃反应2小时，6000转/分钟离心分离，乙醇清洗并将产物分散在环己烷中。加入1M盐酸溶液充分混合，16000转/分钟离心分离收集产物，产物为NaGdF4：Tb，将其分散在去离子水中保存。
实施例二：热共沉淀法合成了铽离子掺杂的纳米颗粒NaGdF4：Tb@NaGdF4。将醋酸钆(0.4mmol)，和油酸(4mL)，十八烯(6mL)充分混合，150℃保持1小时后降至80℃，加入分散在环己烷中的NaGdF4：Tb纳米颗粒保持15分钟，降至室温加入3.3mL氟化氨(0.4M)和2mL氢氧化钠(0.5M)，室温下搅拌两个小时，升温到100℃抽真空，充入氮气环境下升温到280℃反应2小时，6000转/分钟离心收集产物，乙醇清洗三次，分散在环己烷中。加入1M盐酸溶液充分混合，16000转/分钟离心收集产物NaGdF4：Tb@NaGdF4，并分散在去离子水中保存。
实施例三：多巴胺储备液的配制，称取9.48mg的盐酸多巴胺，用蒸馏水定容至25mL的容量瓶中，配制成2mM的溶液。
实施例四：取实施例一中的纳米颗粒NaGdF4：Tb加入Tris缓冲溶液(2.5mM，pH＝8.5)配置成浓度0.5mg/mL的分散液，取2mL加至比色皿中。加入2mM的多巴胺储备液，形成多巴胺浓度为1，2，3，5，10，20，30，40，50，100μM的分散液。在此过程中分别记录272nm和297nm激发波长下，543nm波长的荧光发射强度。并以多巴胺浓度除以荧光强度差值与浓度作出符合Langmuir行为的标准曲线，以三倍噪音法推算检测限，得出纳米颗粒NaGdF4：Tb作为检测探针在272nm激发波长下在0-100μM范围内呈线性关系，对多巴胺的检测限为3.0*10-8M，在297nm激发波长下0-100μM范围内呈线性关系，对多巴胺的检测限为3.9*10-8M。
实施例五：选择性实验。选择的干扰物有抗坏血酸，尿酸，柠檬酸，葡萄糖，谷胱甘肽，半胱氨酸，苏氨酸，脯氨酸，丝氨酸，组氨酸，精氨酸，丙氨酸，Ca2+，Zn2+等，其中多巴胺的检测浓度为20μM，干扰物的浓度为200μM。
实施例六：取实施例二中的纳米颗粒NaGdF4：Tb@NaGdF4加入Tris缓冲溶液(2.5mM，pH＝8.5)配置成浓度0.5mg/mL的分散液，取2mL加至比色皿中。加入2mM多巴胺储备液，形成多巴胺浓度为1，2，3，5，10，20，30，40，50，100μM的分散液。在此过程中分别记录272nm和297nm激发波长下，543nm波长的荧光发射强度。并以多巴胺浓度除以荧光强度差值与浓度作出符合Langmuir行为的标准曲线，以三倍噪音法推算检测限，得出纳米颗粒NaGdF4：Tb@NaGdF4作为检测探针在272nm激发波长下0-100μM范围内呈线性关系，对多巴胺的检测限为7.74*10-9M。
实施例七：取实施例一中的纳米颗粒NaGdF4：Tb加入Tris缓冲溶液(2.5mM，pH＝8.5)配置成浓度0.5mg/mL的分散液，取2mL加至比色皿中。加入2mM多巴胺储备液，形成多巴胺浓度为1，2，3，5，10，20，30，40，50，100μM的分散液。在此过程中分别记录272nm和297nm激发波长下，488nm波长的荧光发射强度。并以多巴胺浓度除以荧光强度差值与浓度作出符合Langmuir行为的标准曲线，以三倍噪音法推算检测限，得出纳米颗粒NaGdF4：Tb作为检测探针在272nm激发波长下0-100μM范围呈线性关系，对多巴胺的检测限为1.75*10-7M，在297nm激发波长下0-100μM范围内呈线性关系，对多巴胺的检测限为1.28*10-7M。
实施例八：取实施例一中的纳米颗粒NaGdF4：Tb加入Tris缓冲溶液(2.5mM，pH＝8.5)配置成浓度0.5mg/mL的分散液，取2mL加至比色皿中。加入2mM多巴胺储备液，形成多巴胺浓度为1，2，3，5，10，20，30，40，50，100μM的分散液。在此过程中分别记录272nm和297nm激发波长下，584nm波长的荧光发射强度。并以多巴胺浓度除以荧光强度差值与浓度作出符合Langmuir行为的标准曲线，以三倍噪音法推算检测限，得出纳米颗粒NaGdF4：Tb作为检测探针在272nm激发波长下0-5μM范围内呈线性关系，对多巴胺的检测限为2.36*10-7M，在297nm激发波长下0-10μM范围内呈线性关系，对多巴胺的检测限为1.64*10-7M。
实施例九：取实施例一中的纳米颗粒NaGdF4：Tb加入Tris缓冲溶液(2.5mM，pH＝8.5)配置成浓度0.5mg/mL的分散液，取2mL加至比色皿中。加入2mM多巴胺储备液，形成多巴胺浓度为1，2，3，5，10，20，30，40，50，100μM的分散液。在此过程中分别记录272nm和297nm激发波长下，620nm波长的荧光发射强度。并以多巴胺浓度除以荧光强度差值与浓度作出符合Langmuir行为的标准曲线，以三倍噪音法推算检测限，得出纳米颗粒NaGdF4：Tb作为检测探针在272nm激发波长下0-100μM范围内呈线性关系，对多巴胺的检测限为4.71*10-7M，在297nm激发波长下0-100μM范围内呈线性关系，对多巴胺的检测限为3.15*10-7M。
Claims (7)
1.一种将铽离子掺杂的纳米颗粒用于荧光双波长检测多巴胺的方法，其特征在于：
a.将醋酸钆、醋酸铽和油酸，十八烯混合，150℃保持1小时，降到室温加入氟化氨和氢氧化钠，室温下搅拌两个小时，升温到100℃抽真空，充入氮气条件下升温到280℃反应2小时，离心分离，乙醇清洗并将产物分散在环己烷中，加入盐酸溶液充分混合，离心分离收集产物，产物为NaGdF4：Tb，将其分散在去离子水中保存；
b.纳米颗粒NaGdF4：Tb加入Tris缓冲溶液配置成浓度0.5mg/mL的分散液，加入2mM的多巴胺储备液，形成1-100μM的标准溶液，在272nm和297nm激发波长下，分别将浓度除以荧光强度差值与浓度作出标准曲线，再将荧光强度差值设为三倍噪音法，求出浓度检测限，以供未知样品测量荧光推算浓度。
2.根据权利要求一所述的检测多巴胺的方法，其特征在于醋酸钆、醋酸铽纯度高于99.9％。
3.根据权利要求一所述的检测多巴胺的方法，其特征在于醋酸钆、醋酸铽中钆和铽的摩尔比为12∶88。
4.根据权利要求一所述的检测多巴胺的方法，其特征在于Tris缓冲溶液pH为8.5。
5.根据权利要求一所述的检测多巴胺的方法，其特征在于检测荧光强度的波长为488nm，543nm，584nm，620nm。
6.根据权利要求一所述的检测多巴胺的方法，其特征在于检测荧光强度的波长为543nm。
7.根据权利要求一所述的检测多巴胺的方法，其特征在于步骤a中得到的NaGdF4：Tb颗粒优选可继续通过热共沉淀法增加一层NaGdF4壳，形成NaGdF4：Tb@NaGdF4，并将其替代步骤b中的NaGdF4：Tb纳米颗粒进行检测，步骤为：
将醋酸钆和油酸、十八烯充分混合，150℃保持1小时后降至80℃，加入分散在环己烷中的NaGdF4：Tb纳米颗粒保持15分钟，降至室温加氟化氨和氢氧化钠，室温下搅拌两个小时，升温到100℃抽真空，充入氮气环境下升温到280℃反应2小时，离心收集产物，乙醇清洗三次，分散在环己烷中，加盐酸溶液充分混合，离心收集产物NaGdF4：Tb@NaGdF4，并分散在去离子水中保存。
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| CN109612971B (zh) * | 2018-12-07 | 2022-03-18 | 宁波大学 | 一种铽金属有机骨架材料及其制备与应用 |
| CN111040764A (zh) * | 2019-12-11 | 2020-04-21 | 昆明理工大学 | 一种氟化物高亮度x射线闪烁体及其制备方法 |
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