CN114933899A - 一种稀土发光团簇酶及其制备方法和应用 - Google Patents
一种稀土发光团簇酶及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种稀土发光团簇酶及其制备方法和在催化测定5‑羟基色胺(血清素)中的应用。这种稀土发光团簇酶是由铕离子、铜离子、1,10‑菲啰啉在碱性的聚乙烯吡咯烷酮水溶液中被盐酸羟胺还原形成的铜团簇。这种稀土发光团簇酶既有铜团簇的蓝光发射又有铕离子的红光发射,是一种双重发光的团簇酶;既具有铜团簇的催化功能又有发光检测5‑羟基色胺的传感功能,是一种双重功能的团簇酶。该稀土发光团簇酶的制备无需复杂有机反应,在水相、室温下完成,合成简便。该稀土发光团簇酶超小的尺寸及高催化活性在生物医学领域有良好的应用前景。
Description
技术领域
本发明涉及一种稀土发光团簇酶及其制备方法和在发光检测神经递质分子5-羟基色胺中的应用,属于人造酶、催化、发光传感技术领域。
背景技术
近年来发现的纳米酶已在临床诊疗、生物传感、环境保护等多个领域获得广泛的应用。由几到几百个金属原子构成的团簇,具有超小的尺寸、超高的比表面积和超强的催化活性,是最具潜力的纳米酶。
国内外报道的金属团簇酶的方法和应用有很多。中国专利公布号CN112008091A,2020年,李少光等,一种高灵敏、低毒、具氧化模拟酶活性的金纳米簇制备方法与应用,公开了一种具有氧化酶活性的金纳米簇,检测细胞内过氧化氢水平;中国专利公布号CN113304748 A,2020年,刘爱骅等,一种具有多种仿酶活性的铜纳米团簇及其制备方法与应用,公开了一种具有过氧化物酶、过氧化氢酶、超氧化物歧化酶和抗坏血酸氧化酶活性的铜纳米簇检测谷胱甘肽和抗坏血酸的应用;中国专利公布号CN 113827613 A, 2021年,张晓东等,原子精度的团簇酶和制备及其在神经系统疾病中的应用,公开了一种具有超高的抗氧化活性以及类酶选择性的团簇酶,用于小鼠神经系统疾病的早期干预。中国专利公布号CN 111151300 A,2020年,吴玉清等,一种单磷酸鸟苷保护的铂金合金纳米簇、制备方法及其在催化氧化反应中的应用,公开了一种有较高类过氧化酶活性的铂金纳米簇。邹仁杰报道了一种微生物法合成的银纳米团簇,具有过氧化物酶性质并应用于血糖浓度检测(黑龙江大学自然科学学报,2022,39,1-7)。已公开的方法中金属团簇只有单一的催化功能,用于传感分析、成像等还需要显色剂产生信号。
5-羟基色胺(5-HT),又称血清素,是一种重要的单胺类神经递质分子。5-羟基色胺分布于中枢神经系统、外周组织以及血液中,参与包括内分泌调节、睡眠、情绪、食欲等多个生理过程。体内低水平的5-HT可能引起抑郁症、焦虑神经症、强迫症、高血压、偏头痛等;高水平的5-HT有可能导致5-羟色胺综合征、中毒、甚至致命。5-HT是重要的生物标志物,在生物体内、体液中、排泄物中的含量对癌症、神经性精神疾病、消化系统损伤、肝功能损伤等多种疾病的诊断、治疗及病理研究具有重要意义。
目前,5-HT的测定方法有荧光测定法、薄层层析法、放射免疫分析法、放射酶法、色谱法(GC、HPLC)、色谱-质谱串联法(GC-MS、LC-MS)等。已公开的方法中色谱法是主要的分析方法,色谱法不仅需要昂贵的设备而且分析周期长。已公开的方法中通过单胺氧化酶催化分析是重要的手段,然而天然酶易失活、保质期短、价格昂贵。开发稳定、廉价、活性高的团簇酶对发展灵敏、快速的5-羟基色胺的分析方法,对临床诊断、疾病治疗和科学研究具有重要意义。
发明内容
发明目的:本发明所要解决的第一个技术问题是提供一种稀土发光团簇酶,稀土发光团簇酶既具有酶催化活性又具有发光特性,是一种具有催化和发光双功能的团簇酶。这种稀土发光团簇酶能催化神经递质分子5-羟基色胺的反应,可发光显示5-羟基色胺的含量。
本发明还要解决的第二个技术问题是提供了一种容易合成稀土发光团簇酶的制备方法。
本发明还要解决的第三个技术问题是提供上述发光团簇酶在催化5-羟基色胺反应和测定5-羟基色胺含量方面的应用。
本发明最后要解决的第四个技术问题是将这种发光团簇酶作为一种可视化的发光指示剂,通过颜色的变化,快速鉴定5-羟基色胺。
发明内容:为解决上述第一个技术问题,本发明所采用的技术方案为:一种稀土发光团簇酶,所述稀土发光团簇酶是由铕离子、铜离子、1,10-菲啰啉在碱性的聚乙烯吡咯烷酮水溶液中被盐酸羟胺还原形成的铜团簇。
其中,所述的稀土发光团簇酶是粒径为2-5nm的纳米颗粒。
其中,所述的稀土发光团簇酶具有铜团簇的蓝光发射和铕离子的红光发射,是一种双重发光团簇酶。
其中,所述的稀土发光团簇酶中铜团簇具有催化和发光功能,催化反应敏化铕离子的发光,铕离子具有发光显示底物浓度的功能。
其中,所述的稀土发光团簇酶具有铜胺氧化酶的催化活性,可催化氧化神经递质分子5-羟基色胺的反应。
其中,所述的稀土发光团簇酶具有双重荧光指示功能,通过荧光能量转移敏化铕离子的发光,荧光从蓝色转变为红色快速指示5-羟基色胺的含量,荧光强度与5-羟基色胺的含量成正比。
为解决上述第二个技术问题,本发明所述的稀土发光团簇酶的合成步骤为:
1)将聚乙烯吡咯烷酮的水溶液与1,10-菲啰啉溶液混合,在激烈搅拌下加入氢氧化钠溶液和氯化铜溶液,搅拌后,逐滴加入盐酸羟胺溶液至溶液颜色不再变化,继续搅拌得到溶液;
2)在溶液中继续加入硝酸铕溶液,反应后离心分离,收集上清液即得。
作为本发明的实施例之一,具体的合成步骤为:
第一步将一定量的聚乙烯吡咯烷酮的水溶液与1,10-菲啰啉溶液混合,在激烈搅拌下加入氢氧化钠溶液和氯化铜溶液,搅拌30min后,逐滴加入盐酸羟胺溶液至溶液颜色不再变化,继续搅拌2h;
第二步在溶液中继续加入一定量的硝酸铕溶液,反应30min,离心分离,收集上清液,上清液在4℃下保存备用。
其中,步骤1)中所述氯化铜溶液中的铜离子与1,10-菲啰啉的摩尔比为2-5:10;
其中,所述合成第一步中的盐酸羟胺∶氢氧化钠的摩尔比为1∶1-1.5,1∶1较优。
其中,所述合成第一步中的聚乙烯吡咯烷酮的浓度为0.1-1mM,0.25mM较优,氢氧化钠的浓度为0.01-0.1M,0.05M较优;盐酸羟胺的浓度为0.01-0.1M,0.05M较优,溶液的pH值保持在6-10,pH 7-8较优,1,10-菲啰啉的溶液含20%的二甲基亚砜(DMSO) 或20%的乙醇。
其中,所述合成第二步中的铕离子∶铜离子∶1,10-菲啰啉的摩尔比为1∶2-5∶10。
为解决上述第三个技术问题,本发明提供了一种稀土发光团簇酶在催化5-羟基色胺反应和测定5-羟基色胺含量中的应用。具体地,
1)在催化5-羟基色胺反应中的应用:首先将一定量的稀土发光团簇酶溶液加入到含有5-羟基色胺的水溶液中,混合均匀,然后每隔几分钟后,测定铜团簇的荧光强度,铜团簇的荧光强度与5-羟基色胺降解的量成正比;
2)在分光光度法测定5-羟基色胺含量中的应用:将一定量的稀土发光团簇酶溶液加入到一系列已知浓度的标准5-羟基色胺溶液中,混合均匀,反应30min后,测定616 nm波长下铕离子的发光强度,绘制发光强度与标准5-羟基色胺浓度的工作曲线,通过测定5-羟基色胺样品在相同条件下的发光强度和工作曲线测得5-羟基色胺样品的含量
3)在比色法测定5-羟基色胺含量中的应用:将一定量的稀土发光团簇酶溶液加入到一系列已知浓度的标准5-羟基色胺溶液中,混合均匀,反应30min后,观察溶液在紫外灯下的荧光颜色,通过观察5-羟基色胺样品在相同条件下的荧光颜色,目视比色测定5- 羟基色胺样品的含量。
为解决上述第四个技术问题,本发明提供了发光团簇酶作为一种可视化的发光指示剂,具体地,将一定量的发光团簇酶溶液加入到含有5-羟基色胺的样品中,混合均匀,随着催化反应的进行,催化反应敏化铕离子的发光,发光团簇酶的荧光从蓝色逐渐变为红色,从而目视比色鉴定5-羟基色胺。
有益效果:与现有技术相比,本发明的稀土发光团簇酶具有以下优点:
1)本发明的稀土发光团簇酶是一种具有催化和发光双功能的人造酶,既具有酶的催化活性,又具有双色荧光发射的特性,无需其它试剂的参与,可直接催化氧化5-羟基色胺,并通过发光颜色指示5-羟基色胺的含量。
2)本发明的稀土发光团簇酶利用了稀土离子的发光,稀土离子长的荧光寿命允许通过时间分辨荧光技术消除各种非特异性荧光的干扰,具有高的信噪比,是一种超灵敏的发光检测技术。
3)本发明的稀土发光团簇酶的制备方法简单、成本低,无需复杂的有机合成反应。
4)本发明的稀土发光团簇酶具有超小的尺寸(2-5nm),容许通过大尺寸纳米酶不能通过的组织,在生物医学上具有广阔的应用前景。
附图说明
图1稀土发光团簇酶Eu-CuNCs的高分辨透射电镜图;
图2稀土发光团簇酶Eu-CuNCs催化5-羟基色胺反应不同时间的荧光光谱图;左边箭头从上到下是0min、30min、60min、90min、120min的荧光强度变化,右边箭头从下往上是0min、30min、60min、90min、120min的荧光强度变化。
图3 5-羟基色胺在稀土发光团簇酶Eu-CuNCs催化下的荧光光谱图;
图4稀土发光团簇酶Eu-CuNCs分光光度法检测5-羟基色胺;
图5稀土发光团簇酶Eu-CuNCs比色法检测5-羟基色胺。
具体实施方式
下面结合附图和实施例,对本发明的技术方案进行详细说明。
实施例1稀土发光团簇酶Eu-CuNCs的制备
Cu NCs制备:将0.02克聚乙烯吡咯烷酮(分子量4万)溶于2mL水中,与0.02 克1,10-菲啰啉溶于500μL二甲基亚砜溶液混合生成混合液,在激烈搅拌下向混合液中加入100μL 0.05M的氯化铜水溶液和100μL 0.35M的盐酸羟胺水溶液,搅拌30分钟后,将100μL 0.5M的氢氧化钠水溶液逐滴加入混合溶液中,持续搅拌由蓝色变为黄色,最后变为绿色至溶液颜色不变,加入10μL 0.1M的硝酸铕水溶液,以10000转/分钟速率离心10分钟,取上清液作为稀土发光团簇酶Eu-CuNCs溶液,理论浓度为0.3mM。图1为所制备的稀土发光团簇酶Eu-CuNCs的透射电镜图,Eu-CuNCs的尺寸为2-5nm。
实施例2稀土发光团簇酶Eu-CuNCs的制备
将0.02克聚乙烯吡咯烷酮(分子量4万)溶于2mL水中,与0.02克1,10-菲啰啉溶于500μL乙醇溶液混合生成混合液,在激烈搅拌下向混合液中加入200μL 0.05M的氯化铜水溶液和100μL 0.35M的盐酸羟胺水溶液,搅拌30分钟后,将100μL 0.5M的氢氧化钠水溶液逐滴加入混合溶液中,持续搅拌由蓝色变为黄色,最后变为绿色至溶液颜色不变,加入10μL0.1M的硝酸铕水溶液,以10000转/分钟速率离心10分钟,取上清液作为稀土发光团簇酶Eu-CuNCs溶液。
实施例3稀土发光团簇酶Eu-CuNCs的制备
将2mL水与0.02克1,10-菲啰啉溶于500μL乙醇溶液混合生成混合液,在激烈搅拌下向混合液中加入200μL 0.05M的氯化铜水溶液和100μL 0.35M的盐酸羟胺水溶液,搅拌30分钟后,将100μL 0.5M的氢氧化钠水溶液逐滴加入混合溶液中,持续搅拌由蓝色变为黄色,最后变为绿色至溶液颜色不变,加入10μL 0.1M的硝酸铕水溶液。以10000转/分钟速率离心10分钟,取上清液作为稀土发光团簇酶Eu-CuNCs溶液。
实施例4稀土发光团簇酶Eu-CuNCs催化5-羟基色胺(5-HT)反应
取0.5mL稀土发光团簇酶Eu-CuNCs溶液加入到1.5mL HEPES(N-2-羟乙基哌嗪 -N′-2-乙磺酸)缓冲溶液(10mM,pH 7.0)中,加入10μL 5-羟基色胺水溶液(5mM),混合均匀后放置30分钟,用荧光分光光度计测定溶液的荧光强度。图2是稀土发光团簇酶Eu-CuNCs催化5-HT反应不同时间(0-120min)的荧光光谱图,随着催化反应的进行,团簇酶Eu-CuNCs中的CuNCs在440nm的发射峰逐渐下降,Eu3+在590nm、616 nm、693nm的发射峰逐渐上升,说明催化反应敏化了Eu3+的发光。图3是在稀土发光团簇酶Eu-CuNCs存在下5-HT的荧光光谱图,随着催化反应的进行(0-2h)5-HT在336 nm的发射峰逐渐下降,说明5-HT被降解,同时反应过程中有气泡生成,能使加入的无色TMB(3,3’,5,5’-四甲基联苯胺)溶液变兰色(氧化型的TMB),说明生成H2O2。在生物体内,5-羟基色胺在铜胺氧化酶的作用下生成产物和H2O2,说明团簇Eu-CuNCs 具有铜胺氧化酶的活性。
实施例4稀土发光团簇酶Eu-CuNCs分光光度法测定5-羟基色胺
取0.5mL Eu-CuNCs溶液加到1.5mL HEPES缓冲溶液(10mM,pH 7.0)中,将 10μL不同浓度的5-HT溶液依次加入到上述缓冲液中配制成含有5-HT 0、0.1、0.5、1、2.5、5和10μM的标准溶液,混合均匀反应20分钟后,在360nm激发下测定Eu3+在 616nm波长处的荧光强度。图4是Eu3+荧光强度与5-HT浓度的关系图(工作曲线), Eu3+荧光强度与5-HT的浓度成线性正比关系,检测限低达10nM。根据5-HT样品溶液在相同条件下的荧光强度和工作曲线,可测定样品溶液中5-HT浓度。
实施例5稀土发光团簇酶Eu-CuNCs目视比色法测定5-羟基色胺
取0.5mL Eu-CuNCs溶液加到1.5mL HEPES缓冲溶液(10mM,pH 7.0)中,将 10μL不同浓度的5-HT溶液依次加入到上述缓冲液中配制成含有5-羟基色胺1、10和 50μM的混合溶液,混合均匀反应20分钟后,在波长为365nm的紫外灯下观察溶液的发光颜色并且拍照。图5是1、10和50μM 5-HT在稀土发光团簇酶Eu-CuNCs存在下的标准发光颜色图,检测限低达1μM。根据5-HT样品溶液在相同条件下的发光颜色和标准发光颜色图,可目视比色测定样品溶液中5-HT浓度。
实施例6稀土发光团簇酶Eu-CuNCs作为可视化的5-羟基色胺发光指示剂
取0.5mL Eu-CuNCs溶液加到1.5mL HEPES缓冲溶液(10mM,pH 7.0)中,将 10μL 5-HT样品溶液加入到上述缓冲液中,混合均匀反应20分钟后,在波长为365nm 的紫外灯下观察样品溶液能否从发射兰色荧光变成发射红色荧光,以鉴定样品溶液是否含有5-HT,红色荧光的强度与5-HT的量成正比。
Claims (10)
1.一种稀土发光团簇酶,其特征在于,所述稀土发光团簇酶是由铕离子、铜离子、1,10-菲啰啉在碱性的聚乙烯吡咯烷酮水溶液中被盐酸羟胺还原形成的铜团簇。
2.根据权利要求1所述的一种稀土发光团簇酶,其特征在于,所述稀土发光团簇酶的粒径为2-5 nm。
3.权利要求1或2所述的一种稀土发光团簇酶的制备方法,其特征在于,包括以下步骤:
1)将聚乙烯吡咯烷酮的水溶液与1,10-菲啰啉溶液混合,在激烈搅拌下加入氢氧化钠溶液和氯化铜溶液,搅拌后,逐滴加入盐酸羟胺溶液至溶液颜色不再变化,继续搅拌得到溶液;
2)在溶液中继续加入硝酸铕溶液,反应后离心分离,收集上清液即得。
4.根据权利要求3所述的一种稀土发光团簇酶的制备方法,其特征在于,步骤1)中所述氯化铜溶液中的铜离子与1,10-菲啰啉的摩尔比为2-5:10;所述盐酸羟胺溶液与氢氧化钠的摩尔比为1:1-1.5。
5.根据权利要求3所述的一种稀土发光团簇酶的制备方法,其特征在于,所述聚乙烯吡咯烷酮的浓度为0.1-1 mM,氢氧化钠的浓度为0.01-0.1 M;盐酸羟胺的浓度为0.01-0.1 M,溶液的pH值保持在6-10,1,10-菲啰啉的溶液含20%的二甲基亚砜或20%的乙醇。
6.根据权利要求3所述的一种稀土发光团簇酶的制备方法,其特征在于,步骤2)中加入的硝酸铕溶液中铕离子与溶液中的铜离子、1,10-菲啰啉的摩尔比为1:2-5:10。
7.权利要求1或2所述的一种稀土发光团簇酶在催化5-羟基色胺反应和测定5-羟基色胺含量中的应用。
8.根据权利要求7所述的一种稀土发光团簇酶在催化5-羟基色胺反应中的应用,其特征在于,所述催化5-羟基色胺反应的步骤如下:将稀土发光团簇酶溶液加入到含有5-羟基色胺的水溶液中,混合均匀即可。
9.根据权利要求7所述的一种稀土发光团簇酶在测定5-羟基色胺含量中的应用,其特征在于,所述测定5-羟基色胺含量采用目视比色法或分光光度法,所述目视比色法步骤如下:首先将稀土发光团簇酶溶液加入到一系列已知浓度的标准5-羟基色胺溶液中,混合均匀,反应完全后,观察溶液在紫外灯下的荧光颜色,通过观察5-羟基色胺样品在相同条件下的荧光颜色,目视比色测定5-羟基色胺样品的含量;所述分光光度法的步骤如下:首先将稀土发光团簇酶溶液加入到一系列已知浓度的标准5-羟基色胺溶液中,混合均匀,反应完全后,测定616 nm波长下铕离子的发光强度,绘制发光强度与标准5-羟基色胺浓度的工作曲线,通过测定5-羟基色胺样品在相同条件下的发光强度和工作曲线测得5-羟基色胺样品的含量。
10.权利要求1或2所述的一种稀土发光团簇酶作为一种可视化的发光指示剂的应用,其特征在于,将发光团簇酶溶液加入到含有5-羟基色胺的样品中,混合均匀,随着催化反应的进行,催化反应敏化铕离子的发光,发光团簇酶的荧光从蓝色逐渐变为红色,从而目视比色鉴定5-羟基色胺。
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