CN115287331A - 类酶活性铁掺杂硼量子点及其级联催化响应型谷氨酸荧光探针的制备方法 - Google Patents
类酶活性铁掺杂硼量子点及其级联催化响应型谷氨酸荧光探针的制备方法 Download PDFInfo
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Abstract
本发明公开了一种基于类酶活性的铁掺杂硼量子点及其级联催化响应型谷氨酸荧光探针的制备方法。采用超声辅助液相剥离和溶剂热处理,制得铁掺杂硼量子点Fe‑BQDs,将其作为具有类过氧化物酶活性的纳米酶参与级联催化反应,构建谷氨酸荧光探针。在Fe‑BQDs、罗丹明RhB和谷氨酸氧化酶GLOD混合体系中,加入的谷氨酸被GLOD催化生成过氧化氢H2O2,而H2O2被Fe‑BQDs催化产生羟基自由基·OH,而·OH与RhB反应引起RhB脱色和荧光淬灭。测定不同谷氨酸浓度所对应的混合体系荧光发射光谱,拟合谷氨酸浓度与荧光峰强度之间的线性关系,构建基于类酶活性铁掺杂硼量子点和级联催化反应的谷氨酸荧光探针。
Description
技术领域
本发明属于金属掺杂硼量子点和谷氨酸荧光探针的制备技术领域,具体涉及一种基于类过氧化物酶活性的铁掺杂硼量子点作为纳米酶参与级联催化反应所构建的谷氨酸荧光探针的制备方法,该制备的探针用于水样和生物流体中谷氨酸的高选择性和高灵敏性定量及荧光可视化检测。
背景技术
谷氨酸是一种酸性氨基酸,是生物体内氮代谢的基本氨基酸之一,其化学名为α-氨基戊二酸。谷氨酸广泛存在于谷类蛋白质中,在动物的脑组织中含量较高,在生物体内蛋白质代谢过程和许多机体化学反应中都起着重要作用。在食品工业,谷氨酸钠是味精主要成分,是重要鲜味剂,具有增香作用。在日用品上,谷氨酸用作生发剂和营养剂,能扩张血管,增强血液循环,对皮肤和毛发有保健作用。在医药行业,用于治疗肝性昏迷,改善儿童智力,降低及消除血氨,改善脑病症状。谷氨酸还用于下游产品的开发,如L-谷氨酸钠、聚谷氨酸等。
在生命医学领域,谷氨酸作为一种重要的兴奋性神经递质,对生物体中枢神经系统的功能活动、神经调节、发育、记忆等生命行为起着关键作用。在生理条件下,谷氨酸在人体脑组织中的浓度及分布可得到严格调控以维持稳定;而在病理条件下,谷氨酸转运体的再摄取受到抑制,需要通过大量的载体进行自身的转运和信号传导。在神经性疾病的发病过程中,谷氨酸在细胞外大量滞留,这过度激活了神经元上的谷氨酸受体,进而产生神经兴奋毒性,甚至引发神经元坏死。谷氨酸浓度升高预示了某些神经病变的可能,尤其是急性缺血性中风、癫痫、帕金森氏病、阿尔茨海默病等,故谷氨酸可作为神经性疾病的一种重要生物标志物。通过精准检测体内谷氨酸的浓度变化,可对神经疾病的进行及时监控、早期预防和诊断。
有关谷氨酸检测的研究工作已有文献和专利报道,如RumeiCheng等制备5-氨基荧光素改性的氧化石墨烯用作检测谷氨酸的荧光探针,谷氨酸引起了激发态5-氨基荧光素发生动态淬灭(Rumei Cheng,Lingling Cheng,Shengju Ou,A graphene oxide-basedfluorescent sensor for recognition of glutamate in aqueous solutions andbovine serum,Spectrochimica Acta Part A:Molecular and BiomolecularSpectroscopy,2019,221,117204)。崔元靖等制备了一种稀土-有机多孔材料,将稀土发光作为内标和有机分子发光作为检测信号,实现对神经疾病标记物谷氨酸的检测(崔元靖,夏体锋,杨雨,钱国栋.一种稀土-有机多孔材料及其制备方法和在神经疾病标记物谷氨酸检测中的应用.中国发明专利.申请公布号CN 108456218 A)。截止目前,尚未有关于采用类过氧化物酶活性的铁掺杂硼量子点作为纳米酶参与级联催化反应来构建谷氨酸荧光探针的国内外文献和专利报道。
发明内容
本发明之目的在于发展一种设计新颖和简单高效的基于类酶活性铁掺杂硼量子点及其级联催化响应型谷氨酸荧光探针的制备方法,该制备的探针用于水样和生物流体中谷氨酸的高选择性和高灵敏性定量及荧光可视化检测。
为实现上述目的,本发明涉及的一种基于类酶活性铁掺杂硼量子点及其级联催化响应型谷氨酸荧光探针,其制备方法具体包括以下步骤:
(1)称取10~50毫克硼粉末,加入到25~100毫升氮甲基吡咯烷酮溶剂中,在充分搅拌下形成硼粉末的均质分散液,然后滴加氯化铁水溶液形成混合液,调节其中硼和铁的浓度分别为0.1~2.0毫克/毫升和0.01~0.2毫克/毫升;
(2)采用探针超声处理上述混合液,在400~800瓦功率下先超声5秒后暂停10秒,重复这样先超声和后暂停的循环操作,整个循环操作过程维持60~120分钟;然后滴加5毫升蒸馏水稀释的0.1~0.5毫升聚乙二醇PEG-400,所得反应混合液在800~1200瓦功率下,采用浴池超声连续处理2~8小时;
(3)将浴池超声处理后的反应混合液,转入含100毫升聚四氟乙烯内衬微型磁力高压反应釜中,在氮气保护、100~200℃和搅拌下,反应混合液在釜中连续反应6~12小时;产物混合液冷却至室温,在3500转/分钟转速下离心10~30分钟,取上层液在12000转/分钟转速下离心5~15分钟,沉淀物经乙醇和蒸馏水洗涤2~4次,然后在60℃下真空干燥1小时,制得铁掺杂硼量子点Fe-BQDs;
(4)配制浓度为0.2~1.0毫克/毫升Fe-BQDs的乙醇和水混合分散液50毫升,在搅拌下依次加入10~50毫克罗丹明RhB和5~25毫克谷氨酸氧化酶GLOD以形成Fe-BQDs/RhB/GLOD混合体系探针;然后加入谷氨酸,调节谷氨酸在混合体系中的浓度,分别测定不同谷氨酸浓度所对应的混合体系荧光发射光谱,拟合荧光发射峰强度与谷氨酸浓度之间的线性关系,从而构建基于Fe-BQDs/RhB/GLOD混合体系的谷氨酸荧光探针;该探针对谷氨酸的线性检测范围为0~500毫克/升,检测限为0.05~1.0毫克/升;该探针用于水样和生物流体中谷氨酸的高选择性和高灵敏性定量及荧光可视化检测。
本发明的效果是公开了一种基于类酶活性铁掺杂硼量子点及其级联催化响应型谷氨酸荧光探针的制备方法。采用超声辅助的液相剥离和溶剂热处理方法,制得铁掺杂硼量子点Fe-BQDs,将其作为具有类过氧化物酶活性的纳米酶参与级联催化反应,构建检测谷氨酸的荧光探针。在Fe-BQDs、罗丹明RhB和谷氨酸氧化酶GLOD混合体系中,加入谷氨酸后,谷氨酸被GLOD催化生成过氧化氢H2O2,而H2O2被具有类过氧化物酶活性的Fe-BQDs催化产生羟基自由基·OH,而·OH与RhB反应引起RhB脱色反应和荧光淬灭。这说明目标物谷氨酸可引起Fe-BQDs/RhB/GLOD探针混合体系,发生级联催化反应,先是GLOD催化谷氨酸生成H2O2,再是Fe-BQDs催化H2O2生成·OH。基于此,针对该探针混合体系,测定不同谷氨酸存在浓度下所对应的荧光发射光谱,拟合谷氨酸浓度与荧光峰强度之间的线性关系,进而构建基于类酶活性的铁掺杂硼量子点和级联催化反应的谷氨酸荧光探针。
附图说明
图1是一种基于类酶活性铁掺杂硼量子点及其级联催化响应型谷氨酸荧光探针的制备方法及其检测谷氨酸的过程示意图。
具体实施方式
下面结合附图并通过具体实施例对本发明进行详细说明。
实施例1
本实施例涉及的一种基于类酶活性铁掺杂硼量子点及其级联催化响应型谷氨酸荧光探针的制备方法及其检测谷氨酸的过程如图1所示,具体制备步骤如下:
称取20毫克硼粉末,加入到30毫升氮甲基吡咯烷酮溶剂中,在充分搅拌下形成硼粉末的均质分散液,然后滴加氯化铁水溶液形成混合液,调节其中硼和铁的浓度分别为0.5毫克/毫升和0.05毫克/毫升。采用探针超声处理上述混合液,在780瓦功率下先超声5秒后暂停10秒,重复这样先超声和后暂停的循环操作,整个循环操作过程维持70分钟;然后滴加5毫升蒸馏水稀释的0.2毫升聚乙二醇PEG-400,所得反应混合液在1050瓦功率下,采用浴池超声连续处理3小时。将浴池超声处理后的反应混合液,转入含100毫升聚四氟乙烯内衬微型磁力高压反应釜中,在氮气保护、120℃和搅拌下,反应混合液在釜中连续反应7小时。产物混合液冷却至室温,在3500转/分钟转速下离心15分钟,取上层液在12000转/分钟转速下离心5分钟,沉淀物经乙醇和蒸馏水洗涤2次,然后在60℃下真空干燥1小时,制得铁掺杂硼量子点Fe-BQDs。配制浓度为0.4毫克/毫升Fe-BQDs的乙醇和水混合分散液50毫升,在搅拌下依次加入20毫克罗丹明RhB和10毫克谷氨酸氧化酶GLOD以形成Fe-BQDs/RhB/GLOD混合体系探针。加入谷氨酸,调节谷氨酸在混合体系中的浓度,分别测定不同谷氨酸浓度所对应的混合体系荧光发射光谱,拟合荧光发射峰强度与谷氨酸浓度之间的线性关系,从而构建基于Fe-BQDs/RhB/GLOD混合体系的谷氨酸荧光探针。该探针对谷氨酸检测的线性范围为0.1~100毫克/升,检测线为0.05毫克/升;该探针用于水样和生物流体中谷氨酸的高选择性和高灵敏性定量及荧光可视化检测。
实施例2
本实施例涉及的一种基于类酶活性铁掺杂硼量子点及其级联催化响应型谷氨酸荧光探针的制备方法及其检测谷氨酸的过程如图1所示,具体制备步骤如下:
称取30毫克硼粉末,加入到60毫升氮甲基吡咯烷酮溶剂中,在充分搅拌下形成硼粉末的均质分散液,然后滴加氯化铁水溶液形成混合液,调节其中硼和铁的浓度分别为1.0毫克/毫升和0.1毫克/毫升。采用探针超声处理上述混合液,在600瓦功率下先超声5秒后暂停10秒,重复这样先超声和后暂停的循环操作,整个循环操作过程维持90分钟;然后滴加5毫升蒸馏水稀释的0.3毫升聚乙二醇PEG-400,所得反应混合液在1000瓦功率下,采用浴池超声连续处理5小时。将浴池超声处理后的反应混合液,转入含100毫升聚四氟乙烯内衬微型磁力高压反应釜中,在氮气保护、150℃和搅拌下,反应混合液在釜中连续反应9小时。产物混合液冷却至室温,在3500转/分钟转速下离心20分钟,取上层液在12000转/分钟转速下离心10分钟,沉淀物经乙醇和蒸馏水洗涤3次,然后在60℃下真空干燥1小时,制得铁掺杂硼量子点Fe-BQDs。配制浓度为0.6毫克/毫升Fe-BQDs的乙醇和水混合分散液50毫升,在搅拌下依次加入30毫克罗丹明RhB和15毫克谷氨酸氧化酶GLOD以形成Fe-BQDs/RhB/GLOD混合体系探针。加入谷氨酸,调节谷氨酸在混合体系中的浓度,分别测定不同谷氨酸浓度所对应的混合体系荧光发射光谱,拟合荧光发射峰强度与谷氨酸浓度之间的线性关系,从而构建基于Fe-BQDs/RhB/GLOD混合体系的谷氨酸荧光探针。该探针对谷氨酸检测的线性范围为0.5~200毫克/升,检测线为0.2毫克/升;该探针用于水样和生物流体中谷氨酸的高选择性和高灵敏性定量及荧光可视化检测。
实施例3
本实施例涉及的一种基于类酶活性铁掺杂硼量子点及其级联催化响应型谷氨酸荧光探针的制备方法及其检测谷氨酸的过程如图1所示,具体制备步骤如下:
称取40毫克硼粉末,加入到90毫升氮甲基吡咯烷酮溶剂中,在充分搅拌下形成硼粉末的均质分散液,然后滴加氯化铁水溶液形成混合液,调节其中硼和铁的浓度分别为1.5毫克/毫升和0.15毫克/毫升。采用探针超声处理上述混合液,在700瓦功率下先超声5秒后暂停10秒,重复这样先超声和后暂停的循环操作,整个循环操作过程维持110分钟;然后滴加5毫升蒸馏水稀释的0.4毫升聚乙二醇PEG-400,所得反应混合液在1100瓦功率下,采用浴池超声连续处理7小时。将浴池超声处理后的反应混合液,转入含100毫升聚四氟乙烯内衬微型磁力高压反应釜中,在氮气保护、180℃和搅拌下,反应混合液在釜中连续反应11小时。产物混合液冷却至室温,在3500转/分钟转速下离心25分钟,取上层液在12000转/分钟转速下离心15分钟,沉淀物经乙醇和蒸馏水洗涤4次,然后在60℃下真空干燥1小时,制得铁掺杂硼量子点Fe-BQDs。配制浓度为0.8毫克/毫升Fe-BQDs的乙醇和水混合分散液50毫升,在搅拌下依次加入40毫克罗丹明RhB和20毫克谷氨酸氧化酶GLOD以形成Fe-BQDs/RhB/GLOD混合体系探针。加入谷氨酸,调节谷氨酸在混合体系中的浓度,分别测定不同谷氨酸浓度所对应的混合体系荧光发射光谱,拟合荧光发射峰强度与谷氨酸浓度之间的线性关系,从而构建基于Fe-BQDs/RhB/GLOD混合体系的谷氨酸荧光探针。该探针对谷氨酸检测的线性范围为1~500毫克/升,检测线为0.5毫克/升;该探针用于水样和生物流体中谷氨酸的高选择性和高灵敏性定量及荧光可视化检测。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (1)
1.类酶活性铁掺杂硼量子点及其级联催化响应型谷氨酸荧光探针的制备方法,其特征在于,该制备方法具体包括以下步骤:
(1)称取10~50毫克硼粉末,加入到25~100毫升氮甲基吡咯烷酮溶剂中,在充分搅拌下形成硼粉末的均质分散液,然后滴加氯化铁水溶液形成混合液,调节其中硼和铁的浓度分别为0.1~2.0毫克/毫升和0.01~0.2毫克/毫升;
(2)采用探针超声处理上述混合液,在400~800瓦功率下先超声5秒后暂停10秒,重复这样先超声和后暂停的循环操作,整个循环操作过程维持60~120分钟;然后滴加5毫升蒸馏水稀释的0.1~0.5毫升聚乙二醇PEG-400,所得反应混合液在800~1200瓦功率下,采用浴池超声连续处理2~8小时;
(3)将浴池超声处理后的反应混合液,转入含100毫升聚四氟乙烯内衬微型磁力高压反应釜中,在氮气保护、100~200℃和搅拌下,反应混合液在釜中连续反应6~12小时;产物混合液冷却至室温,在3500转/分钟转速下离心10~30分钟,取上层液在12000转/分钟转速下离心5~15分钟,沉淀物经乙醇和蒸馏水洗涤2~4次,然后在60℃下真空干燥1小时,制得铁掺杂硼量子点Fe-BQDs;
(4)配制浓度为0.2~1.0毫克/毫升Fe-BQDs的乙醇和水混合分散液50毫升,在搅拌下依次加入10~50毫克罗丹明RhB和5~25毫克谷氨酸氧化酶GLOD以形成Fe-BQDs/RhB/GLOD混合体系探针;然后加入谷氨酸,调节谷氨酸在混合体系中的浓度,分别测定不同谷氨酸浓度所对应的混合体系荧光发射光谱,拟合荧光发射峰强度与谷氨酸浓度之间的线性关系,从而构建基于Fe-BQDs/RhB/GLOD混合体系的谷氨酸荧光探针;该探针对谷氨酸的线性检测范围为0~500毫克/升,检测限为0.05~1.0毫克/升;该探针用于水样和生物流体中谷氨酸的高选择性和高灵敏性定量及荧光可视化检测。
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