CN113528117A - 一种功能化金纳米簇比率型多巴胺荧光探针及其制备方法 - Google Patents
一种功能化金纳米簇比率型多巴胺荧光探针及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种功能化金纳米簇比率型多巴胺荧光探针及其制备方法。所述制备方法为:将4‑(((2,5‑二氧吡咯烷‑1‑基)氧基)羰基苯硼酸与儿茶素进行酯化反应,反应产物通过与谷胱甘肽的氨基酰化反应修饰在金纳米簇表面,即得到功能化金纳米簇比率型多巴胺荧光探针。本发明制备的功能化金纳米簇比率型多巴胺荧光探针中,金纳米簇于560nm处有一个荧光发射峰,通过修饰后的配体中引入了间二苯酚结构,会与多巴胺特异性反应产生一新的461nm处的荧光发射,从而形成比率型荧光发射。本发明的荧光探针,具有良好的水溶性和生物相容性,以及高稳定性、高选择性和高灵敏度,并且制备方法简单,原料易得,产率高,可投入大规模生产。
Description
技术领域
本发明属于荧光探针制备技术领域,特别涉及一种功能化金纳米簇比率型多巴胺荧光探针及其制备方法。
背景技术
多巴胺(DA)作为人体当中一种重要的神经递质,影响着人体的思考、工作、运动等行为,多巴胺的异常含量变化昭示着精神疾病的发生。目前对多巴胺的检测方法主要有电化学分析、高效液相色谱法、毛细管电泳法、荧光光谱法等。高效液相色谱法虽在分离中有明显的优势,但分析成本高、分析时间长,毛细管电泳法分离时间短、成本低,但分离能力较弱、重现性差。电化学分析方法操作简便、灵敏度高,但选择性低,重复性和稳定性差。荧光光谱法的特异性、生物相容性、高时空分辨率的优点弥补了其他方法的低时空分辨率、仪器昂贵的局限性,但仍存在聚集荧光淬灭的局限性需要解决。而且多巴胺荧光检测方法多以单一荧光强度作为指标,存在一定局限性,因此,灵敏且有选择性地检测多巴胺具有重要的意义。
发明内容
为了灵敏、特异性地检测多巴胺,本发明提供一种功能化金纳米簇比率型多巴胺荧光探针及其制备方法。该方法将间二苯酚结构通过配体修饰的方法引入金纳米簇,合成了具有良好水溶性的金纳米簇探针。制得的荧光探针尺寸分布在2-5nm,分散性良好。该荧光探针利用多巴胺与间二苯酚结构所引起的荧光发射的变化,从而在原有金纳米簇橙黄色荧光发射之上产生一个新的蓝色荧光发射。即经过配体修饰的金纳米簇探针不仅具有金纳米簇的荧光性质,同时还具有间二苯酚的功能性。
本发明所述的功能化金纳米簇比率型多巴胺荧光探针的制备方法为:将4-(((2,5-二氧吡咯烷-1-基)氧基)羰基苯硼酸与儿茶素进行酯化反应,反应产物通过与谷胱甘肽的氨基酰化反应修饰在金纳米簇表面,即得到功能化金纳米簇比率型多巴胺荧光探针。
所述的酯化反应的条件为:将4-(((2,5-二氧吡咯烷-1-基)氧基)羰基苯硼酸和儿茶素分别溶解于醇溶剂中,然后将配制的两种醇溶液和缓冲溶液及水混合,得到的混合溶液的pH为9.0-11.0,于振荡器中反应0.5-4h。
所述的醇溶剂选自甲醇、乙醇、丙醇中的一种或几种。
所述的金纳米簇的合成方法为:取谷胱甘肽水溶液和氯金酸水溶液混合均匀,于60-80℃条件下搅拌反应20-24小时后,在无水乙醇或乙腈中沉淀,离心纯化后分散于pH=7-8的PBS缓冲溶液中,得到金纳米簇分散液。
所述的酰化反应的条件为:将4-(((2,5-二氧吡咯烷-1-基)氧基)羰基苯硼酸与儿茶素的酯化反应产物和金纳米簇分散液混合,用氢氧化钠水溶液或碳酸钠水溶液调节溶液pH为8-10,搅拌反应8-15h后置于3KD截止超滤管离心超滤纯化,最后加入超纯水得到功能化金纳米簇比率型多巴胺荧光探针分散液。
所述的功能化金纳米簇比率型多巴胺荧光探针的使用方法为:在缓冲溶液中加入功能化金纳米簇比率型多巴胺荧光探针分散液,然后加入待测液,得到的混合溶液的pH值为9-11,常温下反应30-60min,用荧光分光光度计测定荧光发射强度的变化,以400-440nm为激发波长,得到比率型荧光。
所述的缓冲溶液为碳酸钠-碳酸氢钠缓冲溶液、硼砂-氢氧化钠缓冲溶液、甘氨酸-氢氧化钠缓冲溶液或硼酸-硼砂缓冲溶液。
本发明制备的功能化金纳米簇比率型多巴胺荧光探针中,金纳米簇于560nm处有一个荧光发射峰,通过修饰后的配体中引入了间二苯酚结构,会与多巴胺特异性反应产生一新的461nm处的荧光发射,从而形成比率型荧光发射。本发明的荧光探针采用谷胱甘肽(GSH)作为配体的金纳米簇(AuNCs)具有稳定的化学性质,并且具备聚集诱导发光效应,能够有效弥补聚集荧光淬灭探针的缺陷。本发明的荧光探针检出限可达1nM,同时在阳离子、糖类、氨基酸类物质中均无特异性荧光响应,具有良好的选择性。本发明的荧光探针,具有良好的水溶性和生物相容性,以及高稳定性、高选择性和高灵敏度,并且制备方法简单,原料易得,产率高,可投入大规模生产。
附图说明
图1为实施例1中,在DA存在下,BE、儿茶素、BE-儿茶素的荧光发射光谱。
图2为实施例1中,在DA存在下,金纳米簇、功能化金纳米簇比率型多巴胺荧光探针的荧光发射光谱。
图3为实施例1中,BE-儿茶素、金纳米簇、功能化金纳米簇比率型多巴胺荧光探针的紫外吸收光谱。
图4为实施例1中,金纳米簇、功能化金纳米簇比率型多巴胺荧光探针的红外光谱。
图5为实施例1中,金纳米簇、功能化金纳米簇比率型多巴胺荧光探针的X射线光电子能谱。
图6为应用例1中,功能化金纳米簇比率型多巴胺荧光探针在不同浓度多巴胺存在下的荧光变化线以及荧光比值图。
图7为应用例2中,功能化金纳米簇比率型多巴胺荧光探针用于多巴胺及干扰物检测的荧光比值图。
具体实施方式
为了更清楚的说明本发明,列举如下实施例,但本发明的保护范围并不局限于以下实施例。
实施例1
(1)4-(((2,5-二氧吡咯烷-1-基)氧基)羰基苯硼酸-儿茶素反应物合成步骤:分别将4-(((2,5-二氧吡咯烷-1-基)氧基)羰基苯硼酸(BE)和儿茶素溶解于无水乙醇中,配制得到0.5mL 10mM BE的乙醇溶液及0.25mL 20mM儿茶素乙醇溶液,将两种醇溶液与0.2mL 50mMpH=10碳酸钠-碳酸氢钠缓冲溶液及0.05mL超纯水混合,于振荡器中反应0.5h,无需进一步纯化,得到BE-儿茶素反应产物。反应式如下:
(2)金纳米簇的合成:取5mL浓度为0.1M的谷胱甘肽溶液和5mL浓度为0.05M的氯金酸溶液混合均匀,于70℃条件下搅拌反应24小时后,在无水乙醇中沉淀后离心纯化,分散于10mL 10mM pH=7.4的PBS缓冲溶液中,得到金纳米簇分散液,记为GSH-Au NCs;
(3)荧光探针的合成:取0.5mL步骤(1)的产物与5mL 步骤(2)制得的GSH-Au NCs于反应瓶中均匀混合,用1M氢氧化钠水溶液调节溶液pH为8.0,搅拌反应12h,所得溶液置于3KD的截止超滤管离心超滤,以除去未反应的BE-儿茶素反应物。超滤所得液体加入超纯水,使终体积为5mL,即得功能化金纳米簇比率型多巴胺荧光探针分散液。
BE-儿茶素特异性反应的可行性研究:通过荧光光谱,测定BE、儿茶素及两者反应产物的荧光光谱,均没有荧光发射。在1μM多巴胺的存在下,如图1所示,在410nm激发波长下,儿茶素及反应产物于461nm处产生间二苯酚类与多巴胺特异性产物荧光,而在BE溶液中仍无荧光。证明特异性荧光反应的可行性,且BE的加入对该反应不产生影响。
BE-儿茶素特异性反应引入金纳米簇的可行性研究:通过荧光光谱,在410nm激发波长下,测定Au NCs、探针的荧光光谱,两者均在560nm处有荧光发射,且强度峰型无明显变化,证明BE-儿茶素反应物的引入对金纳米簇的发光没有影响。在1μM多巴胺的存在下,如图2所示,在410nm激发波长下,探针在461nm处产生新发射,形成比率型荧光,证明特异性荧光反应引入金纳米簇形成比率型探针的可行性,且金纳米簇的引入对该反应不产生影响。
BE-儿茶素特异性反应引入金纳米簇的可行性研究:为了证明BE-儿茶素反应物成功的修饰到了金纳米簇的配体上,测试了BE-儿茶素反应物、金纳米簇及探针的紫外吸收。如图3所示,探针的紫外吸收保留了金纳米簇原有的特征峰,并且新出现了BE-儿茶素反应物的苯环特征峰,证明BE-儿茶素的修饰成功。
荧光探针的红外表征:为了进一步证明BE-儿茶素反应物成功的修饰到了金纳米簇的配体上,测试了金纳米簇及探针的红外光谱。如图4所示,探针的红外光谱特征峰保留了金纳米簇的特征峰,并且新出现了1630、1538cm-1处苯环的骨架振动特征峰,以及1078cm-1处C–B键伸缩振动,1359cm-1处O-B键拉伸振动峰,1260cm-1处的芳醚伸缩振动峰,860cm-1处1,2,3,5四取代苯环弯曲振动峰等,证明BE-儿茶素的修饰成功。
荧光探针的x射线光电子能谱表征:为了进一步证明BE-儿茶素反应物成功的修饰到了金纳米簇的配体上,测试了金纳米簇及探针的x射线光电子能谱(XPS)。如图5所示,与金纳米簇相比,探针的光电子能谱中C1s和O1s的特征峰增强,且出现了B1s特征峰,证明BE-儿茶素的修饰成功。
应用例1
荧光探针对不同浓度多巴胺的检测:在0.2mL碳酸钠-碳酸氢钠缓冲溶液(pH=10,50mM)中,加入100μL实施例1的荧光探针分散液;然后分别加入多巴胺使得溶液中多巴胺的终浓度分别为0nM、1nM、2.5nM、5nM、10nM、25nM、50nM、80nM、100nM、150nM、200nM、250nM、500nM、1000nM、2500nM,加入超纯水保持溶液终体积为1mL。在900rpm条件于振荡器中25℃反应30min,然后在激发波长410nm下,用荧光分光光度计测定在461nm和560nm下的荧光发射强度的变化。如图6所示,由荧光测试结果可以看出,本发明的金纳米簇探针对多巴胺有很好的比率型响应,且检出限可达1nM。
应用例2
荧光探针的选择性测试:与应用例1的测试条件相同,在0.2mL碳酸钠-碳酸氢钠缓冲溶液中(pH=10,50mM)中,加入100μL实施例1的荧光探针分散液,然后向溶液中加入0.1mL 100μM氯化镁(MgCl2)水溶液,加入多巴胺,最后加入超纯水使溶液终体积为1mL,多巴胺(DA)的终浓度为1μM。以相同的方法分别加入氯化锌(ZnCl2)、L-丙氨酸(Ala)、精氨酸(Arg)、甘氨酸(Gly)、组氨酸(His)、蛋氨酸(Met)、脯氨酸(Pro)、丝氨酸(Ser),苏氨酸(Thr)、酪氨酸(Tyr)、缬氨酸(Val)、抗坏血酸(VC)、尿酸(UA)、蔗糖(sucrose)、葡萄糖(glucose)替换氯化镁(MgCl2)配制不同干扰溶液。在900rpm条件于振荡器中25℃反应30min,然后在激发波长410nm下,用荧光分光光度计测定在461nm和560nm下的荧光发射强度的变化。如图7所示,由结果可以看出,除DA外,其他金属离子和有机生物小分子均无明显比率型荧光产生,说明本发明制备的金纳米簇荧光探针对多巴胺具有较高的选择性。
Claims (8)
1.一种功能化金纳米簇比率型多巴胺荧光探针的制备方法,其特征在于,所述制备方法的具体操作为:将4-(((2,5-二氧吡咯烷-1-基)氧基)羰基苯硼酸与儿茶素进行酯化反应,反应产物通过与谷胱甘肽的氨基酰化反应修饰在金纳米簇表面,即得到功能化金纳米簇比率型多巴胺荧光探针。
2.根据权利要求1所述的制备方法,其特征在于,所述的酯化反应的条件为:将4-(((2,5-二氧吡咯烷-1-基)氧基)羰基苯硼酸和儿茶素分别溶解于醇溶剂中,然后将配制的两种醇溶液和缓冲溶液及水混合,得到的混合溶液的pH为9.0-11.0,于振荡器中反应0.5-4h。
3.根据权利要求2所述的制备方法,其特征在于,所述的醇溶剂选自甲醇、乙醇、丙醇中的一种或几种。
4.根据权利要求2所述的制备方法,其特征在于,所述的缓冲溶液为碳酸钠-碳酸氢钠缓冲溶液、硼砂-氢氧化钠缓冲溶液、甘氨酸-氢氧化钠缓冲溶液或硼酸-硼砂缓冲溶液。
5.根据权利要求1所述的制备方法,其特征在于,所述的金纳米簇的合成方法为:取谷胱甘肽水溶液和氯金酸水溶液混合均匀,于60-80℃条件下搅拌反应20-24小时后,在无水乙醇或乙腈中沉淀,离心纯化后分散于pH=7-8的PBS缓冲溶液中,得到金纳米簇分散液。
6.根据权利要求1所述的制备方法,其特征在于,所述的酰化反应的条件为:将4-(((2,5-二氧吡咯烷-1-基)氧基)羰基苯硼酸与儿茶素的酯化反应产物和金纳米簇分散液混合,用氢氧化钠水溶液或碳酸钠水溶液调节溶液pH为8-10,搅拌反应8-15h后置于3KD截止超滤管离心超滤纯化,最后加入超纯水得到功能化金纳米簇比率型多巴胺荧光探针分散液。
7.根据权利要求1-6任意一项所述的方法制备得到的功能化金纳米簇比率型多巴胺荧光探针的使用方法,其特征在于所述使用方法的具体操作为:在缓冲溶液中加入功能化金纳米簇比率型多巴胺荧光探针分散液,然后加入待测液,得到的混合溶液的pH值为9-11,常温下反应30-60min,用荧光分光光度计测定荧光发射强度的变化,以400-440nm为激发波长,得到比率型荧光。
8.根据权利要求7所述的使用方法,其特征在于,所述的缓冲溶液为碳酸钠-碳酸氢钠缓冲溶液、硼砂-氢氧化钠缓冲溶液、甘氨酸-氢氧化钠缓冲溶液或硼酸-硼砂缓冲溶液。
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