CN107163068A - 一种新型荧光探针及其制备方法和在检测尿酸中的应用 - Google Patents
一种新型荧光探针及其制备方法和在检测尿酸中的应用 Download PDFInfo
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Abstract
本发明公开了一种新型荧光探针及其制备方法和在检测尿酸中的应用。取二乙三胺五乙酸,乙酸酐,吡啶,在65℃下搅拌加热24h,冷却,过滤,洗涤,干燥,得到的二乙三胺五乙酸二酐与三乙胺,DMF,2,6‑二氨基嘌呤,混合均匀,恒温100℃加热搅拌24h,冷却,过滤,干燥,得到的二乙三胺五乙酸‑双(2,6‑二氨基嘌呤)溶解于pH=7.4的Tris‑HCl缓冲溶液中,与Tb(NO3)3·6H2O混合,过滤,滤液加热或长时间放置,得目标产物。将TbШ‑dtpa‑bis(2,6‑Diaminopurine)作为探针结合荧光方法检测尿酸。本发明方法简单新颖,效率高,成本低,且可应用在实际尿样当中。
Description
技术领域
本发明属于分析化学领域,尤其涉及新型荧光探针的合成及其对尿酸的检测。
背景技术
尿酸在人体中是嘌呤的最终代谢产物,在人体尿液以及血液中,尿酸含量的检测都是非常重要的指标。通常而言,在人体尿液中尿酸的正常浓度应该为1.49-4.46mmol/L,通过各种生物过程维持自身平衡。当尿酸的生成量和排泄量不平衡时,其尿酸的氧化产物嘌呤碱就会发生代谢紊乱,从而导致高尿酸症、痛风、Lesch-Nyhan综合症,如肾脏损伤、白血病、高血压、糖尿病以及高尿酸症等等。白血病患者在1d内尿酸最大排泄量可达12g,而正常人1d的尿酸排泄量只有600mg左右。更加可怕的是,最近一些报道证实,高尿酸血症与妊娠高血压疾病相关联,将导致孕妇的死亡。由于缺乏运动,不合理的饮食以及药物的滥用,导致尿酸含量在体内不断升高。高尿酸症在中国已经引起了很高的重视。高尿酸症是即高血压、高血脂、高血糖之后被称为“第四高”的疾病。并且高尿酸症是与高血压、高血脂、高血糖等密切相关的代谢综合征,近年来,此病的发病率呈上升趋势。因此对尿酸的检测是至关重要的。
荧光探针就是以荧光物质作为指示剂,并在一定波长光的激发下使指示剂产生荧光,通过检测所产生的荧光实现对被检测物质的定性或者定量分析。荧光探针具有灵敏度高,选择性好,使用方便,成本低,不需预处理,不受外界干扰等优点。特别是在分子生物学、生物化学、医学等领域中有较广泛的应用。
发明内容
本发明的目的在于设计合成一种可用于有效检测尿液当中的尿酸的新型荧光探针TbIII-dtpa-bis(2,6-Diaminopurine)。本发明所涉及化合物属于新型荧光探针,将其应用于检测尿酸操作简单,成本低,无污染,且选择性好。
本发明采用的技术方案是:一种新型荧光探针,所述的新型荧光探针是TbIII-dtpa-bis(2,6-Diaminopurine)。
上述的新型荧光探针的制备方法,方法如下:
1)二乙三胺五乙酸(dtpa)、乙酸酐和吡啶,混合均匀,在60-70℃下,搅拌加热22-25h,冷却至室温,过滤,用乙酸酐和无水乙醚洗涤,抽滤,干燥,得二乙三胺五乙酸二酐(dtpaa);
2)二乙三胺五乙酸二酐(dtpaa)、三乙胺、无水DMF和2,6-二氨基嘌呤,混合均匀,于95-105℃下,搅拌加热22-25h,静置,冷却到室温,过滤,真空干燥,得二乙三胺五乙酸-双(2,6-二氨基嘌呤)(dtpa-bis(2,6-Diaminopurine));
3)将二乙三胺五乙酸-双(2,6-二氨基嘌呤)用pH=7.4的Tris-HCl缓冲溶液溶解,得dtpa-bis(2,6-Diaminopurine)溶液,与Tb(NO3)3·6H2O混合,过滤,滤液于70-80℃下加热20-30min或于室温下放置1-2天,得TbIII-dtpa-bis(2,6-Diaminopurine)。
优选的,上述的新型荧光探针的制备方法,步骤1),用于反应过程的乙酸酐的加入量为,按摩尔比,二乙三胺五乙酸:乙酸酐:吡啶=1:4:6。
优选的,上述的新型荧光探针的制备方法,按摩尔比,二乙三胺五乙酸二酐:三乙胺:2,6-二氨基嘌呤=1:3:2。
优选的,上述的新型荧光探针的制备方法,按质量比,二乙三胺五乙酸-双(2,6-二氨基嘌呤):Tb(NO3)3·6H2O=1:0.8-0.85。
本发明的新型荧光探针可应用于检测尿样中的尿酸。定性分析方法如下:取尿液,加入上述的新型荧光探针TbШ-dtpa-bis(2,6-Diaminopurine)的pH=7.4的Tris-HCl缓冲溶液,搅拌均匀,在241nm的激发波长下观察荧光光谱的变化。
定量分析方法如下:取尿液0.5mL于50mL容量瓶中,加入5mL浓度为5.0×10-4mol/L的上述新型荧光探针TbШ-dtpa-bis(2,6-Diaminopurine)的pH=7.4的Tris-HCl缓冲溶液,用Tris-HCl缓冲溶液定容,在241nm的激发波长下观察荧光光谱的变化。
本发明的有益效果是:
1.本发明,对dtpa进行了修饰,在dtpa两端接上2,6-二氨基嘌呤,由于结构的相似性,通过氢键和π-π堆积以及配位键的作用抓取目标物,从而达到检测尿酸的目的。
2.本发明,针对尿酸的结构特点,设计了一种新型的荧光探针。通过本发明的方法,该探针可以对尿酸进行特异性检测并且应用在实际尿样中。与其它检测尿酸的荧光探针相比,具有简单,快速,成本低等特点。
附图说明
图1是荧光探针TbIII-dtpa-bis(2,6-Diaminopurine)的合成反应的流程图。
图2a是dtpa的傅里叶变换红外光谱(FT-IR)图。
图2b是dtpaa的傅里叶变换红外光谱(FT-IR)图。
图2c是2,6-二氨基嘌呤的傅里叶变换红外光谱(FT-IR)图。
图2d是dtpa-bis(2,6-Diaminopurine)的傅里叶变换红外光谱(FT-IR)图。
图3是Tb3+,TbIII-dtpa-bis(2,6-Diaminopurine)(Tb-dtpa-Bdap)的紫外吸收光谱图。
图4a是荧光探针对尿酸检测的荧光光谱图。
图4b是荧光探针对尿酸检测的荧光光谱对比柱状图(350nm)。
图5是荧光探针对尿酸分别与不同物质混合的干扰荧光光谱对比图。
图6是荧光探针对真实尿样中尿酸检测的荧光光谱对比图。
具体实施方式
实施例1新型荧光探针TbIII-dtpa-bis(2,6-Diaminopurine)
(一)制备方法
1、二乙三胺五乙酸二酐(dtpaa)的制备
称取dtpa 7.8100g(0.02mmol),乙酸酐16.0mL(0.08mmol),吡啶10.0mL(0.12mmol)置于三颈圆底烧瓶中,混合均匀,在65℃下,搅拌加热24h。冷却至室温,将反应混合物过滤,并用少量乙酸酐和无水乙醚洗涤两次,用真空泵抽滤,所得物于真空干燥箱中80℃真空干燥,即得dtpaa。
2、二乙三胺五乙酸双2,6-二氨基嘌呤(dtpa-bis(2,6-Diaminopurine))的制备
取1.9610g的dtpaa(5.5mmol),三乙胺8.0mL(16.5mmol),无水DMF(50mL),2,6-二氨基嘌呤1.6515g(11mmol),于三颈圆底烧瓶,混合均匀。恒温100℃加热,快速搅拌24h。反应完全后静置,冷却到室温后,得浅黄色固体物质,过滤,50℃真空干燥,即得dtpa-bis(2,6-Diaminopurine)。
3、荧光探针TbIII-dtpa-bis(2,6-Diaminopurine)的制备
称取0.0677g的dtpa-bis(2,6-Diaminopurine)于200.0mL的pH=7.4的Tris-HCl缓冲溶液中溶解。称取0.0566g的Tb(NO3)3·6H2O置于烧杯中,然后用上面配制的dtpa-bis(2,6-Diaminopurine)溶液溶解,过滤,滤液移入250mL容量瓶中,用pH=7.4的Tris-HCl缓冲溶液定容。把容量瓶中的溶液加热或长时间放置,形成TbIII-dtpa-bis(2,6-Diaminopurine),此时浓度为5.0×10-4mol/L,作为荧光探针储备液。合成过程如图1所示。
(二)检测
1.Dtpa,dtpaa,2,6-Diaminopurine(dap),dtpa-bis(2,6-Diaminopurine)(dtpa-Bdap)的FT-IR图如图2a,图2b,图2c和图2d所示。对比发现,在图2a和图2b中,1821cm-1和1774cm-1的峰归于C=O,1118cm-1的峰吸收峰来自于C-O,2979cm-1的峰来自于-CH2-CH2-,通过这个比较可以确定dtpaa已经合成。图2d展示了dtpa-Bdap的红外光谱图,通过与图2a和图2c比较可以观察到,图2d没有出现1774cm-1-1821cm-1的酸酐的吸收峰而新的吸收峰出现在1630cm-1处。1630cm-1和3108cm-1是源于C=O和N-H的震动。此外,在1210cm-1处出现了C-N的震动吸收峰,由此可以确定dtpa-Bdap已经合成。值得注意的是,图2c和图2d相比较,937cm-1处-NH2的峰没有发生位移,这说明,dtpa-Bdap的合成仅仅占用了dap的一个-NH2,还有一个-NH2被保留下来,这为以后的研究奠定了理论基础。
2.稀土Tb3+,TbIII-dtpa-bis(2,6-Diaminopurine)(Tb-dtpa-Bdap)的紫外吸收光谱图如图3所示。由紫外吸收光谱可以看出,Tb3+的溶液在225nm-250nm处几乎没有吸收峰,而Tb-dtpa-Bdap有两个吸收峰分别在241nm和281nm处。这两个峰与Tb3+相比较,明显增强。说明dtpa-Bdap可以形成新的配位场使Tb3+电子结构发生变化。这表明Tb-dtpa-Bdap具有作为荧光探针检测尿酸的潜在能力。
实施例2荧光探针TbIII-dtpa-bis(2,6-Diaminopurine)在检测尿酸中的应用
1.荧光探针尿酸检测的荧光光谱
实验条件:取一定量的尿酸(UV)用pH=7.4的Tris-HCl缓冲液配制成浓度为5.0×10-4mol/L的溶液,作为尿酸储备液。
分别取5.0mL尿酸储备液于50mL容量瓶中,再分别加入实施例1制备的5.0mL的荧光探针储备液,用pH=7.4的Tris-HCl缓冲液定容。此时探针及各检测物质浓度都为5.0×10-5mol/L。在241nm的激发波长下观察荧光光谱的变化。
结果如图4a、图4b所示。如图4a所示,在241nm激发下,荧光探针在350nm处有较强发射峰。而尿酸(UV)在350nm处几乎没有荧光。当把尿酸加入到探针溶液时,荧光强度在350nm处被明显猝灭。在图4b中这个现象更加能够直观的看到在350nm处荧光强度的变化。
2.尿酸与不同检测物混合对荧光探针TbIII-dtpa-bis(2,6-Diaminopurine)检测的影响
实验条件:分别取浓度为5.0×10-4mol/L的肌酸酐,抗坏血酸,酪氨酸,马尿酸,色氨酸,组氨酸储备液5.0mL于50mL容量瓶中,分别加入5.0mL的尿酸储备液,再分别加入5.0mL的荧光探针储备液,用pH=7.4的Tris-HCl缓冲液定容。此时探针,尿酸,各检测物质浓度都为5.0×10-5mol/L的溶液。在241nm的激发波长下观察荧光光谱的变化。
结果如图5所示。如图5,在350nm处除了色氨酸与尿酸的混合液对荧光探针的检测有一定的影响,使荧光强度升高外,其他混合物质对探针检测几乎没有影响。但是根据调查结果显示,色氨酸在尿液中的量为2.3×10-5mol/L–1.49×10-4mol/L,而尿酸在尿液中的量为1.49×10-3mol/L–4.46×10-3mol/L,经过对尿液的稀释处理,在尿液中色氨酸对该荧光探针检测尿酸的影响使非常小的。并且有相关报道也曾提及,可以对色氨酸进行掩蔽,以达到检测尿酸的目的。此现象在表1中更加直观的看到荧光强度的具体数值。由此可见,荧光探针在有其他物质干扰的情况下,对尿酸依然具有特异性。
表1
3.荧光探针TbIII-dtpa-bis(2,6-Diaminopurine)在真实尿样中对尿酸的检测
实验条件:分别取尿液(urine)0.5mL于5支50mL的容量瓶中,第一支用pH=7.4的Tris-HCl缓冲液定容。第二支中加入荧光探针储备液5mL,然后用pH=7.4的Tris-HCl缓冲液定容。其余三支分别加入1mL,3mL,5mL尿酸储备液,再分别加入荧光探针储备液5mL,用pH=7.4的Tris-HCl缓冲液定容。再取一支容量瓶,加入5mL荧光探针贮备液,用pH=7.4的Tris-HCl缓冲液定容。在241nm的激发波长下观察荧光光谱的变化。
结果如图6所示。如图6,在350nm处,稀释后的尿液具有一定的荧光强度,但是极其微弱,而探针溶液具有较强的荧光。当把尿液放入探针溶液后,探针的荧光强度明显被猝灭。继续加入不同浓度的尿酸,荧光强度继续被猝灭。由此可以判断,该探针可以在真实尿样中对尿酸进行检测,并且不受到尿液中其他物质干扰。此现象在表2中更加直观的看到荧光强度的具体数值。
表2
Claims (9)
1.一种新型荧光探针,其特征在于,所述的荧光探针是TbШ-dtpa-bis(2,6-Diaminopurine)。
2.权利要求1所述的新型荧光探针的制备方法,其特征在于,方法如下:
1)二乙三胺五乙酸、乙酸酐和吡啶,混合均匀,在60-70℃下,搅拌加热22-25h,冷却至室温,过滤,用乙酸酐和无水乙醚洗涤,抽滤,干燥,得二乙三胺五乙酸二酐(dtpaa);
2)二乙三胺五乙酸二酐、三乙胺、无水DMF和2,6-二氨基嘌呤,混合均匀,于95-105℃下,搅拌加热22-25h,静置,冷却到室温,过滤,真空干燥,得二乙三胺五乙酸-双(2,6-二氨基嘌呤)(dtpa-bis(2,6-Diaminopurine));
3)将二乙三胺五乙酸-双(2,6-二氨基嘌呤)用pH=7.4的Tris-HCl缓冲溶液溶解,得dtpa-bis(2,6-Diaminopurine)溶液,与Tb(NO3)3·6H2O混合,过滤,滤液于70-80℃下加热20-30min或于室温下放置1-2天,得TbIII-dtpa-bis(2,6-Diaminopurine)。
3.如权利要求2所述的新型荧光探针的制备方法,其特征在于:步骤1),用于反应过程的乙酸酐的加入量为,按摩尔比,二乙三胺五乙酸:乙酸酐:吡啶=1:4:6。
4.如权利要求2所述的新型荧光探针的制备方法,其特征在于:按摩尔比,二乙三胺五乙酸二酐:三乙胺:2,6-二氨基嘌呤=1:3:2。
5.如权利要求2所述的新型荧光探针的制备方法,其特征在于:按质量比,二乙三胺五乙酸-双(2,6-二氨基嘌呤):Tb(NO3)3·6H2O=1:0.8-0.85。
6.权利要求1所述的新型荧光探针在检测尿样中的应用。
7.如权利要求6所述的应用,其特征在于,所述的新型荧光探针应用于尿样中尿酸的检测。
8.如权利要求7所述的应用,其特征在于,方法如下:取尿液,加入权利要求1所述的新型荧光探针TbШ-dtpa-bis(2,6-Diaminopurine)的pH=7.4的Tris-HCl缓冲溶液,搅拌均匀,在241nm的激发波长下观察荧光光谱的变化。
9.如权利要求7所述的应用,其特征在于,方法如下:取尿液0.5mL于50mL容量瓶中,加入5mL浓度为5.0×10-4mol/L的权利要求1所述的新型荧光探针TbШ-dtpa-bis(2,6-Diaminopurine)的pH=7.4的Tris-HCl缓冲溶液,用Tris-HCl缓冲溶液定容,在241nm的激发波长下观察荧光光谱的变化。
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