CN107652220B - 一种检测半胱氨酸的荧光探针的制备方法及应用技术 - Google Patents
一种检测半胱氨酸的荧光探针的制备方法及应用技术 Download PDFInfo
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Abstract
本发明专利的技术领域属于分析化学中分析检测领域,涉及一种新型荧光探针的制备方法及其在检测半胱氨酸(Cys)中的应用。所述制备方法包括探针由3‑氰基乙酰吲哚与氯代的苯基丙烯醛衍生物通过克诺维纳盖尔缩合反应得到,其制备方法简单、探针结构稳定。该探针通过荧光分光光度计可应用于高选择性、高灵敏度、快速地可视化检测半胱氨酸。此外,该探针还成功的应用于生物活细胞的成像。该发明提供了一种新型的检测半胱氨酸的荧光探针的制备方法,具有很好的应用前景。
Description
技术领域
本发明涉及分析化学中的荧光探针传感技术领域,涉及一种快速、高选择性、高灵敏度检测半胱氨酸的荧光探针的制备方法及其应用。
背景技术
随着生命科学研究的发展,生物小分子的检测显得尤其重要。例如生物巯基小分子,包括半胱氨酸(Cys)、同型半胱氨酸(Hcy)和谷胱甘肽(GSH)在生理和病理过程中都扮演着十分重要的作用,如:Cys与机体的解毒作用、新陈代谢和蛋白质合成有着密切的联系,而异常的Cys含量可能引起各种疾病,例如:生长受阻、皮肤病变、造血功能障碍、毛发褪色等。作为Cys的前驱体,Hcy则与心血管疾病、阿尔茨海默症、精神障碍等疾病有关。GSH与艾滋病、癌症、心血管疾病等疾病有关。另外,Cys在医药、化妆品和食品中还有重要的运用,比如:用于丙烯腈、芳香族酸中毒后的解毒;用于治疗支气管炎的药物;化妆品方面主要用于烫发液、美容水、防日晒的护肤膏霜等;食品方面主要用于面包料和天然果汁中。因此,对于这些生物巯基小分子的检测是十分重要的。但是由于结构的相似性,很难将Cys/Hcy与GSH区分开。因为体内Cys的含量是Hcy含量的十倍以上,所以目前主要集中于Cys和GSH的区分检测。传统的测定方法主要是依赖于液相色谱法、分光光度法、同位素法、免疫学法等。这些方法虽然能够实现定性或定量的测定,但是也存在一定的缺陷,例如样品制备复杂、操作复杂、仪器昂贵、测试时间冗长、不易普及应用。因此,开发一种能够高选择性、高灵敏度、快速便捷地检测Cys的方法对一些重大疾病的初期临床诊断具有十分重要的意义。
本发明涉及一种检测半胱氨酸的荧光探针的制备方法及其应用技术。我们采用3-氰基乙酰吲哚与氯代的苯基丙烯醛衍生物通过克诺维纳盖尔缩合反应得到荧光探针,其制备方法简单、结构稳定。通过体外测试,实验表明该荧光探针可用于高选择性、高灵敏度、快速地可视化检测半胱氨酸。此外,该荧光探针还成功地应用于生物活细胞的成像,实验表明它可监测细胞内源性与外源性的Cys。因此,该探针具有潜在的应用前景。
发明内容
本发明的目的是提供一种检测半胱氨酸的荧光探针的制备方法及其应用技术。
根据本发明,所述通过3-氰基乙酰吲哚与氯代的苯基丙烯醛衍生物缩合反应得到荧光探针,其合成步骤具体包括:(1)将吲哚和氰基乙酸溶于酸酐中,氩气保护下加热回流反应4小时,待反应结束,处理得到白色固体产物3-氰基乙酰吲哚;(2)将N,N-二甲基甲酰胺置于冰浴中预冷却至0-5℃,然后缓慢加入三氯氧磷,冰浴下搅拌1小时,然后缓慢加入3,4,5-三甲氧基苯乙酮,再在室温下搅拌3小时,待反应结束,将反应液倒入碎冰中,用碳酸钠调节pH值至7~8,反应液中有黄色沉淀物生成,过滤、分离提纯得到黄色固体产物氯代的苯基丙烯醛衍生物;(3)将(1)和(2)中的3-氰基乙酰吲哚与氯代的苯基丙烯醛衍生物溶于无水乙醇中,再加入碱性催化剂,室温下搅拌反应,反应液中有黄色固体析出,过滤、分离提纯得到最终产物,即本发明所述的检测半胱氨酸的荧光探针。
与现有技术相比本专利技术具有以下优点:
1.高选择性,相关物质的对其几乎没有影响;
2.高灵敏度,可实现对微量目标物的检测;
3.快速响应,可实现快速检测;
3.合成简单,成本低,无污染。
附图说明
图1为探针检测半胱氨酸的荧光滴定图
图2为探针检测半胱氨酸的响应时间荧光光谱图
图3为探针的选择性实验荧光光谱图
图4为检测环境的pH优化荧光光谱图
具体实施方式
以下将结合实施例对本发明做进一步说明,本发明的实施例仅用于说明本发明的技术方案,并非限定本发明。
实施例1
将吲哚(2.00g,17.02mmol)和氰基乙酸(1.59g,18.69mmol)溶于15mL的酸酐中,在氩气保护下加热回流反应4小时,反应结束,待反应液冷却至室温,将反应液倒入100mL水中,乙酸乙酯萃取,收集有机相溶液,无水硫酸钠干燥,旋蒸得到粗产物,再经过柱层析分离得到最终产物即3-氰基乙酰吲哚(2.13g,产率68%)。核磁表征数据:1H NMR(400MHz,DMSO-d6,TMS):δ12.18(s,1H),8.38(d,J=4.0Hz,1H),8.14(dd,J1=2.0Hz,J2=7.1Hz,1H),7.51(m,1H),7.24(m,2H),4.50(s,2H)。
实施例2
将5mL的N,N-二甲基甲酰胺置于冰浴中预冷却至0-5℃,然后缓慢加入2mL的三氯氧磷,冰浴下搅拌1小时,然后缓慢加入3,4,5-三甲氧基苯乙酮(1.00g,4.76mmol),再在室温下搅拌3小时,待反应结束,将反应液倒入碎冰中,用碳酸钠调节pH值为7,有黄色沉淀物生成,过滤得到粗产物,然后柱层析分离得到最终产物即氯代的苯基丙烯醛衍生物(588mg,产率48%)。核磁表征数据:1H NMR(400MHz,CDCl3,TMS):δ10.20(d,J=7.0Hz,1H),6.98(s,2H),6.63(d,J=6.9Hz,1H),3.91(s,9H)。
实施例3
将上述的3-氰基乙酰吲哚(150mg,0.82mmol)与氯代的苯基丙烯醛衍生物(209mg,0.82mmol)溶于10mL的无水乙醇中,再加入三滴吡啶,室温下搅拌反应,反应液中有黄色固体析出,过滤、分离提纯得到最终产物(152mg,产率44%),即本发明所述的检测半胱氨酸的荧光探针。核磁表征数据:1H NMR(400MHz,CDCl3,TMS):δ8.85(s,1H),8.60(d,J=11.52Hz,1H),8.53(d,J=3.2Hz,1H),8.47(d,J=5.0,1H),7.46(d,J=4.0Hz,1H),7.36(m,3H),7.04(s,2H),3.94(d,J=7.2Hz,9H)。高分辨质谱:[M+H]+=423.1102(Calcd=423.1111)。
应用实例1:如图1,探针检测半胱氨酸的荧光滴定图。在二甲基亚砜与水的混合溶液中加入5μM的探针,在波长430nm的光激发下探针自身的荧光非常弱,然而探针与不同浓度的半胱氨酸响应之后,在655nm处荧光强度有明显的变化,随着半胱氨酸浓度的递增,荧光强度也随之增强,并呈良好的线性关系(R2=0.994),检测限达到240nmol/L。实验证明该探针可以高灵敏地应用于半胱氨酸的检测。
应用实例2:如图2,探针检测半胱氨酸的响应时间荧光谱图。在二甲基亚砜与水的混合溶液中加入探针(5μM),在波长430nm的光激发下探针自身的荧光非常弱,但加入50μM半胱氨酸之后,随着时间的推移,在480nm处的荧光强度开始逐渐增强,并在10分钟之内响应完全,荧光强度不再变化。以此证明该探针可实现对半胱氨酸的快速检测。
应用实例3:如图3,探针的选择性实验荧光光谱图。在相同的测试体系和测试条件下分别加入不同的干扰物(1~27分别为:探针本身、丙氨酸、精氨酸、天冬氨酸、甘氨酸、谷氨酸、脯氨酸、苯丙氨酸、苏氨酸、亮氨酸、组氨酸、丝氨酸、色氨酸、蛋氨酸、S2O3 2-、SO4 2-、Mg2 +、Ca2+、Ba2+、K+、CO3 2-、NO3 -、NO2 -、HS-、GSH、Cys、Hcy)。实验结果证明,探针对半胱氨酸/同型半胱氨酸具有高选择性,表现为480nm处的荧光强度发生明显的增强。然而对于其他的相关干扰物几乎不响应,表现为荧光强度保持不变
应用实例4:如图4,检测环境的pH优化荧光光谱图。分别在不同pH(pH=3~10)的二甲基亚砜/水缓冲液中加入5μM的探针和50μM的半胱氨酸,保持其他的测试条件一致,测得不同pH下探针响应半胱氨酸的情况。由图可知,探针自身在这不同pH环境中保持较高稳定性,荧光强度几乎不变。另外,在pH=3~7之间探针响应半胱氨酸之后的荧光强度逐渐增强,在pH为中性时达到最佳效果,而在pH=7~10之间探针响应半胱氨酸之后的荧光强度则逐渐降低。综合实验数据可知:探针在不同pH环境下能保持自身稳定性,在较大的pH范围内都能较好的响应半胱氨酸,并且在中性环境下达到测量的最佳效果,有利于其在生物样品检测中的应用。
Claims (8)
2.一种根据权利要求1所述的检测半胱氨酸的荧光探针的制备方法,其特征在于制备过程包括如下步骤:
a:将吲哚和氰基乙酸溶于酸酐中,氩气保护下加热回流反应4小时,待反应结束,处理得到白色固体产物3-氰基乙酰吲哚;
b:将N,N-二甲基甲酰胺置于冰浴中预冷却至0-5℃,然后缓慢加入三氯氧磷,冰浴下搅拌1小时,然后缓慢加入3,4,5-三甲氧基苯乙酮,再在室温下搅拌3小时,待反应结束,将反应液倒入碎冰中,用碳酸钠调节pH值,反应液中有黄色沉淀物生成,过滤、分离提纯得到黄色固体产物氯代的苯基丙烯醛衍生物;
c:将a,b中的3-氰基乙酰吲哚与氯代的苯基丙烯醛衍生物溶于无水乙醇中,再加入碱性催化剂,室温下搅拌反应,反应液中有黄色固体析出,过滤、分离提纯得到最终产物,即权利要求1所述的检测半胱氨酸的荧光探针。
3.根据权利要求1所述的荧光探针在检测半胱氨酸中的应用。
4.一种根据权利要求2所述的检测半胱氨酸的荧光探针的制备方法,其特征在于,步骤a中原料吲哚与氰基乙酸的化学计量比为1:0.1~10。
5.一种根据权利要求2所述的检测半胱氨酸的荧光探针的制备方法,其特征在于,步骤b中调节后的pH值为7~8。
6.一种根据权利要求2所述的检测半胱氨酸的荧光探针的制备方法,其特征在于,步骤c中用到的碱性催化剂种类为吡啶或者三乙胺。
7. 一种根据权利要求3所述的荧光探针在检测半胱氨酸中的应用,其特征在于,检测时所选用最佳激发光的波长在420 nm~440 nm之间。
8.根据权利要求3所述的荧光探针在检测半胱氨酸中的应用,其特征在于,检测时所选用的溶剂体系为二甲基亚砜与水的混合溶液。
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