CN104535552B - 一种丙烯醛衍生物的新用途 - Google Patents
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Abstract
本发明公开了一种丙烯醛衍生物的新用途，具体为3‑[3'‑(4″‑氟苯基)‑1'‑异丙基‑1H‑吲哚‑2″‑基]‑2‑丙烯醛衍生物在制备检测细胞内pH的荧光探针中的应用，以及在细胞成像中的应用。该衍生物在水溶液中，以365nm激发，528nm处有发射，随着pH值的降低，528nm处的发射峰降低，478nm处出现新的发射峰，溶液荧光由绿色变为蓝色。该探针具有较大的Stokes位移，对H+灵敏度高，选择性好，对pH变化成比率型响应。该探针细胞穿透性强，很适合检测细胞内pH变化和用于细胞成像。
Description
技术领域
本发明涉及丙烯醛衍生物，具体属于3-[3'-(4″-氟苯基)-1'-异丙基-1H-吲哚-2″-基]-2-丙烯醛衍生物的新用途。
背景技术
细胞环境的pH值是一项非常重要的生理参数，它在细胞、酶和很多生理活动中起着关键的作用，如细胞增殖、细胞凋亡、耐药抗药性、离子传输、肌肉收缩等。因此监测细胞内的pH值的变化对研究细胞行为和神经系统传输具有重要意义。在正常生理条件下，细胞环境存在两种不同的pH范围，一种是pH 6.8～7.4，如细胞液。pH的波动范围为0.1～0.2，变化幅度过大往往和一些疾病相关联，比如癌症、阿尔兹海默症。另一种是pH 4.5～6.0，即酸性细胞器，如溶酶体。其保持在酸性环境下有利于在细胞新陈代谢过程中促进蛋白质的降解。因此，监测细胞环境中的pH变化对进一步理解生理功能的规则机制和解决病变过程中的问题有重要意义。
pH玻璃电极和pH试纸是测定pH值的传统方法，pH玻璃电极不适合监测生物体内pH变化，因为其存在电化学干扰和机械损伤。pH试纸不够精确并且不能测定生物体内的pH值。和测定pH值的其他方法相比，如比色法、微电极、核磁共振，荧光光谱法在监测细胞内pH变化具有时空分布的优势。除此之外，荧光探针操作简单、灵敏度高，对细胞无损伤、可实现快速实时监测。因此pH荧光探针法受到研究人员广泛关注。目前报道的许多测定细胞内pH的荧光探针大多为荧光增强型或猝灭型荧光探针，即其荧光强度随待测物浓度的变化而变化。这种类型的荧光探针在测量过程中，会受到仪器本身、外界环境和探针浓度等因素的干扰。比率型荧光探针是通过两个不同波长下的荧光强度的比值来检测待测物的浓度变化，可以避免由探针浓度、发射强度以及光漂白带来的干扰。因此发展灵敏度高、选择性好、具有大的Stokes位移的比率型荧光探针具有很大的应用前景。
3-[3'-(4″-氟苯基)-1'-异丙基-1H-吲哚-2″-基]-2-丙烯醛衍生物，目前主要用于抗菌药物的合成前体，也有人用其作为原料合成荧光探针。但是将该衍生物用于pH荧光探针监测细胞内pH变化和用于细胞成像还未见报道。
发明内容
本发明的目的在于提供丙烯醛衍生物的新用途，即提供3-[3'-(4″-氟苯基)-1'-异丙基-1H-吲哚-2″-基]-2-丙烯醛衍生物的新用途。
所述的3-[3'-(4″-氟苯基)-1'-异丙基-1H-吲哚-2″-基]-2-丙烯醛衍生物，其结构式为：
其中，R1，R2为氢原子或烷基。
该衍生物对H+高度敏感，选择性好，具有大的Stokes位移，分散性好，细胞渗透性强，可避免温度、激发强度、探针浓度及生物组织背景光的干扰。其可在制备检测细胞内pH的比率型荧光探针中应用。该衍生物分散性好，细胞渗透性强，也可用于细胞成像。
与现有技术相比，本发明中所涉及的3-[3'-(4″-氟苯基)-1'-异丙基-1H-吲哚-2″-基]-2-丙烯醛衍生物作为荧光探针属于比率型荧光探针，以发射波长的比率变化来检测pH，可以避免因为光学路径长度、光漂白以及探针浓度、生物样品负染不均等带来的干扰。并且该探针具有较大的Stokes位移，有利于减小造影中激发光的干扰。本发明所涉及的荧光探针分子在质子化与去质子化过程中，在紫外灯下，荧光颜色明显改变，可通过肉眼观察。该探针分散性好，细胞渗透性强，通过激光共聚焦显微成像技术可进行细胞成像，也可检测细胞中的pH值。
附图说明
图1.3-[3'-(4″-氟苯基)-1'-异丙基-1H-吲哚-2″-基]-2-丙烯醛随pH变化的紫外吸收光谱图。
图2.3-[3'-(4″-氟苯基)-1'-异丙基-1H-吲哚-2″-基]-2-丙烯醛随pH变化的荧光发射光谱图。
图3 3-[3'-(4″-氟苯基)-1'-异丙基-1H-吲哚-2″-基]-2-丙烯醛结合H+前后紫外灯下颜色变化，颜色由绿色变为蓝色。
图4 3-[3'-(4″-氟苯基)-1'-异丙基-1H-吲哚-2″-基]-2-丙烯醛成比率的荧光强度I528/I478随pH值变化的sigmoidal拟合曲线。
图5 3-[3'-(4″-氟苯基)-1'-异丙基-1H-吲哚-2″-基]-2-丙烯醛对常见金属离子的响应情况。
图6 3-[3'-(4″-氟苯基)-1'-异丙基-1H-吲哚-2″-基]-2-丙烯醛在pH＝7.4、pH＝4、pH＝3.1时，分别加入到培养好的膀胱癌细胞BIU-87中的激光共聚焦成像图。
具体实施方式
实施例1
将3-[3'-(4″-氟苯基)-1'-异丙基-1H-吲哚-2″-基]-2-丙烯醛溶于乙醇中制得0.001M的母液，再用超纯水稀释至60μM,用0.001～1当量的HCl溶液调节pH值，记录其吸收光谱(图1)，随着pH值的降低，365nm处的吸收峰逐渐下降。
实施例2
将3-[3'-(4″-氟苯基)-1'-异丙基-1H-吲哚-2″-基]-2-丙烯醛溶于乙醇中制得0.001M的母液，再用超纯水稀释至25μM，用0.001～1当量的HCl溶液调节pH值，以365nm为激发波长，记录其荧光发射光谱(图2)。随着pH值的降低，528nm处的发射峰逐渐下降，478nm出现了新的发射峰，同时在478nm处出现新的等发射点。紫外灯下，溶液颜色由原来的绿色变为蓝色(图3)。利用发射的荧光强度比I528/I478对pH值作图，并利用sigmoidal进行拟合得到该探针的pKa为3.86(图4)。
实施例3
将3-[3'-(4″-氟苯基)-1'-异丙基-1H-吲哚-2″-基]-2-丙烯醛溶于乙醇中制得0.001M的母液，再用水稀释至25μM，考察该探针对常见金属离子的响应情况。如图5所示，在H+存在与否的条件下，常见的金属离子不会对该探针荧光性能产生干扰。证明该探针对H+有良好的选择性。图5中物质的顺序及浓度依次为：pH＝7时，1、探针；2、Na+(100mM)；3、K+(133mM)；4、Ca2+(10mM)；5、Mg2+(3.3mM)；6、Zn2+(77.33mM)；7、Al3+(43.3mM)；8、Ba2+(33mM)；9、Ni2+(16.67mM)；10、Cu2+(13.3mM)；11、Co2+(3.3mM)；12、Cd2+(2mM)；13、Pb2+(mM)；14、Cr3+(0.7mM)；15、Fe3+(0.2mM)，pH＝3.2时1、探针；2、Na+(66.67mM)；3、K+(133.3mM)；4、Ca2+(16.67mM)；5、Mg2+(10mM)；6、Zn2+(100mM)；7、Al3+(60mM)；8、Ba2+(46.67mM)；9、Ni2+(23.3mM)；10、Cu2+(53.3mM)；11、Co2+(3.3mM)；12、Cd2+(4.5mM)；13、Pb2+(27μM)；14、Cr3+(0.73mM)；15、Fe3+(0.1mM)。
实施例4
将3-[3'-(4″-氟苯基)-1'-异丙基-1H-吲哚-2″-基]-2-丙烯醛在pH＝7.4、pH＝4和pH＝3的条件下分别加入到培养好的膀胱癌细胞BIU-87中，放到激光共聚焦显微镜下观察，固定激发波长为405nm，分别在绿光和蓝光通道收集细胞发出的荧光。实验结果表明(图6)，在pH＝7.4条件下，细胞发出明亮的绿色荧光，蓝色荧光很弱。pH＝4条件下，绿色荧光减弱，蓝色荧光稍微增强。pH＝3条件下，绿色荧光明显减弱，蓝色荧光增强。
Claims (2)
1.3-[3'-(4”-氟苯基)-1'-异丙基-1H-吲哚-2”-基]-2-丙烯醛作为荧光探针在检测细胞内pH中的应用。
2.3-[3'-(4”-氟苯基)-1'-异丙基-1H-吲哚-2”-基]-2-丙烯醛在细胞成像中的应用。
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