CN106518860A - 一种基于荧光共振能量转移机理的靶向线粒体的次氯酸比例荧光探针及其应用 - Google Patents
一种基于荧光共振能量转移机理的靶向线粒体的次氯酸比例荧光探针及其应用 Download PDFInfo
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Abstract
本发明公开了一种基于荧光共振能量转移机理的靶向线粒体的次氯酸比例荧光探针，其特征在于：所述比率荧光探针是以香豆素荧光团作为能量供体，苯乙烯基苯并噻唑盐作为能量受体，构成荧光共振能量转移组合对，其化学结构式如式(I)所示。本发明还公开了所述比例荧光探针在检测PBS/DMF缓冲液及生物细胞中的次氯酸的应用。实验证实本发明探针可以选择性地与次氯酸作用，溶液颜色由橘红色变为无色，可肉眼辨别，荧光由橘红色变为绿色，荧光比例响应明显，也可以通过紫外吸收和荧光光度法进行分析。可实现对体外次氯酸盐的微量高灵敏度检测，以及细胞内的比率成像，有望在临床医学及工业生产中发挥作用，具有重要的应用价值。
Description
技术领域
本发明涉及一种比例荧光探针及其应用，尤其涉及一种基于荧光共振能量转移(FRET)机理的靶向线粒体的次氯酸比例荧光探针及其应用；属于有机小分子荧光探针技术领域。
背景技术
次氯酸是生物体中各种生物过程的重要的活性氧之一，内源性的次氯酸主要由双氧水与氯离子经过氧化酶催化产生。次氯酸作为生物体内的杀菌剂，在免疫***中有重大作用，内源性次氯酸的过量或缺乏都可能导致疾病发生，比如关节炎、动脉硬化以及肿瘤等疾病。因此，实现生物体内次氯酸快速、灵敏、高选择性的检测至关重要。
线粒体是细胞内活性氧的主要来源，线粒体内次氯酸的检测对细胞生物学以及生理病理学研究非常重要。中国专利如【201510794522】、【201610323154】、【201610263094】涉及次氯酸的选择性检测，但缺乏结构多样性，存在应用方面的局限，至今尚未见靶向线粒体的次氯酸比例荧光探针专利。
发明内容
针对现有技术的不足，本发明要解决的问题是提供一种基于荧光共振能量转移机理的靶向线粒体的次氯酸比例荧光探针及其应用。
本发明技术方案的核心在于利用香豆素为能量供体，苯乙烯基苯并噻唑盐为能量受体构筑FRET体系。当用香豆素的激发波长激发探针时，发生FRET过程，探针发射苯乙烯基苯并噻唑盐荧光团的荧光；当加入次氯酸时，苯乙烯基苯并噻唑盐的双键部位被氧化成醛，共轭体系断裂，FRET***被破坏。当同样用香豆素的激发波长激发时，仅发出香豆素荧光团的荧光。两个发射波长的荧光强度比与次氯酸浓度呈现线性关系，实现比率荧光检测次氯酸，避免探针浓度以及环境等干扰，提高检测精确度。
本发明所述的基于荧光共振能量转移机理的靶向线粒体的次氯酸比例荧光探针，其特征在于：所述比率荧光探针是以香豆素荧光团作为能量供体，苯乙烯基苯并噻唑盐作为能量受体，构成荧光共振能量转移组合对，其化学结构式如式(I)所示：
本发明所述的基于荧光共振能量转移机理的靶向线粒体的次氯酸比例荧光探针的制备方法是：4-(4-(7-(二乙胺基)-2-氧代-2H-苯并吡喃-3-羰基)哌嗪基-1)苯甲醛【4-(4-(7-(diethylamino)-2-oxo-2H-chromene-3-carbonyl)piperazin-1-yl)benzaldehyde】与3-甲基苯并噻唑盐反应得到所述比例荧光探针。
所述基于荧光共振能量转移机理的靶向线粒体的次氯酸比例荧光探针在检测PBS/DMF缓冲液及生物细胞中的次氯酸的应用。
本发明所述的探针的结构可以被次氯酸氧化成醛，释放出具有荧光的4-(4-(7-(二乙胺基)-2-氧代-2H-苯并吡喃-3-羰基)哌嗪基-1)苯甲醛，从而达到检测的效果，见图1。
配制此比例荧光探针的PBS/DMF缓冲液(pH＝7.0，v/v＝9:1)的溶液，分别定量加入微升级的KNO3,Ba(NO3)2,AlCl3,OCl-,t-BuO·,H2O2,O2 -,OONO-,NO·,1O2,t-BuOOH,NaNO2,Na2CO3,NaCH3CO2,Na2S and NaSO4的水溶液。通过紫外可见分光光度法和荧光分光光度法测试对不同活性氧、人体常见金属离子的选择性和响应能力，结果见图2-3。在加入OCl-前后体系在580nm与480nm处的荧光强度比值(I580/I480)以及在483nm处的吸收强度变化明显。
在分别加入上述探针的RAW264.7活细胞中，对照不加LPS和加入LPS的细胞蓝色荧光显微成像变化。结果见图4.
本发明所述基于荧光共振能量转移机理的靶向线粒体的次氯酸比例荧光探针在检测线粒体中的次氯酸的应用。
通过探针在RAW264.7活细胞中与线粒体深红的共定位实验，表明所述探针能定位于线粒体。结果见图5。预示其在检测线粒体中的次氯酸的应用具有开发前景。
本发明设计合成的基于FRET的比例荧光探针可以与OCl-发生氧化反应，随着次氯酸浓度的增加，580nm处荧光强度逐渐减弱，480nm处荧光强度逐渐增强；二者比值与次氯酸浓度在一定范围内呈线性关系。该探针实现了对OCl-的微量高灵敏度检测，具有重要的应用价值。
附图说明
图1：本发明探针与次氯酸氧化产物的高分辨质谱。
图2：本发明探针的PBS/DMF缓冲液(pH＝7.0，v/v＝9:1，10μM)中加入不同的活性氧、常见金属离子的紫外吸收光谱，选择性识别次氯酸。
图3：本发明探针的PBS/DMF缓冲液(pH＝7.0，v/v＝9:1，2.5μM)中加入不同的活性氧、常见金属离子的荧光发射谱，选择性识别次氯酸。
其中：(A)荧光发射谱图，(B)两个波长下荧光强度比值量化图。
图4：本发明探针在RAW264.7cells中的荧光显微成像图。
其中：(A)荧光成像图，(B)量化图。
图5：本发明探针在RAW264.7活细胞中与线粒体深红染料的共定位。
其中：(a)探针的蓝光405-550nm；(b)探针的绿光(560-700nm)；(c)Mito TrackerDeepRed，激发波长(Ex)644nm，发射波长(Em)665nm；(d)明场图像；(e)为(a)，(b)和(c)的叠加图；(f)探针和Mito Tracker Deep Red的共定位分析(共定位系数：0.91)。
具体实施方式
实施例1
用已知方法合成4-(4-(7-(二乙胺基)-2-氧代-2H-苯并吡喃-3-羰基)哌嗪基-1)苯甲醛(1)，然后化合物1与3-甲基苯并噻唑盐反应得到本发明所述的基于荧光共振能量转移机理的靶向线粒体的次氯酸比例荧光探针。该探针可以与次氯酸发生氧化反应，释放出有荧光的7-二乙胺基香豆素衍生物1。
反应式如下：
将4-(4-(7-(二乙胺基)-2-氧代-2H-苯并吡喃-3-羰基)哌嗪基-1)苯甲醛(1,20mmol)，2-甲基苯并噻唑盐(2,20mmol)至50mL无水乙醇中回流，反应3小时后冷至室温，浓缩，硅胶柱层析，得到红色粉末本发明的探针(3)，熔点：182℃，产率87％。
核磁共振氢谱测定：1H NMR(300MHz,DMSO-d6):δ＝8.34(d,J＝7.5Hz,1H),8.15-8.04(m,3H),7.95(d,J＝8.7Hz,2H),7.84-7.69(m,3H),7.52(d,J＝9.0Hz,1H),7.09(d,J＝9.0Hz,2H),6.76(dd,J＝2.1,9.0Hz,1H),6.57(d,J＝2.1Hz,1H),4.26(s,3H),3.74-3.32(m,12H),1.14(t,J＝6.9Hz,6H)；
核磁共振碳谱测定：13C NMR(75MHz,DMSO-d6):δ＝171.60,164.23,158.45,156.65,153.18,151.30,149.40,144.17,141.92,132.44,130.15,128.96,127.68,127.04,123.89,123.39,116.17,115.58,113.83,109.42,108.11,107.10,93.26,46.55,45.81,44.15,35.79,12.27；
高分辨质谱：[M]+calcd for C36H35N6O4,579.2430,found 579.2469。
实施例2
用微量注射器向10mL配好的本发明所述的基于荧光共振能量转移机理的靶向线粒体的次氯酸比例荧光探针的PBS/DMF缓冲液(pH＝7.0，v/v＝9:1，10μM)中，分别定量加入10μM的KNO3,Ba(NO3)2,AlCl3,OCl-,t-BuO·,H2O2,O2 -,OONO-,NO·,1O2,t-BuOOH,NaNO2,Na2CO3,NaCH3CO2,Na2S,NaSO4的水溶液，作用3小时后进行荧光分光光度法和紫外分光光度计测试，显示探针对次氯酸有较好的选择性，加入次氯酸前后对照显示紫外吸收以及两个发射波长的荧光强度发生明显变化，见图2，3。
实施例3
细胞内荧光成像：
RAW264.7细胞先用脂多糖(LPS，1μg/ml)培养12h，随后在无血清培养基中加入佛波酯(PMA，1μg/mL)继续孵育30min，再用本发明所述探针(2μM)孵育30min。PBS(pH 7.20)冲洗细胞3次后，用激光共聚焦显微镜(LSM 700)进行成像，收集405-550nm(蓝光)和560-700nm(红光)波长范围内的荧光，得到明场图像、荧光图像和二者的叠加图像，然后进行统计(*p<0.05vs.control,**p<0.01vs.control,n＝3)，见图4。
实施例4
本发明所述探针与线粒体的共定位分析：
利用本发明所述探针(2μM)孵育RAW264.7细胞15min，再加入线粒体标记物MitoTracker Deep Red(0.4μM)继续孵育15min，PBS洗3次，然后进行共聚焦成像观察。
结果见图5：(a)探针的蓝光405-550nm；(b)探针的绿光(560-700nm)；(c)MitoTrackerDeep Red，激发波长(Ex)644nm，发射波长(Em)665nm；(d)明场图像；(e)为(a)，(b)和(c)的叠加图；(f)探针和Mito Tracker Deep Red的共定位分析(共定位系数：0.91)。
通过探针在RAW264.7活细胞中与线粒体深红的共定位实验，表明所述探针能定位于线粒体。
Claims (3)
1.一种基于荧光共振能量转移机理的靶向线粒体的次氯酸比例荧光探针，其特征在于：所述比率荧光探针是以香豆素荧光团作为能量供体，苯乙烯基苯并噻唑盐作为能量受体，构成荧光共振能量转移组合对，其化学结构式如式(I)所示：
2.权利要求1所述基于荧光共振能量转移机理的靶向线粒体的次氯酸比例荧光探针在检测PBS/DMF缓冲液及生物细胞中的次氯酸的应用。
3.权利要求1所述基于荧光共振能量转移机理的靶向线粒体的次氯酸比例荧光探针在检测线粒体中的次氯酸的应用。
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