CN111175262A - 氟硼荧光探针在线粒体定位中的应用 - Google Patents
氟硼荧光探针在线粒体定位中的应用 Download PDFInfo
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Abstract
本发明涉及有机化工及线粒体用途领域,公开了一种氟硼荧光探针在线粒体定位中的应用,该氟硼荧光染料组成探针后,在聚集时表现出深红色到近红外的发射,并且具有高度生物相容性能够特别“点亮”细胞线粒体,通过一种超快速且易于操作的染色方法对活细胞进行染色。更重要的是,它们在连续强激光照射下都表现出很高的光稳定性,显示出它们在生物医学和材料科学中的潜在应用。
Description
技术领域
本发明涉及有机化工及线粒体用途领域,具体地,涉及一种氟硼荧光探针在线粒体定位中的应用。
背景技术
线粒体,作为为几乎所有真核细胞的动力,是重要的亚细胞器,为各种生命活动提供能量。例如,线粒体可产生三磷酸腺苷(ATP),涉及一系列电子传递过程,与活性氧的产生有关。线粒体功能紊乱会导致活性氧水平异常,最终导致许多疾病,如人类心血管疾病,神经退行性疾病、雷氏综合征或肿瘤。荧光生物成像作为一种无创的生物系统过程可视化方法,具有高分辨率、高对比度、高灵敏度的实时监测能力,在早期图像引导诊断中具有广阔的应用前景,可监测或可视化其形态或生物活性,为研究细胞凋亡和退行性变状况,从而在疾病诊断中发挥不可或缺的作用。为了观察活体样品的长期动态跟踪,光稳定性是荧光成像的一个特别重要的参数。然而,传统的荧光探针,特别是商业上可用的线粒体特异性染料,如Green FM和Deep Red FM,在连续激光照射下,光稳定性差。此外,当这些商用探针的浓度逐渐增加时会出现聚集猝灭(ACQ)现象,它们的荧光强度会减弱。因此,由于伴随着ACQ效应,荧光团浓度的增加不能提高发光强度。
发明内容
本发明的目的是提供一种氟硼荧光探针在线粒体定位中的应用,解决了传统的荧光探针,特别是商业上可用的线粒体特异性染料,在连续激光照射下,光稳定性差,伴随着ACQ效应,荧光团浓度的增加不能提高发光强度的问题。
为了实现上述目的,本发明提供一种氟硼荧光探针在线粒体定位中的应用。
所述氟硼荧光探针的结构式为:
其中,所述R为H或者乙基。
优选地,所述应用包括将氟硼荧光染料在不良溶剂中自组装形成纳米探针,利用纳米探针对细胞线粒体进行荧光染色。
优选地,将氟硼荧光染料和二甲基亚砜混合,之后加入1640完全培养基,形成包含探针的溶液。
通过上述技术方案,本发明提供了一种氟硼荧光探针在线粒体定位中的应用,该氟硼荧光染料组成探针后,在聚集时表现出深红色到近红外的发射,并且具有高度生物相容性能够特别“点亮”细胞线粒体,通过一种超快速且易于操作的染色方法对活细胞进行染色。更重要的是,它们在连续强激光照射下都表现出很高的光稳定性,显示出它们在生物医学和材料科学中的潜在应用。
本发明的其它特征和优点将在随后的具体实施方式部分予以详细说明。
附图说明
附图是用来提供对本发明的进一步理解,并且构成说明书的一部分,与下面的具体实施方式一起用于解释本发明,但并不构成对本发明的限制。在附图中:
图1a,1e分别为探针1和2的归一化吸收光谱(黑色虚线为探针二氯甲烷溶液)、归一化荧光光谱(曲线为不同溶剂)和固态光谱(紫色虚线)中的荧光发射,以及在日光和365nm紫外线灯照射下的粉末照片(插入小图);
图1b,1f分别为探针1和2(30μM)在DMSO/去离子水(DI)中的荧光光谱(去离子水在在DMSO/去离子水体系中所占比例为fw),激发波长490nm,以及插图分别为探针1和2(30μM)在DMSO和DMSO/DI体系(95%和99%含水量)中紫外光照射下的照片;
图1c,1g分别为探针1和2(30μM)在不同水组分DMSO/DI中的吸收光谱;
图1d,1h分别为探针1和2在635nm或643nm处荧光强度与不同水组分的关系图(λex=490nm);
图2为用CCK-8检测不同浓度的探针1(左)和2(右)处理HeLa细胞24小时的细胞毒性;
图3a为Hela细胞(人宫颈癌细胞)摄取探针1和探针2(20μM)的时间依赖性CLSM图像;
图3b为图像分析荧光强度定量;
图4表示Hela细胞中探针2(20μM)的线粒体靶向特性;其中,图4a为商品化细胞核定位试剂4',6-二氨基-2-苯基吲哚(DAPI,0.08μg/mL)的细胞成像;图4b为孵育0.5小时后,使用488nm的激发波长激发探针2,并收集500-800nm波段发射光谱区域;图4c为商品化线粒体定位试剂深红色FM(0.5μM)细胞成像;图4d为图4a、图4b和图4c的合并图像;图4e为深红色FM和探针2在Hela细胞中的细胞区域内荧光强度定量分布折线图;图4f为深红色FM和探针2强度的散点图,比例尺:25μm,皮尔逊相关系数R=0.92,重叠系数R=0.94;
图5a和5b分别为探针1和探针2在488nm激光连续照射下,不同时间的细胞荧光图像和荧光强度折线图,图像每60秒拍摄一次,比例尺:25μm。
具体实施方式
以下对本发明的具体实施方式进行详细说明。应当理解的是,此处所描述的具体实施方式仅用于说明和解释本发明,并不用于限制本发明。
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值,这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说,各个范围的端点值之间、各个范围的端点值和单独的点值之间,以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围,这些数值范围应被视为在本文中具体公开。
实施例1
配制探针1的DMSO溶液(10000μM)。在超声波作用下,将一定体积的母液注入去离子水或1640完全培养基中稀释500倍。由于在不良溶剂中溶解度突然降低,导致探针1自组装形成纳米颗粒。将30000个HeLa细胞接种到玻璃底皿中,在37℃、5%CO2和95%加湿的培养基(RPMI-1640,含10%FBS)中培养24小时。移除培养基后,用探针1(20μM,含<1%二甲基亚砜的1640完全培养基)在37℃,5%CO2的培养箱中孵化0.5h,PBS洗涤两次后,用4%甲醛固定细胞20min,随后加入细胞器示踪剂4',6-二氨基-2苯基吲哚(DAPI,0.08μg/mL),孵育30min,对细胞核染色。最后,用PBS再次清洗培养皿,并使用共聚焦荧光显微镜(分别在405、488和638nm处激发DAPI、探针1和深红色FM)观察HeLa细胞的形态。Hela细胞随机选择区域的荧光强度分布显示得出高的皮尔逊系数0.95,证实探针1能够选择性地靶向活细胞中的线粒体。
将30000个HeLa细胞接种到玻璃底皿中,在37℃、5%CO2和95%湿度下培养24小时。用含探针1的溶液(20μM、含<1%DMSO的1640完全培养基)培养Hela细胞,培养30分钟。上述细胞用PBS洗涤两次。然后用共聚焦荧光显微镜测量荧光强度。可以看出光照300秒后,MTDR荧光强度下降约75%。在相同条件下,探针1的荧光强度仅降低了12%。如图5所示,在相同的电压条件下,MTDR的荧光信号明显降低,而探针1在强激光照射5min后荧光强度几乎没有降低。
实施案例2
操作方法同实例1,只需要将将实例1中相应的探针1改为探针2。
绿色(探针2)通道和红色(MTDR)通道的相关图也给出了很高的Pearson相关系数(0.92)和很高的重叠系数(0.94),证实探针2选择性地定位活细胞中的线粒体。
光照300秒后,MTDR荧光强度下降约75%。在相同条件下,探针2的荧光强度仅降低了13%。
通过上述实施例1和实例2可知,我们可以看出探针1和2能够选择性点亮细胞线粒体亚细胞器,同时具有极强的高稳定性,这是目前氟硼荧光染料体系中极少的例子,具有潜在的应用前景。
以上详细描述了本发明的优选实施方式,但是,本发明并不限于上述实施方式中的具体细节,在本发明的技术构思范围内,可以对本发明的技术方案进行多种简单变型,这些简单变型均属于本发明的保护范围。
另外需要说明的是,在上述具体实施方式中所描述的各个具体技术特征,在不矛盾的情况下,可以通过任何合适的方式进行组合,为了避免不必要的重复,本发明对各种可能的组合方式不再另行说明。
此外,本发明的各种不同的实施方式之间也可以进行任意组合,只要其不违背本发明的思想,其同样应当视为本发明所公开的内容。
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