靶向到内质网的Three-input“AND型”逻辑门荧光探针的设计、合成及对次氯酸根的比例成像

发布时间：2018-06-02 16:15

本文选题：活性氧 + 分子逻辑门　；参考：《河南师范大学》2017年硕士论文

【摘要】：在生物体不同的生命活动中,会自发产生适量的活性氧,诸如神经传递中的信号转换,免疫系统控制,细胞正常生长,机体生命化合物的合成与新陈代谢等生命活动,活性氧都发挥了极为重要的作用。而当机体内活性氧过量产生时,活性氧就会造成细胞的损坏,甚至凋亡,以及其他机体的氧化损伤。经过研究表明,人体内活性氧(ROS)是导致各种疾病产生的主要因素。而次氯酸是活性氧中含量最多并且氧化性强的一种,在机体内大部分的次氯酸会分解成次氯酸根(ClO-),因此,如何对细胞中特别是活细胞器内的次氯酸根(ClO-)的实时监测,已逐渐成为有机化学和化学生物学研究领域中极其挑战性的课题之一。近年来,荧光探针分子也经常被应用于化学传感器和分子逻辑门中。分子逻辑门是将传统上的集成电路功能实现在分子尺度上的一门新兴科学。传统意义上,以硅为材料的半导体的逻辑运算(也称为布尔运算)是通过电压的变化来实现的,超分子化学上的逻辑运算是通过客体与主体之间的相互作用、相对应的光谱信号变化,与逻辑计算过程中的输入和输出一一相对应。而荧光探针的逻辑运算是利用以化学方法合成的荧光染料作为荧光探针分子,利用荧光探针分子中的官能团对某一种物质的特定识别原理,来实现对特定物质的检测。我们设计合成了靶向到内质网的Three-input“AND型”逻辑门荧光探针NPA-CN。探针分子是以6-溴-2-萘酚为发光团及骨架,在分子结构中引入N-乙基酰胺键,以及一个强的吸电子基团丙二腈,构成了荧光分子逻辑门NPA-CN。在NPA-CN探针分子中,由于丙二腈的强吸电子和酰胺键的推电子作用,使分子中存在“推-拉”电子体系。研究发现,当环境中同时存在水、次氯酸根、和OH-时,官能团丙二腈会被氧化为醛基,既生成分子NPA-CHO。探针分子NPA-CN还具有高光稳定性,细胞低的毒性,以及良好的选择性。因此该分子探针可以成功的应用于活细胞成像以及活体成像。当荧光分子NPA-CN应用于生物成像,利用激光共聚焦显微成像技术,细胞中同时存在水、次氯酸根、和OH-时,该探针分子会发生明显的光谱变化,长波长(583 nm)荧光强度减弱,短波长(499 nm)的荧光强度增强。但是,当三输入改成单输入或者双输入时,短波长(499 nm)和长波长(583 nm)的荧光都没有明显变化,通过与商业内质网染料(ER-Tracker Red)细胞共定位成像对比,NPA-CN在糙面内质网上成像。同时在活体成像中,该探针分子可以很好用于斑马鱼的成像分析。在本工作中,通过引入新的检测机制,构建了一系列逻辑门分子探针体系,实现了NPA-CN探针分子对次氯酸根的检测。本工作报道的探针分子为监测相关疾病中内源性次氯酸根提供了一个潜在的可能。
[Abstract]:In different biological activities, reactive oxygen species (Ros) are produced spontaneously, such as signal switching in nerve transmission, immune system control, normal cell growth, biosynthesis and metabolism of biological compounds, and so on. Reactive oxygen species play an extremely important role. When the reactive oxygen species in the body is excessive, the reactive oxygen species will cause cell damage, even apoptosis, as well as other oxidative damage. The results show that Ros is the main cause of various diseases. Hypochlorite is one of the most abundant and highly oxidized species of reactive oxygen species. Most of the hypochlorite in the body is decomposed into hypochlorite, so how to monitor the hypochlorite in cells, especially in living organelles, in real time. It has gradually become one of the most challenging subjects in the field of organic chemistry and chemical biology. In recent years, fluorescent probe molecules are often used in chemical sensors and molecular logic gates. Molecular logic gate is a new science that realizes the function of integrated circuit on molecular scale. In the traditional sense, the logic operation of silicon semiconductor (also called Boolean operation) is realized by the change of voltage, and the logic operation of supramolecular chemistry is through the interaction between the object and the host. The corresponding spectral signal changes correspond to the input and output in the logical calculation process. The logical operation of the fluorescence probe is to use the fluorescent dye synthesized by chemical method as the fluorescence probe molecule and the specific recognition principle of the functional group in the fluorescence probe molecule to realize the detection of the specific substance. We have designed and synthesized a Three-input "AND" logic gate fluorescent probe NPA-CNtargeted to endoplasmic reticulum (ER). The fluorescent molecular logic gate NPA-CNS is composed of 6-bromo-2-naphthol as luminescent group and skeleton, N- ethyl amide bond and a strong electron absorbent group malonitrile. In the NPA-CN probe molecule, due to the strong electron absorption of malonitrile and the push electron of amide bond, there is a "push-pull" electron system in the molecule. It was found that when water, hypochlorite, and OH- were present in the environment, the functional group malonitrile was oxidized to aldehyde group, which formed the molecule NPA-CHO. The probe molecule NPA-CN also has high light stability, low cytotoxicity and good selectivity. Therefore, the molecular probe can be successfully used in living cell imaging and in vivo imaging. When fluorescent molecule NPA-CN was used in biological imaging and laser confocal microscopy was used, water, hypochlorite, and OH- existed simultaneously in the cell, the fluorescence intensity of the probe molecule decreased and the fluorescence intensity of the probe molecule decreased. The fluorescence intensity of short wavelength (499 nm) was enhanced. However, when the three inputs are changed to single input or double input, the fluorescence of short wavelength (499 nm) and long wavelength (583 nm) do not change obviously. NPA-CN is compared with commercial endoplasmic reticulum dye ER-Tracker Redacker cell co-localization imaging in rough endoplasmic reticulum. At the same time, in vivo imaging, the probe molecule can be well used in the imaging analysis of zebrafish. In this work, a series of logic gate molecular probe systems were constructed by introducing a new detection mechanism, and the detection of hypochlorite by NPA-CN probe molecule was realized. The probe molecule reported in this work provides a potential for monitoring endogenous hypochlorite in related diseases.
【学位授予单位】：河南师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657.3

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