反应型荧光探针的构建及生物传感研究

发布时间：2018-09-03 17:51

【摘要】：与基于主客体间弱相互作用的荧光探针相比,反应型荧光探针利用了分析物特异性的化学反应来调节荧光的变化,因此在选择性和灵敏性方面具有一定优势,目前已是荧光传感领域研究的热点,并在生物相关的小分子或离子的实时监测和影像方面获得了广泛应用。本论文针对能调控生物重要生理病理功能的信使分子硫化氢、生物硫醇、生物活性氧-次氯酸,结合特异性的化学反应以及螺环开环、光诱导的电子转移机理,设计合成了相关荧光探针,在模拟生理条件下系统评价了探针的传感性能,并在细胞水平实现了该系列生物小分子的特异性荧光成像。此外,本论文也利用邻胺基苯乙炔衍生物与Au~(3+)发生的特异性化学反应,构建了一个能高选择性识别Au~(3+)的荧光探针,为该离子药理和病理角色的研究提供了可视化研究工具。相关内容总结如下:(1)以5(6)-羧基-罗丹明为信号单元、7-硝基苯恶二唑为反应单元,结合螺环“开-关”原理和光诱导电子转移(PET)机理设计合成了信使分子-硫化氢(H_2S)荧光探针2-1a和生物硫醇荧光探针2-1b。在PBS缓冲溶液(10 mM,pH=7.4,1 m M CTAB)中,探针2-1a主要以非荧光的螺环内酯的形式存在,与H_2S发生巯解反应后,螺环打开,溶液由无色变为粉色,同时荧光增强近160倍;鉴于良好的细胞渗透性,该探针已在HaLa细胞内实现了对外源性H_2S的荧光影像。在PBS缓冲溶液(10mM,pH=7.4)中,探针2-1b则主要以开环的形式存在;由于存在由罗丹明荧光团到NBD基团的PET过程,探针本身没有荧光;然而与半胱氨酸反应后,探针的NBD单元发生裂解反应,释放出罗丹明荧光团,导致明显荧光增强,检出限可达2.8×10-8mol/L。(2)以萘二酰亚胺为荧光团、硒吗啉为反应基团、三苯基膦阳离子为靶向基团,设计合成了一个线粒体靶向的,并且能可逆检测次氯酸(HOCl)的荧光探针3-1。在PBS缓冲溶液(20 mM,pH=7.4)中,由于PET过程,该探针几乎没有荧光;与HOCl作用后,探针的硒原子被氧化为硒亚砜,该过程抑制了PET猝灭,导致了明显的荧光增强。值得注意的是,该探针对HOCl具有极高的灵敏性,1当量HOCl即可引起40倍的荧光增强,检出限可达4.2 nM。此外,该探针对HOCl表现出良好的选择性,其它生物活性氧化物均不会引起明显的荧光响应。该探针已成功用于RAW246.7细胞线粒体内外源和Qg源性HClO的检测。(3)以氟硼二吡咯甲川(BODIPY)荧光染料为母体、2-(苯乙炔基)苯胺为反应基团,构建了能选择性识别Au~(3+)的荧光探针4-1。在EtOH-PBS缓冲溶液(20 mM,pH=7.4,v/v=1:1)中,由于PET过程,该探针几乎没有荧光;在Au~(3+)存在下,该探针发生了分子内环化反应,导致2-(苯乙炔基)苯胺反应基团转变为2-苯基吲哚,该过程抑制了PET猝灭,导致了荧光的明显增强。选择性研究表明,其它金属阳离子几乎不会引起该探针的荧光变化。该探针已在水样中Au~(3+)的检测以及HeLa细胞内Au~(3+)的荧光影像方面得以应用。
[Abstract]:Compared with the fluorescence probe based on the weak interaction between the host and the guest, the reactive fluorescence probe makes use of the specific chemical reaction of the analyte to regulate the change of fluorescence, so it has some advantages in selectivity and sensitivity. At present, it has been a hot spot in the field of fluorescence sensing, and has been widely used in real-time monitoring and imaging of biologically related small molecules or ions. In this paper, the mechanism of electron transfer, such as hydrogen sulfide, biological mercaptan, biological active oxygen species and hypochloric acid, which can regulate the important physiological and pathological functions of biology, is combined with specific chemical reactions and the mechanism of electron transfer induced by snail ring opening and photoinduced electron transfer. The related fluorescent probes were designed and synthesized, and the sensing performance of the probes was systematically evaluated under simulated physiological conditions, and the specific fluorescence imaging of this series of biological small molecules was realized at the cellular level. In addition, using the specific chemical reaction between o-aminophenylacetylene derivatives and Au~ (3), a fluorescence probe with high selectivity to recognize Au~ (3) was constructed, which provides a visual tool for the study of the role of the ion pharmacology and pathology. The related contents are summarized as follows: (1) with 5 (6) -carboxyl-rhodamine as the signal unit, 7-nitrobenzoxadiazole as the reaction unit, Based on the "on-off" principle of the snail ring and the photoinduced electron transfer (PET) mechanism, the 2-1a and 2-1b fluorescence probes of messenger molecule hydrogen sulfide (H 2S) and biological mercaptan were designed and synthesized. In the PBS buffer solution (10 mM,pH=7.4,1 m M CTAB), the probe 2-1a mainly existed as non-fluorescent spirolide. After captopolysis with H _ 2S, the snail ring was opened, the solution changed from colorless to pink, and the fluorescence increased nearly 160-fold. In view of the good cell permeability, the probe has realized the fluorescence image of exogenous H2S in HaLa cells. In the PBS buffer solution (10 mMg pH = 7.4), the probe 2-1b mainly exists in the form of ring opening, and the probe itself has no fluorescence due to the existence of PET from Rhodamine fluorescence group to NBD group. However, after reaction with cysteine, the NBD unit of the probe cleavage. Rhodamine fluorescence group was released, resulting in obvious fluorescence enhancement, and the detection limit was up to 2.8 脳 10 ~ (-8) mol / L. (2) A mitochondrial targeting group was designed and synthesized with naphthalene diimide as fluorescence group, selenomorpholine as reaction group and triphenylphosphine cation as targeted group. The fluorescence probe 3-1 can be used to detect hypochlorite (HOCl). In PBS buffer solution (20 mM,pH=7.4), the probe has almost no fluorescence due to the PET process, and the selenium atom of the probe is oxidized to selenium sulfoxide after the reaction with HOCl, which inhibits the quenching of PET and leads to obvious fluorescence enhancement. It is worth noting that the probe has a very high sensitivity to HOCl and can cause 40 times fluorescence enhancement with a detection limit of 4.2 nM.. In addition, the probe showed good selectivity to HOCl, and no other bioactive oxides could induce obvious fluorescence response. The probe has been successfully used for the detection of mitochondrial and Qg derived HClO in RAW246.7 cells. (3) the fluorescence probe 4-1, which can selectively recognize Au~ (3), was constructed with fluoroborboron dipyrrolidine-methylpyrrolidene (BODIPY) as the reactive group and phenylethynyl aniline as the reaction group. In EtOH-PBS buffer solution (20 mM,pH=7.4,v/v=1:1), the probe has almost no fluorescence due to the PET process, and in the presence of Au~ (3), the probe has an intramolecular cyclization reaction, resulting in the conversion of 2- (phenylethynyl) aniline to 2-phenylindole. This process inhibits the quenching of PET and leads to a marked enhancement of fluorescence. Selective studies show that other metal cations can hardly cause the fluorescence changes of the probe. The probe has been applied to the detection of Au~ (3) in water samples and fluorescence imaging of Au~ (3) in HeLa cells.
【学位授予单位】：山西大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：O657.3;TP212.3

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