【作者】 谭轶群；

【导师】 钱国栋；

【作者基本信息】 浙江大学 ， 材料学， 2014， 博士

【摘要】 近年来,荧光探针由于在环境保护、生物成像以及医学诊断等领域显示出重要的应用价值而越来越多地受到材料和化学研究工作者的重视。尽管目前用于检测环境和生物体常见金属离子和化学物质的荧光探针有很多,但是针对过渡族重金属离子、有毒中性化学物质的荧光探针还较少,并且多数不能在生物体外的极端环境下应用。此外,由于荧光探针的工作环境复杂性,许多荧光探针会与多种物质结合并产生相同的荧光响应,选择性较差。这一现象在金属离子,尤其是过渡族重金属离子的探测过程中十分普遍。针对上述问题,本文开展了可用于过渡族金属离子和中性化学物质以及极端环境下pH检测的荧光探针的研究：设计并合成了一系列具有高灵敏度和高选择性,可用于环境检测和生物成像的荧光探针,探讨了荧光探针的构效关系以及溶剂效应对荧光探针性能的影响；改进了Job’s曲线法,以测定淬灭型荧光探针的配位比；研究了共轭体系对于荧光探针识别基团的性能影响及其在扩大探测范围方面的应用；深入研究了利用分子内电荷转移(Intramolecular Charge Transfer, ICT)机理使荧光探针具有区分相似金属离子的方法,探讨了溶剂极性、探针结构对于荧光探针选择性探测能力的影响,并总结出一般规律；通过生物成像实验探讨了ZC-F系列荧光探针的生物相容性和膜透性及其在生物体内定位和离子识别的能力。设计合成了一种基于ICT机理的新型Co2+荧光探针2,6-双(4-二苯氨基苯乙烯羰基)吡啶(PhPy)。通过紫外-可见吸收光谱和荧光光谱等分析手段,系统研究了该探针的光学性能及其对Co2+的选择性探测能力。研究发现,该探针可实现对Co2+高灵敏度、高选择性的荧光识别,而对其他常见的或与Co2+具有相似化学结构的金属离子几乎没有荧光响应,可用于Co2+的定量检测和实时监控；通过对Job’s曲线法进行改进,使其可用于淬灭型荧光探针的配位比的测定,确定了PhPy与Co2+的化学计量比；此外,PhPy还表现出了显著的溶剂显色效应,随溶剂极性的变化,该探针的荧光发射谱具有较大的位移,表明其在极性探测中有潜在应用价值。基于联氨对丙二腈基团的消去作用设计并合成了一种反应型荧光探针2-(4-(4-苯并噻唑基苯乙炔基)苯乙烯基)丙二腈(BP)。研究表明该探针溶剂显色效应显著,对联氨具有极高的灵敏度,检出限低至0.11ppb,优于美国国家标准,并且其他自然界常见离子对其没有干扰,选择性良好；该探针对联氨的检测范围宽泛,响应速度快,荧光信号可在2.5分钟内达到稳定,具备实时、定量检测痕量联氨的能力；此外,BP自身荧光信号极弱,为增强型荧光响应,可以有效降低探针自身的干扰,提高了检测的可信度。设计合成了一种可用于强酸性环境下pH检测的荧光探针4-(4-(苯并噻唑基苯乙炔基)苯胺(BA)。通过偶联反应将具有较大共轭平面和适中吸电子能力的苯并噻唑与氨基共轭连接,合成了基于ICT机理的荧光探针BA；紫外-可见吸收光谱和荧光发射谱研究表明,溶剂极性对该探针的荧光性能影响极大,但是对吸收谱影响较小,并且随着溶剂极性的增加,荧光探针BA出现明显的荧光发射谱红移、斯托克斯位移增大以及荧光寿命和量子效率降低的现象,表明BA分子内部的ICT效应显著；该探针对强酸性环境下pH值的变化较为敏感,其pH定量检测范围低至0.5～2.5,并且具有较高的灵敏度和良好的选择性；通过理论计算对荧光探针BA进行了分子构型优化,结果表明,受到ICT效应与溶剂效应双重影响,质子化前后探针分子BA的电荷排布与电荷转移情况发生了较大变化,HOMO-LUMO能隙增大,进而导致了荧光增强和发光峰蓝移。选用三联吡啶为识别基团,以水溶性的香豆素衍生物为发光基团,通过一系列化学反应,设计合成了两种可用于探测Zn2+的荧光探针(E)-3-(3-(4-(2,2’：6’,2”-三联吡啶基)-4’-苯基)丙烯酰基)-7-二乙胺基香豆素(ZC-F4)和3-(4-(2,2’：6’,2”-三联吡啶)-4’-苯乙烯基)-7-甲氧基香豆素(ZC-F7)；两种荧光探针性能对比发现ZC-F4具有更长波长的吸收和荧光发射,这是由于该探针引入了给电子能力更强的二乙胺基；荧光探针ZC-F4和ZC-F7均展现了较高的灵敏度和良好的选择性,在Zn2+含量低至10nM (0.65ppb)时即发出明显的荧光响应信号,并且几乎不受其他金属离子干扰。pH滴定实验表明ZC-F4在pH为6-9之间具有良好的稳定性,适用于人体生理环境,而Job’s曲线分析则证明这两种探针与Zn2+的配比关系均为1：1；通过理论计算探究了ZC-F4的探测机理,发现三联吡啶与Zn2+结合后吸电子能力增强,导致ZC-F4内部的电荷重排增强,而三联吡啶末端的两个吡啶环起到了扩大共轭面积,增强ICT效应的作用；细胞成像实验发现与ZC-F4和ZC-F7共培养的细胞生长正常,表明这两种探针探针具有膜透性,可用于细胞内的Zn2+标记和荧光成像。将识别基团三联吡啶与基于苯并噻唑的荧光团共轭相连,获得了两种具有较高量子效率、对Zn2+和Cd2+具有不同荧光响应信号的荧光探针2-(4-((4-(2,2’,6’,2”-三联吡啶基)-4’-苯基)乙炔基)苯基)苯并噻唑(ZC-F1)和2-(4-((7-((4-(2,2’：6’,2”-三联吡啶)4’-苯基)乙炔基)-9,9-双(2-(2-乙氧基乙氧基)乙基)-9氢-芴)-2-乙炔基)苯基)-苯并噻唑(ZC-F2)。这两种探针与Zn2+和Cd2+均形成了1：1化学计量比的配合物,都可以通过荧光颜色的不同区分Zn2+和Cd2+；滴定实验表明ZC-F1与ZC-F2的灵敏度极高,可用于浓度在ppb级别的Zn2+和Cd2+区分探测,而对比选择性实验则证明这两种探针具有良好的选择性,不受其他金属离子的干扰；通过理论计算深入研究这两种探针区分Zn2+和Cd2+的机理,结果发现由于Zn2+和Cd2+之间电负性的差异,使得其对荧光探针的发射谱位移具有不同影响,进而导致不同波长的荧光发射；双光子测试研究发现ZC-F2具有较高的双光子吸收截面,并且ZC-F2-Zn具有比ZC-F2-Cd更高的双光子吸收截面,这也是由于Zn2+具有较高的电负性造成的；荧光探针ZC-F2在细胞内Zn2+和Cd2+成像实验表明ZC-F2具有细胞膜透性,与探针共培养的细胞生长正常,可以用于细胞内的离子定位和成像,在细胞内主要集中在细胞器表面。通过对比ZC-F1和ZC-F2的性能,研究总结了基于ICT机理设计可区分探测相似结构金属离子的一般规律,发现扩大共轭面积可以提高探针的光学性能,得到长波长的荧光发射,利于生物应用,但是会降低分辨效果；同时,溶剂极性对于区分效果有明显的影响,极性越大的溶剂里,Zn2+和Cd2+对应的发射谱峰位差别越大,因此选择共轭面积恰当的发光基团和合理的溶剂具有重要意义。更多还原

【Abstract】 Rescently, fluorescent probes has attracted much more attention for its significant useage in environmental protection, bio-imaging and medical treatment. However, although there are many probes towards metal ions and chemicals common for biology and environmental use, little studies on probes for trasition-metal ions and toxic neutral chemicals have been reported, and most of the which can not be used in vitro under extreme environment. In addition, many fluorescent probes will coordinate with two or more chemicals, resulting in similar fluorescence signal responses and poor selectivity, because of their complex nature and complicated working environments.This phenomenon can be observed in the probes for metal ions, especiallythose for transition-metal ions.To solve these problems, studies on fluorescence probes for transition-metal ions, neutral chemicals and strong acidic pH detection are performed in this paper. A series of fluorescent probes for environmental detection and bio-imaging with high sensitivity and selectivity are designed and synthesized and their structure-property relationship and solvent effect are studied. Job’s plot analysis are improved for "ON-OFF" probes To get insight into the sensing mechanism of fluorescent probes based on ICT effect for recognizing similar metal ions and and summarize a common rule, the effect of conjugated system and solvent molecules on the sensing properties of fluorescence probes are also discussed. In addition, the biocompatibility and membrane permeability of ZC-F series fluorescent probes have been confirmed through bio-imaging experiments.A novel fluorescence chemosensor based on intramolecular charge transfer (ICT) mechanism was designed and synthesized and its photophysical properties were characterized. By coordinating of Co2+and central2,6-dicarbonylpyridinyl function group, the chemosensor2,6-bis(4-diphenylamino-styrylcarbonyl)pyridine (PhPy) showed nearly complete fluorescence quenching towards Co2+while no fluorescence response to other competing cations, suggesting its potential usage in quantitative and real-time detection of Co2+. By improving Job’s plot analysis to make it suitable for "ON-OFF" fluorescent probes, the stoichiometry between PhPy and Co2+was determined. Furthermore, PhPy also exhibited significant solvatochromic effect depending on the polarity of solvent, indicating its potential usage as polarity probes.A new both fluorescent and colorimetric probe2-(4-((4-(benzo[d]thiazol-2-yl) phenyl)ethynyl)benzylidene)malononitrile (BP) based on reaction and ICT effect is designed and synthesized. The probe responds rapidly towards hydrazine and exhibits an apparent color changes from yellow to a colorless state, indicating that this probe can be used as a color indicator for hydrazine. Meanwhile, the probe also shows a significant enhancement on fluorescence by around120-fold after the addition of hydrazine in a broad band (410-700nm). With a detection limit as low as0.11ppb, the probe can detect hydrazine in a wide concentration range because of the blunted sensing functional group. The contrast test shows nearly no interruption from some common elements in water, suggesting the high selectivity of this probe towards hydrazine. Moreover, the theoretical calculation based on density functional theory (DFT) is also performed to get insight into the sensing mechanism and two different ICT modes are found. This hydrazine probe would be a promising candidate for the applications in environment protection, water treatment and safety inspection.A fluorescent pH sensor4-((4-(benzothiazol-2-yl)phenyl)ethynyl)aniline (BA) for strong acid environment based on ICT effect was realized to exhibit high stability, sensitivity and selectivity. The absorption and fluorescent emission spectra suggest that solvent molecules have significant influence on the fluorescent properties of BA, while minor effect on absorption can be observed, indicating strong ICT effect in this probe. This probe can be used to detect acidity within the range of0.5～2.5with high sensitivity and selectivity. Theoretical calculations are carried out to find that great changes of electronic locations before and after protonation lead to larger HOMO-LUMO energy gap and fluorescence enhancement. The design strategy may help to develop probes for targets at high concentration.By introducing terpyridine as recognizing function and water soluble coumarin as fluorophore, two new fluorescent probes (E)-3-(3-(4-([2,2’:6’,2"-terpyridin]-4’-yl)phenyl)acryloyl)-7-(diethylamino)-2H-chromen-2-one (ZC-F4) and3-((4-([2,2’:6’,2"-terpyridin]-4’-yl)phenyl)ethynyl)-7-methoxy-2H-chromen-2-one (ZC-F7) are designed and synthesized. Comparing with ZC-F7, ZC-F4has absorption and emission spectra located at longer wavelength because of its stronger electronic donor. Both these two probes exhibit good selectivity and sensitivity towards Zn2+even at10nM (0.65ppb) with significant variation of emission wavelength (more than100nm shifts). One can observe the emission colour converted from green to red. pH titration suggest good stability of ZC-F4under pH6-9for biology use. Job’s plot test suggests a1:1stoichiometry between probes and Zn2+, and the theoretical calculation based on density functional theory has been carried out to get insight into the sensing mechanism. Furthermore, the imaging of Zn2+in cells are also applied to test their feasibility in biology. These two fluorescence probes would be a promising candidate for the applications in cell-imaging, environment protection, water treatment and safety inspection.By conjugating terpyridine as receptor and fluorophore based on benzothiazole, two new fluorescent probes2-(4-((4-([2,2’,6’,2"-terpyridin]-4’-yl)phenyl)ethynyl) phenyl)benzo-[d]thiazole (ZC-F1) and2-(4-((7-((4-([2,2’:6’,2"-terpyridin]-4’-yl) phenyl)ethynyl)-9,9-bis(2-(2-ethoxyethoxy)ethyl)-9H-fluoren-2-yl)ethynyl)phenyl)-be nzo[d]thiazole (ZC-F2) with different fluorescence response towards Zn2+and Cd2+are designed and synthesized. Baring high fluorescence quantum yield, both of these probes recognize Zn2+and Cd2+with stoichiometry of1:1. Titration experiments suggest high sensitivity of these probes even at ppb level without interuption from other metal ions. To get insight into the sensing mechanism of these probes, theoretical calculations are carried out and the difference between the electronegativity of Zn2+and Cd2+is confirmed to be the root cause, which induces different emission spectra shift, leading to different emission color. Studies on two-photon absorption (TPA) suggest high TPA cross-section (8) of ZC-F2, while ZC-F2-Zn has higher8, which can be ascribed to higher electronegativity of Zn2+. In addition, cell imaging experiments of ZC-F2towards Zn2+and Cd2+certify its membrane permeability and biocompatibility, suggest its potential use in metal ions positioning in vivoSome rules for designing fluorescent probes which can be used to distinguish similar metal ions based on ICT mechanism are summarized through comparison of ZC-F1and ZC-F2. By enlarging the conjugating system, a probe with higher optical properties, longer emission wavelength for biology use can be expected, accompany with lower recognizing ability. Meanwhile, solvent polarity is found to be influential in the distinguishment. larger distinction between Zn2+and Cd2+can be observed in solvents with higher polarity. Thus, rational choice of conjugation system and solvents is of great importance.更多还原