【作者】 齐海萍；

【导师】 杨小峰；

【作者基本信息】 西北大学 ， 分析化学， 2010， 硕士

【摘要】 荧光比率法是通过测量两个不同波长处的荧光强度,以其比值作为信号参量,从而来测定目标物的分析方法。荧光比值信号不受光源强度和仪器灵敏度的影响,因此与传统的荧光探针相比,比率型荧光探针可以提高方法的选择性、灵敏度和动态响应范围。比率型荧光探针的设计机理主要有荧光共振能量转移、激发态分子内质子转移、分子内电荷转移等。2-(2-羟基苯基)苯并咪唑(2-(2-hydroxyphenyl) benzimidazole, HPBI)由于存在激发态分子内质子转移(excited state intramolecular proton transfer, ESIPT)过程,发射双重荧光峰。如果我们对HPBI结构中的羟基进行修饰,则该分子的ESIPT过程不能有效发生,从而使HPBI的荧光光谱发生相应改变。本文以HPBI为母体,将其结构中的羟基作为比率型荧光探针的开关,构建了对F-,碱性磷酸酶(alkaline phosphatase, ALP)和P2O74-具有选择性响应的比率型荧光探针。同时本文以2-苯基苯并咪唑为母体,在苯并咪唑的对位引入醛基,生成2-(4-醛基苯基)苯并咪唑。该化合物结构中含有供电子基团苯并咪唑基和强吸电子基团醛基,受紫外光激发,可有效发生分子内电荷转移机理(intramolecular charge transfer, ICT),因此,2-(4-醛基苯基)苯并咪唑发射双重荧光峰。如果我们利用此化合物结构中的醛基与HSO3-反应,则分子的吸电性能力降低,导致ICT过程不能有效地发生,从而使体系的荧光特性发生变化。因此,我们以2-(4-醛基苯基)苯并咪唑实现了对HSO3-的比率型响应。本论文包括以下五部分内容：第一章绪论。主要介绍了比率型荧光探针的设计机理,在此基础上提出本论文的设想。第二章基于ESIPT机理测定F-的比率型荧光探针。本章通过HPBI分子结构中羟基的保护／去保护反应,设计合成了一种测定F-的比率型荧光探针1。化合物1由于不能发生ESIPT过程,仅仅在360 nm处发射单一荧光峰。在DMF水溶液中加入F-,化合物1结构中的TBS官能团被F-选择性的切割,从而恢复了母体荧光分子HPBI的ESIPT过程,导致反应体系在360 nm处的荧光发射减弱,同时454 nm处的荧光发射增强。利用反应前后体系在360 nm与454 nm处的荧光强度的比值变化(I454／I360)可对F-的含量进行定量。第三章荧光比率法测定ALP的性质研究。本章通过三氯氧磷与HPBI分子中的羟基反应,然后水解,生成荧光探针分子2。化合物2由于阻断了ESIPT过程的正常进行,于363 nm处发射单一荧光峰。在2的溶液中加入ALP,ALP可选择性地使2分子中的磷酸基团水解,恢复了原来的母体荧光分子结构HPBI,从而使体系的ESIPT过程有效发生,导致体系在430 nm处出现了一个新的荧光发射峰。随着ALP浓度的增加,反应体系在430nm处的荧光发射强度逐渐增强,同时363 nm的荧光发射逐渐减弱。利用反应前后体系的荧光强度比值I430／I363可对ALP进行定量分析。该方法测定ALP的线性上限至0.050 UmL-1,检出限为0.0013 U mL-1。第四章同步荧光法测定焦磷酸根的性质研究。本章以HPBI为母体,将其与Zn2+络合,反应生成金属配合物(化合物3)。由于HPBI分子中羟基上的氢被束缚住,因此化合物3不能有效发生ESIPT过程,其荧光发射波长为418 nm。当向体系中加入P2O74-后,P2O74-中的磷酸基可有效地与HPBI竞争Zn2+离子,从而使3离解,恢复了HPBI分子本身的ESIPT过程。然而化合物3和反应产物HPBI的最大发射波长(454 nm)相差较近,二者的荧光发射光谱容易重叠,因此实验考虑采用同步荧光法。HPBI在364 nm处存在另一荧光发射峰,其斯托克斯位移Δλ与3的发射峰的从很相近,实验中采用同步荧光法并且选用Δλ为40 nm,可以将HPBI在364 nm处的荧光发射峰和化合物3的荧光发射峰很好地区分开来。在3的溶液中加入P2O74-后,反应体系在364 nm处的荧光发射增强,同时在418 nm处的荧光发射减弱。基于此原理,本章建立了一种测定P2O74-的同步荧光比率法。第五章一种表征HSO3-的比率型荧光探针的合成及性质研究。本章设计合成了一种测定HSO3-的新型比率型荧光探针4。化合物4由于存在分子内电荷转移过程,分别于368 nm和498 nm发射荧光峰。当在化合物4中加入HSO3-后,4中的醛基与HSO3-发生亲核加成反应,从而阻断ICT过程的正常进行,使4在368 nm处的荧光发射增强,同时498 nm处的荧光强度减弱,并且这两个荧光峰的强度比值(I368／I498)与HSO3-浓度在2×10-6-2.0×10-4mol L-1范围内呈线性关系。该探针对HSO3-响应速度快,选择性好。更多还原

【Abstract】 Ratiometric fluorescent probes allow the measurement of emission intensities at two different wavelengths, which can overcome the drawbacks of intensity-based measurements due to a built-in correction for environmental effects (i.e. canceling artifacts due to instrumental efficiency and light intensity) and increase the selectivity, sensitivity and dynamic ranges of the method. The design mechanisms for ratiometric fluorescent probes are based on fluorescence resonance energy transfer (FRET), excited-state intramolecular proton transfer (ESIPT), intramolecular charge transfer (ICT) and so on.2-(2-hydroxyphenyl) benzimidazole (HPBI) could undergo an ESIPT process upon light excitation and displayed fluorescence emission at 360 and 454 nm. If the hydroxyl group of HPBI was modified, ESIPT process of HPBI would be switched off and its spectral characteristics should change. Based on the consideration, three ratiometric fluorescent probes for F-, alkaline phosphatase (ALP) and P2O74- were designed based on modulation of ESIPT process via modification of the functional hydroxyl group of HPBI in the thesis. In addition, we developed 2-(4-aldehydephenyl)benzimidazole (4) as a novel ratiometric fluorescent probe for HSO3-. Compound 4 could undergo an intramolecular charge transfer (ICT) from electron rich benzimidazole moiety to electron deficient aldehyde group upon photoexcitation. Thus,4 could emit two emission bands. If the aldehyde group on 4 interacted with HSO3-, the electron deficient capability of compound 4 decreased and the ICT process would be switched off, which resulted fluorescent spectral characteristics of the system changed. Based on the mechanism,4 displayed a ratiometric response to HSO3-. The thesis composes of five chapters as follows:In chapter 1, several mechanisms for designing ratiometric fluorescent probes were introduced, and research content of the thesis was presented.In chapter 2, a ratiometric fluorescent probe for fluoride ion employing ESIPT process. The probe 1 was developed based on modulation of ESIPT process of HPBI through the hydroxyl group protection/deprotection reaction. Because the ESIPT process was switched off, the probe 1 showed only fluorescence emission maximum at 360 nm. Upon treatment with fluoride in aqueous DMF solution, the TBS protective group of probe 1 was removed readily and ESIPT of the probe was switched on. Accordingly, it was observed that the fluorescence emission at 360 nm showed "turn-off", while the fluorescence emission at 454 nm showed "turn on". The proposed probe showed excellent selectivity toward fluoride ion.In chapter 3, an ESIPT-based approach to ratiometric fluorescent detection of ALP. The probe 2 was readily prepared by the reaction of HPBI with phosphorus oxychloride (POCl3) and then hydrolyzed to give the desired product. In the absence of ALP, free 2 exhibited one typical emission peak at 363 nm. However, addition of ALP to the solution of 2 induced formation of a new emission peak at 430 nm because 2 was hydrolyze into HPBI by ALP and ESIPT of the probe was switched on. Moreover, with an increasing amount of ALP, the emission band of probe 2 at 360nm gradually decreased with the concomitant growth of emission band at 430 nm. The fluorescent intensity ratio at 430 and 363 nm (I430/I363) increased linearly with the activity of ALP up to 0.050 U mL-1 with a detection limit of 0.0013 UmL-1.In chapter 4, a new P2O74--selective ratiometric synchronous fluorescent probe was designed and synthesized. The probe 3 was prepared by the reaction of HPBI with Zn2+ Because its hydroxyl group was bound, the ESIPT process of HPBI was switched off and probe 3 showed fluorescence emission maximum at 418 nm. Upon introducing P2O74- to a solution of probe 3, P2O74- could compete effectively Zn2+ with HPBI, which resulted in the decomposition of 3 into HPBI and restoration of the ESIPT process. However, the emission wavelength of 3 (418 nm) was close to the maximum emission wavelength (454 nm) of HPBI and their fluorescence spectra were prone to overlap. Therefore, synchronous fluorescence technique was applied in this experiment. HPBI showed another fluorescence emission at 364 nm and its stockes shift (△λ) was very close to that of 3. Therefore, fluorescence emission spectra of 3 could distinguish from the emission band of HPBI at 364 nm by synchronous fluorescence technique with a△λof 40 nm. Addition of P2O74- to a solution of 3 induced a decrease of the emission band at 418 nm and an increase of a new fluorescence peak at 364 nm simultaneously. Based on above mechanism, a ratiometric synchronous fluorescent probe for P2O74- was developed. The proposed probe showed excellent selectivity toward P2O74-over other common anions.In chapter 5, a novel ratiometric fluorescent probe for HSO3- was developed based on ICT process. The probe 4 showed two fluorescence emission bands centered at 368 nm and 498 nm, respectively. Upon addition of HSO3-, the aldehyde of 4 reacted with bisulfite and produced an aldehyde-bisulfite adduct, which resulted in a decrease of the emission band at 498 nm and an increase of a fluorescence peak at 368 nm due to switching off ICT process of probe 4. The fluorescent intensity ratio at 368 and 498 nm (I368/I498) increased linearly with HSO3- concentration in the range 2×10-6-2.0×10-4mol L-1. The proposed method showed high sensitivity and excellent selectivity toward HSO3-.更多还原