湖南大学
基于IR-780荧光团的Zn<'2+>近红外荧光探针研究 姓名：***
申请学位级别：硕士
专业：分析化学
指导教师：***
20070501
硕士学位论文
摘要
锌离子是多种酶和转录因子的必需组成成分，广泛的分布于人体的细胞和体液中，直接参与体内细胞的
生长发育、生殖和组织修复等各种生命代谢过程。Zn2+在细胞的生命活动中起着非常重要的作用，在基因转录、金属酶催化、神经传递等过程中都有Zn2+的参与。随着人们对Zn2+在生命活动中的作用认识越来越深，细胞内Zn2+的研究成为化学、生物学和临床医学等学科重要的前沿热点研究课题。荧光检测方法具有操作简便、灵敏度高等优点，近红外荧光探针更是具有激发波长较长，可避免对活体细胞的损伤及其减小背景干扰的优势，因此设计合成Zn2+的近红外荧光探针具有重要意义。碱性磷酸酯酶（ALP）是一种金属锌酶，它广泛的存在于各种生物体内，在磷酸盐的代谢中起着重要的作用。ALP在组织中的含量和活性还是肝胆疾病和骨骼疾病的一个指示剂，在临床诊断和分析中具有十分重要的意义。因此设计合成高灵敏度的ALP荧光探针具有十分重要的意义。本论文主要完成了以下研究工作：
1、以三碳菁染料IR-780为荧光团，三(2-氨乙基)胺（TAEA）为识别基团合成了一种新型的Zn2+近红外荧光探针TAEA-IR-780，该探针通过配位作用与Zn2+结合后，荧光显著增强。探针的结构用质谱、氢谱进行了表征，并探讨了pH值、其它金属离子对Zn2+检测的影响。该探针的激发波长为683 nm，发射波长为750 nm，可避免对生物活体细胞的损伤，同时生物体内常见的金属离子对其干扰较小。该探针对Zn2+的检测下限达1.00×10-9 mol·L-1，具有较高的检测灵敏度。
2、三碳菁染料IR-780为荧光团，合成了一种以二（2-吡啶甲基）胺（DPA）为识别基团的Zn2+近红外荧光探针DPA-IR-780，该探针与Zn2+结合后，荧光明显增强。探针的结构用质谱、氢谱进行了表
征，并探讨了pH值、其它金属离子对Zn2+检测的影响。该探针的激发波长为686 nm，发射波长为764 nm，可避免对生物活体细胞的损伤，同时生物体内常见的金属离子对其干扰较小。该探针对Zn2+的检测下限可达1.00×10-10 mol·L-1，具有较高的检测灵敏度。
3、合成了一种ALP的荧光探针荧光素单磷酸酯（FMP），探针在ALP的催化作用下水解，荧光显著增强。探针的结构用质谱、氢谱进行了表征，并研究了不同的培育时间，不同浓度的ALP对催化FMP水解情况的影响，探讨了ALP催化FMP 水解的作用机理。该探针的检测下限为1.83×10-9 mg·mL-1，有较高的灵敏度。
关键词: 近红外；荧光探针；荧光素；锌离子；碱性磷酸酯酶；三(2-氨乙基)胺；二（2-吡啶甲基）DPA；
基于IR-780 荧光团的Zn2+ 近红外荧光探针研究
Abstract
Zinc ion (Zn2+) is an essential component of many enzymes and transcription factors, which exists in cells and body fluid. Zn2+ directly participates in various metabolic process, such as cell growth and procreation, tissue repair, genetic transcription, metalloenzyme catalysis and neurotransmission.
It has been reported that Zinc deficiency leads to the dysfunction and defect of immune systems. Moreover, Zn2+ has been reported to induce selective neuronal cell death that is associated with certain acute conditions, including epilepsy, transient global ischemia and brain injure. Because of the significance of Zn2+ in biological system, the research of Zn2+ in cell becomes a hot research topic in chemistry, biology and clinical medicine. Fluorescent detection has the advantages of easy operation and high sensitivity, especially, near-infrared fluorescent probe has the predominance of avoiding cell damage and minishing interference by autofluoresence from biological molecules. It has important significance to design and synthesize the near-infrared fluorescent probe of Zn2+. Alkaline Phosphatase (ALP) is a kind of metalloenzyme, which extensively exists in various tissues of body and plays an important role in metabolizability of phosphate. ALP is an indicator of hepatobiliary and bone disorder.Therefore, it needs exist to detect ALP sensitively and selectively in many diagnostic and clinical assays. So to design the high sensitivity fluorescent probe of ALP also has important significance. The detailed materials are summarized as following:
1. A novel near-infrared fluorescent probe, TAEA-IR-780, was proposed for the measurement of Zn2+, which was obtained by directly attaching an acceptor, tris (2-aminoethyl) amine (TAEA), to the near-infrared fluorophore IR-780. The excitation and emission wavelengths of TAEA-IR-780 were 68
3 nm and 750 nm, respectively, and its fluorescence intensity increased with the addition of Zn2+ under physiological condition (pH=7.4) and the detection limit reached 1.00 × 10-9 mol·L-1. Moreover, the fluorescence intensity of TAEA-IR-780 did not noticeably increase in the presence of other biologically important cations such as Ca2+ and Mg2+. The results showed that the probe could not only effectively minimize cell damage and autofluorescence by the near-infrared excitation light but also afford sufficient sensitivity and selectivity for the biological detection of Zn2+.
2. A near-infrared fluorescent probe for Zn2+, DPA-IR-780, was designed and
硕士学位论文
synthesized, which based on Di-(2-picoylyl)amine (DPA) as the recognition group. The structure of the probe was charactered by 1HNMR, MS. The excitation and emission wavelengths of the as prepared DPA-IR-780 were 686 nm and 764 nm, respectively,
and its fluorescence intensity increased with the addition of Zn2+ under physiological condition (pH=7.40) and the detection limit reached 1.00 × 10-10 mol·L-1. Furthermore, this probe has strong anti-interference ability under the existing other metals. At the same time, because of the long excitation wavelength and emission wavelength, the probe could effectively minimize cell damage an
d minish interference by autofluoresence from biological molecules.
3. A new fluoremic substrate,3-phoshpase-fluorescein (FMP), detecting alkaline phosphatase (ALP) was developed in this part. The synthetic method was sample, convenient, economical and this new fluoremic substrate was sensitive to ALP. The background fluorescent intensity of FMP was very weak, while the fluorescent intensity was strong when the ALP added after incubation for 40 min at 37℃. And the experiment showed that FMP was relative stable in the Tris·Cl buffer (PH=9.10). The fluorescent intensity increased with the increasing of ALP concentration. About 40 min, the fluorescent intesity could reach a plateau. And the detection limit could reach to 1.83 × 10-9 mg·mL.
Key words:Near-Infrared; Fluorescent Probe; Fluorescein; Zn2+; Alkaline Phosphatase; Tris (2-aminoethyl) amine; Di-(2-picoylyl)amine
湖南大学
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