基于荧光共振能量转移的i-motif探针用于细胞内外pH值检测

发布时间：2018-06-18 09:06

  本文选题：FRET + i-motif　； 参考：《湖南大学》2016年硕士论文

【摘要】：pH在生命活动中扮演着非常重要的角色,它密切参与细胞、酶和组织的活动。pH值异常会影响免疫系统或神经系统,甚至与癌症等严重疾病紧密相关。因此,对pH值进行精准测量,能为生理和病理的研究提供至关重要的信息。荧光共振能量转移(FRET)是指在两个荧光基团中,如果一个荧光基团(供体)的荧光发射光谱与另一个荧光基团(受体)的吸收光谱产生一定的重叠,当这两个荧光基团之间的距离合适时(一般小于10 nm),供体就能激发受体发出荧光,同时供体自身的荧光强度衰减。富含胞嘧啶(C)的寡核苷酸序列上,C：C+碱基对在酸性条件下能够以交替排列和互相嵌入的形式形成四聚体结构,这种四聚体称为i-motif结构。Ⅰ-motif对生理范围内的pH值具有很灵敏且可逆的响应。在本论文中,我们基于FRET的原理和i-motif对H+响应的特性,发展了两种比率型探针分别对细胞内和细胞外的pH进行检测,具体内容如下：1.结合i-motif和金纳米颗粒的优势,构建了一种双标记的比率型纳米探针用于细胞内pH的检测。将两端标记了荧光基团的i-motif链与标记了巯基的互补链杂交,通过金-巯键将其修饰到金纳米颗粒表面。以金纳米颗粒作为载体,探针能通过胞吞作用无损伤地进入细胞内。在pH较高的条件下,双链杂交形成刚性的双螺旋结构,i-motif链两端的荧光基团处于分离状态,FRET效应很弱；在pH较低的条件下,i-motif链形成四聚体结构离开金纳米颗粒表面,两端的荧光基团相互靠近,产生较强的FRET效应。通过测定两种荧光基团的荧光强度比值可以实现pH值的定量检测。该探针设计新颖,响应快速,灵敏度高,具有良好的生物相容性,能进入细胞溶酶体并对细胞内的酸性环境进行高分辨率时空成像,pH检测范围为5.5-7.0。2.根据细胞表面氨基易生物素化的特性以及链霉亲和素-生物素之间的强相互作用,首次将i-motif序列固定于细胞表面,构建了一种细胞表面固定化的比率型探针用于细胞外pH检测。将标记了荧光基团的i-motif链与同时标记了荧光基团和生物素的互补链杂交,通过链霉亲和素-生物素相互作用将其固定到细胞表面。在pH较高的条件下,双链杂交形成刚性的双螺旋结构,两条链上的荧光基团彼此远离,FRET效应很弱；在pH较低的条件下,i-motif序列形成四聚体结构,i-motif链上的荧光基团弯曲与互补链上的荧光基团相互靠近,FRET效应增强。该方法操作简单,灵敏度高,可逆性好,具有良好的通用性,pH检测范围为5.2-6.8。
[Abstract]:Ph plays a very important role in life activities. It is closely involved in the activities of cells, enzymes and tissues. The abnormal pH value will affect the immune system or nervous system, and even be closely related to serious diseases such as cancer. Therefore, accurate pH measurement can provide vital information for physiological and pathological studies. Fluorescence resonance energy transfer (fret) means that in two fluorescence groups, if the fluorescence emission spectra of one fluorescence group (donor) overlap with the absorption spectrum of another fluorescence group (receptor), the fluorescence emission spectrum of one fluorescence group (donor) overlaps with the absorption spectrum of the other group (receptor). When the distance between the two groups is suitable (generally less than 10 nm), the donor can excite the fluorescence of the receptor and the fluorescence intensity of the donor itself attenuates. C: C base pairs on the oligonucleotide sequences rich in cytosine C) can form tetramer structures in the form of alternating arrangement and intercalation under acidic conditions. This tetramer is called i-motif structure. I-motif has a very sensitive and reversible response to pH in physiological range. In this thesis, based on the principle of fret and the response of i-motif to H, we developed two ratio probes to detect intracellular and extracellular pH, respectively, as follows: 1. Combined with the advantages of i-motif and gold nanoparticles, a double labeled ratio nanoprobe was constructed for the detection of intracellular pH. The i-motif chains labeled with fluorescent groups at both ends were hybridized with the complementary chains labeled with sulfhydryl groups and modified to the surface of gold nanoparticles by gold-captopril bond. With gold nanoparticles as the carrier, the probe can enter the cells without damage by endocytosis. At high pH value, the fluorescence groups at the ends of the rigid double helix structure of i-motif chain were separated in a weak state, and the tetramer structure was formed to leave the surface of gold nanoparticles at lower pH value, while the fluorescence group at the two ends of the double helix structure was separated from the surface of the gold nanoparticles at high pH value, and the fluorescence group at the two ends of the chain was separated from the surface of the gold nanoparticles. The fluorescence groups at both ends are close to each other, resulting in a strong fret effect. The quantitative determination of pH value can be realized by measuring the ratio of fluorescence intensity of two groups. The probe has the advantages of novel design, rapid response, high sensitivity and good biocompatibility. It can enter the cell lysosomes and detect pH in the acidic environment of cells by high resolution space-time imaging in the range of 5.5-7.0.2. According to the characteristics of amino biotin on cell surface and the strong interaction between streptomycin and biotin, the i-motif sequence was immobilized on the cell surface for the first time, and a ratio probe was constructed for cell surface immobilization to detect extracellular pH. The i-motif strands labeled with fluorescent groups were hybridized with complementary strands labeled with fluorescent groups and biotin at the same time, and were immobilized on the cell surface by the interaction of streptomycin and biotin. Under the condition of high pH, the double strand hybridization formed a rigid double helix structure, and the fluorescence groups on the two chains were far from each other and the fret effect was very weak. At low pH value, the fluorescence group bending on the tetramer structure and the fluorescence group in the complementary chain are close to each other in the tetramer structure, and the FRET effect is enhanced. The method is simple in operation, high in sensitivity, good in reversibility and good in the range of pH detection from 5.2 to 6.8.
【学位授予单位】：湖南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：O657.3
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本文编号：2034945