本文選題：FRET + i-motif��； 參考：《湖南大學(xué)》2016年碩士論文

【摘要】：pH在生命活動(dòng)中扮演著非常重要的角色,它密切參與細(xì)胞、酶和組織的活動(dòng)。pH值異常會(huì)影響免疫系統(tǒng)或神經(jīng)系統(tǒng),甚至與癌癥等嚴(yán)重疾病緊密相關(guān)。因此,對(duì)pH值進(jìn)行精準(zhǔn)測(cè)量,能為生理和病理的研究提供至關(guān)重要的信息。熒光共振能量轉(zhuǎn)移(FRET)是指在兩個(gè)熒光基團(tuán)中,如果一個(gè)熒光基團(tuán)(供體)的熒光發(fā)射光譜與另一個(gè)熒光基團(tuán)(受體)的吸收光譜產(chǎn)生一定的重疊,當(dāng)這兩個(gè)熒光基團(tuán)之間的距離合適時(shí)(一般小于10 nm),供體就能激發(fā)受體發(fā)出熒光,同時(shí)供體自身的熒光強(qiáng)度衰減。富含胞嘧啶(C)的寡核苷酸序列上,C：C+堿基對(duì)在酸性條件下能夠以交替排列和互相嵌入的形式形成四聚體結(jié)構(gòu),這種四聚體稱為i-motif結(jié)構(gòu)。Ⅰ-motif對(duì)生理范圍內(nèi)的pH值具有很靈敏且可逆的響應(yīng)。在本論文中,我們基于FRET的原理和i-motif對(duì)H+響應(yīng)的特性,發(fā)展了兩種比率型探針分別對(duì)細(xì)胞內(nèi)和細(xì)胞外的pH進(jìn)行檢測(cè),具體內(nèi)容如下：1.結(jié)合i-motif和金納米顆粒的優(yōu)勢(shì),構(gòu)建了一種雙標(biāo)記的比率型納米探針用于細(xì)胞內(nèi)pH的檢測(cè)。將兩端標(biāo)記了熒光基團(tuán)的i-motif鏈與標(biāo)記了巰基的互補(bǔ)鏈雜交,通過金-巰鍵將其修飾到金納米顆粒表面。以金納米顆粒作為載體,探針能通過胞吞作用無損傷地進(jìn)入細(xì)胞內(nèi)。在pH較高的條件下,雙鏈雜交形成剛性的雙螺旋結(jié)構(gòu),i-motif鏈兩端的熒光基團(tuán)處于分離狀態(tài),FRET效應(yīng)很弱；在pH較低的條件下,i-motif鏈形成四聚體結(jié)構(gòu)離開金納米顆粒表面,兩端的熒光基團(tuán)相互靠近,產(chǎn)生較強(qiáng)的FRET效應(yīng)。通過測(cè)定兩種熒光基團(tuán)的熒光強(qiáng)度比值可以實(shí)現(xiàn)pH值的定量檢測(cè)。該探針設(shè)計(jì)新穎,響應(yīng)快速,靈敏度高,具有良好的生物相容性,能進(jìn)入細(xì)胞溶酶體并對(duì)細(xì)胞內(nèi)的酸性環(huán)境進(jìn)行高分辨率時(shí)空成像,pH檢測(cè)范圍為5.5-7.0。2.根據(jù)細(xì)胞表面氨基易生物素化的特性以及鏈霉親和素-生物素之間的強(qiáng)相互作用,首次將i-motif序列固定于細(xì)胞表面,構(gòu)建了一種細(xì)胞表面固定化的比率型探針用于細(xì)胞外pH檢測(cè)。將標(biāo)記了熒光基團(tuán)的i-motif鏈與同時(shí)標(biāo)記了熒光基團(tuán)和生物素的互補(bǔ)鏈雜交,通過鏈霉親和素-生物素相互作用將其固定到細(xì)胞表面。在pH較高的條件下,雙鏈雜交形成剛性的雙螺旋結(jié)構(gòu),兩條鏈上的熒光基團(tuán)彼此遠(yuǎn)離,FRET效應(yīng)很弱；在pH較低的條件下,i-motif序列形成四聚體結(jié)構(gòu),i-motif鏈上的熒光基團(tuán)彎曲與互補(bǔ)鏈上的熒光基團(tuán)相互靠近,FRET效應(yīng)增強(qiáng)。該方法操作簡(jiǎn)單,靈敏度高,可逆性好,具有良好的通用性,pH檢測(cè)范圍為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.
【學(xué)位授予單位】：湖南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：O657.3
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本文編號(hào)：2034945