本文選題：熒光探針　切入點：小檗堿二聚體　出處：《南方醫(yī)科大學(xué)》2015年碩士論文

【摘要】：在過去的十年中,研究開發(fā)用于生物大分子檢測的熒光探針受到了越來越廣泛的關(guān)注,主要是因為此類生物熒光探針具有靈敏度和選擇性高、成本低、操作簡便等優(yōu)點,此外,其在臨床疾病診斷與治療方面也有一定的應(yīng)用前景。目前,一些已報道的熒光探針,例如：香豆素、羅丹明等在有機溶劑中對一些陽離子、陰離子以及小分子化合物具有良好的檢測功能。但是,由于這些熒光探針分子缺乏一定的水溶性,限制了它們在生物大分子檢測方面的應(yīng)用。因此,設(shè)計并合成具有一定水溶性的新型熒光分子探針,成為了一個熱門的研究領(lǐng)域。本論文基于具有一定水溶性的天然產(chǎn)物小檗堿,設(shè)計合成了具有較好水溶性的2,2'-聯(lián)吡啶鏈接的小檗堿二聚體1,并系統(tǒng)研究了化合物1對生物體內(nèi)重要的金屬離子、生物大分子G-四鏈體和HIV病毒核酸分子DNA的熒光探針功能。主要研究結(jié)果如下：1.化合物1的合成、表征與性質(zhì)設(shè)計合成了由2,2’-聯(lián)吡啶鏈接的小檗堿二聚體1,通過1H NMR、13C NMR、 ESI-MS和HR-MS進行了結(jié)構(gòu)表征。化合物1具有較好水溶性,能溶于含1‰DMSO的水溶液：化合物1具有聚集誘導(dǎo)發(fā)光性能,在DMSO/H2O溶液中,隨著水的含量升高,熒光逐漸增強。化合物1的熒光強度受pH值影響,當(dāng)pH值從2.0升高到7.0時,熒光逐漸增強,而pH從7.0升高到10.5時,熒光則逐漸減弱,并且化合物的熒光變化具有pH值變化可逆性,說明在不同pH環(huán)境中,骨架結(jié)構(gòu)并未發(fā)生變化,可能是由于其質(zhì)子化程度不同導(dǎo)致熒光強度發(fā)生變化。2.化合物1識別金屬離子的探針功能研究紫外吸收和熒光光譜實驗證明化合物1與Cu2+的結(jié)合比為2：1,因此,化合物1能夠在pH 7.0的5 mM Tris-HCl緩沖溶液中選擇性識別Cu2+,且不受其他金屬離子干擾,檢測極限為1.08μM�；衔�1對金屬離子Cu2+, Ni2+和Co2+表現(xiàn)出了不同的時間響應(yīng),熒光完全淬滅響應(yīng)時間分別為瞬時、2h和10 h�；衔�1與Co2+和Ni2+的的反應(yīng)速率常數(shù)k分別為9.83×10-3min-1和9.60×10-2min-1,Ni2+與化合物1的反應(yīng)速率常數(shù)為Co2+的10倍。3.化合物1識別G-四鏈體的探針功能研究熒光和紫外滴定結(jié)果表明化合物1與G-四鏈體(G4s)、單鏈DNA和雙鏈DNA均有較強的結(jié)合能力,結(jié)合常數(shù)為105～106 M-1,但是G-四鏈體對化合物1的紫外和熒光呈現(xiàn)增強的趨勢,而單、雙鏈DNA則呈現(xiàn)減弱趨勢,熒光變化可以通過肉眼識別。化合物1對G4平行結(jié)構(gòu)的結(jié)合能力高于G4反平行結(jié)構(gòu)。此外,化合物1對不含有金屬離子的人體端粒無序序列22AG具有較強的結(jié)合能力并能誘導(dǎo)其形成反平行結(jié)構(gòu)。CD melting實驗結(jié)果表明化合物1對平行c-kit2、反平行22AG (Na+)和無序22AG三種G4s DNA作用的(△Tm)分別為24℃、1.5℃和14℃,進一步證明了化合物1對平行G4s的穩(wěn)定性能。此外,化合物1對G4/hemin配合物過氧化酶的抑制活性實驗證明,化合物1與平行G-四鏈體主要是通過π-π堆積的方式結(jié)合�；衔�1還可以對Hela細胞內(nèi)的核酸進行熒光成像。4.化合物1對靶向HIV DNA的探針功能研究化合物1的結(jié)構(gòu)中含有芳香環(huán)和季銨鹽正電荷中心,這些官能團可能與DNA產(chǎn)生π-π、靜電、氫鍵等作用。為了驗證我們的想法,我們研究了化合物1檢測HIV DNA的探針功能。研究結(jié)果表明,化合物1能夠通過π-π、靜電、或氫鍵作用將FAM熒光標(biāo)記的單鏈DNA熒光淬滅,淬滅率高達92%,有趣的是,加入與單鏈DNA互補的靶向HIV雙螺旋DNA后,熒光基本恢復(fù),復(fù)蘇率RE為7.4,化合物1能夠特異性的識別HIV DNA,檢測限為0.11nM。
[Abstract]:In the past ten years, the research and development of fluorescent probe for Biomacromolecule detection has attracted more and more attention, mainly because of such biological fluorescent probe with high sensitivity and selectivity, low cost, simple operation and other advantages, in addition, it also has a certain application prospect in clinical diagnosis and treatment of diseases at present. Some have been reported, such as fluorescent probe, coumarin, Luo Danming in organic solvent for cations, anions and small molecule compounds have good detection function. However, due to the lack of a fluorescent probe into water soluble, limit their application in the detection of biological molecules. Therefore, a novel fluorescence molecular probes were designed and synthesized with certain water soluble, has become a hot research field. This paper has some water soluble natural product of berberine based on Design Has a good water solubility of berberine 2,2'- bipyridine links two dimer 1, and studied 1 compounds of metal ions in vivo important, fluorescence probe function of macromolecular chain of G- four and HIV virus nucleic acid molecules of DNA. The main results are as follows: 1. the synthesis of 1 compounds by berberine 2,2 '- bipyridine linked two dimer 1 synthesis and characterization of design, through the 1H NMR, 13C NMR, ESI-MS and HR-MS were used to characterize the structures. Compound 1 has good solubility in water, soluble in aqueous solution containing 1 DMSO%: compound 1 exhibited aggregation induced luminescence properties in DMSO/H2O solution with the increase in water content, fluorescence gradually. The fluorescence intensity of compound 1 is affected by the pH value, when the pH value increased from 2 to 7, the fluorescence gradually increased, while pH increased from 7 to 10.5, the fluorescence decreases, and the fluorescence change of compounds with pH Changes in the value of the reversibility, explains the different pH environment, the skeleton structure has not changed, may be due to the combination of different degree of protonation leads to probe function of purple fluorescence intensity changes of.2. compound 1 recognition of metal ions absorption and fluorescence spectra experiments show that compound 1 and Cu2+ ratio is 2:1, therefore, compound 1 can be selective identification of Cu2+ in pH 7 mM 5 Tris-HCl buffer solution, and no other metal ion interference, the detection limit is 1.08 M. 1 compounds for metal ions Cu2+, Ni2+ and Co2+ showed different fluorescence quenching response time, complete response time were instantaneous, 2h and 10 h. compounds with Co2+ and 1 the Ni2+ of the reaction rate constant K was 9.83 * 10-3min-1 and 9.60 * 10-2min-1, the reaction rate constants of Ni2+ and compound 1 as a probe function 10 times.3. Co2+ 1 G- four compound identification chain research fluorescence And UV titration results showed that compounds 1 and four G- chain (G4s), single stranded DNA and double stranded DNA have strong binding ability, the binding constant was 105~106 M-1, but the G- four chain body shows the trend of compound 1 UV and fluorescence of single, double stranded DNA showed a weakening trend fluorescence, changes can be identified by the naked eye. The binding ability of compound 1 on G4 parallel structure is higher than that of the G4 anti parallel structure. In addition, 1 compounds of metal ion containing human telomeric sequence of random 22AG has stronger binding ability and can induce the formation of anti parallel structure of.CD melting showed that 1 compounds of c-kit2 parallel, anti parallel 22AG (Na+) three G4s and disordered 22AG the role of DNA (delta Tm) respectively for 24 degrees, 1.5 degrees and 14 degrees, further proved that the compound 1 pairs of parallel G4s stable performance. In addition, compound 1 complexes with G4/hemin oxidase inhibitory activity Experiments show that compounds 1 and four parallel G- chain is mainly through the combination of stacking way. Compound 1 can also nucleic acid on Hela cells for fluorescence imaging of.4. compound structure of the compound function of 1 probe targeting HIV DNA 1 containing aromatic ring and quaternary ammonium positive charge center, these functional groups may produce Pi Pi, and DNA electrostatic, hydrogen bond effects. In order to verify our idea, we studied 1 probe compounds for detection of HIV DNA. The results show that the compound 1 can by tt-tt, electrostatic, hydrogen bonding or single stranded DNA fluorescence quenching of FAM fluorescence labeled inactivated, quenching rate 92%, interestingly, adding complementary and single stranded DNA targeting HIV double helix DNA, fluorescence recovered, the recovery rate of RE was 7.4, compound 1 is able to identify HIV specific DNA, the detection limit is 0.11nM.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R914

【共引文獻】

相關(guān)碩士學(xué)位論文 前2條

1 薛珊燕;幾種新型小分子熒光探針的合成及性能研究[D];湖南師范大學(xué);2014年

2 郭林娥;基于重要陰、陽離子的熒光檢測傳感器合成、性質(zhì)及生物學(xué)應(yīng)用研究[D];云南大學(xué);2015年

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本文編號：1685548