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研究生: 許嘉琪
Chia- Chi Hsu
論文名稱: 石英壓電晶體感測器應用於無機化合物與DNA作用力的研究
The Interaction between Inorganic Compounds and Calf Thymus-DNA Studied by Quartz Crystal Microbalance
指導教授: 施正雄
Shih, Jeng-Shong
學位類別: 碩士
Master
系所名稱: 化學系
Department of Chemistry
論文出版年: 2001
畢業學年度: 89
語文別: 中文
論文頁數: 134
中文關鍵詞: 石英壓電晶體去氧核糖核酸金屬離子作用力
英文關鍵詞: QCM, DNA, metal ions, interaction
論文種類: 學術論文
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  • 本研究利用DNA在1-Ethyl-3-(3-dimeyhylaminopropyl) carbodi imide (EDAC)/N-hydroxysulfosuccinimide (NHS)的溶液中與3-carboxy propyl disulfide(CPS)形成醯胺鍵產生共價鍵結,再將小牛胸腺DNA固定於石英壓電晶體的銀電極上。然後利用石英壓電感測系統研究3-carboxypropyl disulfide 與ct-DNA的鍵結位置、DNA的濃度效應及DNA與各種無機物間的作用力。
    在利用石英壓電感測系統研究金屬離子與小牛胸腺DNA間的作用力中,結果顯示:鹼金屬離子及NH4+與DNA的作用力是屬於物理吸引。而鹼土族金屬離子除Mg2+外,對DNA的作用力顯示有化學吸附,其大小為:Ca2+ > Ba2+ ~ Sr2+ > Mg2+,化學鍵結強度則介於過渡金屬離子與鹼金屬離子之間。過渡金屬能與DNA產生化學鍵結,對DNA的作用力大小為:Ag+ > Hg2+ > Cu2+ > Cr3+ ~ Ni2+ > Co2+ >Fe3+ ~Mn2+ ~ Zn2+。過渡金屬離子鍵結於DNA的濃度效應及平均每個核甘酸所鍵結的金屬離子數目也以此系統加以探討。Ag+、Hg2+和Cu2+平均每個核苷酸會有較多的鍵結數目可能因會形成DNA分子內及分子間的交錯鍵結,這些金屬離子能與單股或雙股的DNA形成結構穩定的錯合物。
    本研究亦對DNA與其他無機化合物如:一氧化碳、過氧化氫、無機鹼和無機酸間的作用關係做系統探討。研究發現過氧化氫和無機鹼如NaOH及NH3亦會和雙股DNA作用,而無機酸和一氧化碳僅能與DNA產生相對較弱的化學鍵結。
    在過渡金屬離子和DNA作用研究中,利用石英壓電感測系統探討過渡金屬離子Cu2+與不同的聚核甘酸(polyG、polyC、polyA、polyU)的作用力,來探討Cu2+與DNA的鍵結位置,並可求得不同的鹼基對過渡金屬離子Cu2+鍵結的差異及平均每種核甘酸所能鍵結的Cu2+數目。結果顯示DNA的四種不同的鹼基與Cu2+ 鍵結的強度大小為:G > C > A > U,主要的鍵結位置是鹼基G的N(7)與O(6)、鹼基C的N(3)與O(2)及鹼基A的N(7)與N(6)。此外,Cu2+對雙股配對的聚核甘酸polyG+d-CMP的鍵結數目會小於相對應的polyG及polyC。
    本研究亦探討DNA 的種類、離子強度、pH值及溫度效應對Cu2+鍵結於DNA/CPS-石英壓電晶體的影響。不同的DNA種類對Cu2+鍵結的強度並沒有明顯的差異,可能由於這些種類的DNA其鹼基A=T與GºC的含量比值約略相同。至於單股DNA相對於雙股DNA對Cu2+能有較大的鍵結數目也是因為Cu2+與單股DNA形成分子內或分子間的交錯鍵結時,不須破壞氫鍵的緣故。在有Na+鹽的存在下,DNA與Cu2+的鍵結量較沒有Na+的溶液要少的多,可能由於高濃度的Na+,能穩定DNA的雙螺旋結構。在pH效應研究中發現,在弱鹼性的環境下,Cu2+較容易與DNA鍵結。而在溫度效應研究中發現:低溫時,DNA不易與Cu2+鍵結,而隨著溫度的增高,鍵結量逐漸增加,但在高溫時,DNA與Cu2+的鍵結量則反而會下降。

    ct-DNA was immobilized on the silver electrode surface of piezoelectric quartz crystal by the covalent binding to 3-carboxypropyl disulfide (CPS) in EDAC/NHS solution. The DNA attachment is attributed to the formation of amide bond between the carboxylate groups of CPS and the amino groups on the DNA bases. The binding site and the concentration effect on the interaction between 3-carboxypropyl disulfide and ct-DNA were investigated. Interaction between inorganic compounds and calf thymus-DNA was studied by piezoelectric quartz crystal detection system.
    Alkali metal and NH4+ ions exhibited nonspecific physical binding with ct-DNA. Alkali-earth metal ions, e.q., Mg2+, Ca2+, Sr2+ and Ba2+ showed some chemical binding with ct-DNA and the chemical binding could be arranged in the order: Ca2+ > Ba2+ ~ Sr2+ > Mg2+. The chemical interaction of alkali-earth showed stronger than that of alkali metal ions, but weaker than that of transition metal ions. Transition metal ions showed relatively strong chemical binding with ct-DNA. The binding ability could be arranged in the order: Ag+ > Hg2+ > Cu2+ > Cr3+ ~ Ni2+ > Co2+ >Fe3+ ~ Mn2+ ~ Zn2+. The concentration effect and binding number per nucleotide for the interaction between transition metal ions and ct-DNA were also investigated by the system. Ag+, Hg2+ and Cu2+ binding to DNA could result in intramolecular as well as intermolecular crosslinking and thus have great binding number per nucleotide. These ions react with single or double strands DNA to form a stable metal-DNA complex.
    The interaction of DNA with other inorganic species such as carbon monoxide, hydrogen peroxide, inorganic base and acid were also studied.
    Hydrogen peroxide and inorganic base such as NaOH and NH3 would denature the double strand DNA, while inorganic acid and carbon monoxide showed relative small binding to DNA.
    Interaction of metal ions, e.g., Cu2+ with different polynucleotides (polyG, polyC, polyA, and polyU) was also investigated by the QCM system. Cu2+ showed different affinities with the four kinds of bases, in the order: G > C > A > U. The binding number per nucleotide for Cu2+ was also calculated. The major binding sites could be N(7), O(6) of guanine, N(3), O(2) of cytosine, and N(7), O(6) of adenine.
    Other factors such as (1) different kinds of DNA, (2) double and single strand DNA, (3) ionic strength, (4) pH value, (5) temperature effect on the frequency response for Cu2+ binding to DNA/CPS PZ crystal were also investigated. There is no significant difference for Cu2+ binding to ct-DNA, st-DNA and ht-DNA, which may be attributed to the similar molar percentages of the base pairs (A=T, GºC) in those DNA. Upon forming intramolecular or intermolecular crosslinking, ds-ctDNA has to deprotonate the hydrogen and break a hydrogen binding. Cu2+ binding to ss-ctDNA without beraking hydrogen bond have a greater binding number than ds-DNA. Ionic strength also has a great effect on the frequency response for Cu2+ binding to DNA/CPS immobilized PZ crystal. The existence of Na+ stabilized the double strand helix and resulted in less binding affinity of Cu2+ toward DNA in the presence of Na+ ions. The Cu2+ ion showed a greater binding amount to DNA at high pH than at low pH. At low temperature, the DNA double helix demonstrated low binding affinity to Cu2+. As the temperature was raised, the DNA started to denature and the binding amount for Cu2+ with ct-DNA increased. However, weaker binding of Cu2+ to DNA was observed at higher temperature.

    英文摘要 Ⅰ 中文摘要 Ⅲ 目錄 Ⅴ 圖目錄 Ⅶ 表目錄 Ⅹ 第一章 緒論 1 1-1 DNA概論 1 1-1-1 DNA鹼基的配對與雙螺旋構造 2 1-1-2 DNA分子的大小 6 1-1-3 DNA分子受溫度影響 7 1-1-4 三股DNA 9 1-1-5 DNA的超螺旋結構 10 1-2金屬離子 12 1-2-1生物體中的金屬離子 12 1-2-2金屬離子抑制酵素 13 1-2-3金屬離子與核酸的鍵結 15 1-2-4金屬離子與核酸的可能鍵結位置 16 1-2-5金屬離子對核酸結構的影響 22 1-2-6金屬離子的致癌性 23 1-2-7金屬離子的反致癌性 24 1-2-8 金屬離子造成突變之機制探討 25 1-3壓電晶體 27 1-3-1壓電晶體之壓電性 27 1-3-2 AT-cut石英振盪器 29 1-3-3石英振盪器的線路 32 1-3-4振盪頻率的量測 34 1-3-5石英微量天平 36 1-4石英壓電晶體在分析化學領域上的應用 40 1-4-1石英壓電晶體於氣相上的應用 40 1-4-2石英壓電晶體於液相之振盪的情形 44 1-4-3石英壓電晶體於液相上的應用 46 1-4-4石英晶體微量天平(QCM)應用於DNA的研究 48 1-5本研究動機、目的及實驗項目 52 第二章 實驗部分 54 2-1 藥品及儀器 54 2-1-1藥品: 54 2-1-2儀器: 55 2-2 石英晶體的處理 55 2-3 實驗系統與方法 56 2-3-1液相靜態壓電實驗系統 56 2-3-2石英壓電晶體液相實驗系統之穩定性 58 2-3-3靜相實驗系統-固定化DNA研究 59 2-3-4液相脫附實驗系統-DNA與待測物間的作用力研究 60 2-3-5液相系統之實驗步驟 61 2-3-6氣相實驗系統的建立 62 2-3-7石英壓電晶體氣相實驗系統之穩定性 65 2-3-8氣相系統之實驗步驟 66 2-3-9 一氧化碳氣體濃度的配製 66 第三章、結果與討論 67 3-1 TGC及CPS硫化物於石英晶片的固定 67 3-2 DNA於石英晶片的固定 72 3-2-1 DNA與CPS鍵結位置的探討 72 3-2-2 固定聚核苷酸於石英晶片 75 3-2-3 EDAC/NHS溶液固定DNA於CPS-石英晶片 78 3-2-4 DNA的濃度對CPS-石英壓電晶體之頻率感應效應 81 3-3利用石英壓電感測系統偵測DNA與陰離子的鍵結 81 3-4 DNA與金屬離子的鍵結 85 3-4-1 DNA與鹼金屬離子的鍵結 85 3-4-2 DNA與鹼土金屬離子的鍵結 88 3-4-3 DNA與過渡金屬離子的鍵結 90 3-4-4 DNA與各類重金屬離子鍵結比較 101 3-5 DNA與非金屬無機物的作用 105 3-6金屬離子與DNA之鍵結位置探討 112 3-7比較不同的DNA與金屬離子的鍵結 115 3-8離子強度對DNA與金屬離子鍵結的影響 115 3-9 PH值對DNA與金屬離子鍵結的影響 118 3-10溫度對DNA與金屬離子鍵結的影響 118 第四章、結論 121 附錄一 123 附錄二 125 Reference 128

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