本文選題：DNA自組裝 + 電化學(xué)生物傳感��； 參考：《山東師范大學(xué)》2017年碩士論文

【摘要】：MicroRNA(miRNA)和ATP等重要生物分子是生物體和生命現(xiàn)象的結(jié)構(gòu)基礎(chǔ)和功能基礎(chǔ),它們的含量和活性直接關(guān)系到生物體的健康。其中,miRNA在調(diào)控基因表達中發(fā)揮著重要的生物學(xué)作用。建立這些生物分子快速、簡便、準(zhǔn)確、靈敏的分析方法,對攻克許多疑難病癥以及促進信息科學(xué)的發(fā)展都有重要的意義,是當(dāng)前生物分析化學(xué)研究的前沿和熱點。在本論文中,利用DNA自組裝及核酸外切酶的循環(huán)放大技術(shù)發(fā)展了miRNA及ATP的檢測方法,對腫瘤的早期診斷具有重大意義,主要內(nèi)容如下:1.運用DNA自組裝原理,發(fā)展了一種用于let-7d RNA超敏檢測的無標(biāo)記電化學(xué)生物傳感器,該傳感器是基于切口核酸內(nèi)切酶(NEase)輔助級聯(lián)模板增強雜交過程(TEHP)和滾環(huán)擴增(RCA),誘導(dǎo)形成G-四鏈體-血紅素(hemin)復(fù)合物。發(fā)夾探針(H1)通過Au-S鍵固定在金電極的表面,在輔助DNA的幫助下,引入目標(biāo)RNA let-7d,形成了切口核酸內(nèi)切酶(NEase)的剪切位點,剪開發(fā)夾探針并釋放目標(biāo)RNA。發(fā)夾探針(H_2)的引入引發(fā)了另外兩個級聯(lián)循環(huán)過程。被剪開的片段作為滾環(huán)擴增(RCA)反應(yīng)的引物,產(chǎn)生了大量hemin的適體,適體在鉀離子的幫助下,可以形成G-四鏈體-血紅素(hemin)復(fù)合物,而hemin則是電化學(xué)檢測的響應(yīng)物。最新設(shè)計的超靈敏電化學(xué)檢測方法使得let-7d RNA的檢測限達0.42 fM,并能從let-7RNA家族中特異性的識別目標(biāo)RNA。這種檢測手段在相關(guān)基因疾病的早期診斷應(yīng)用中有著巨大的潛力。2.設(shè)計了ATP適體-羧基熒光素(FAM)/氧化石墨烯納米片(GO-nanosheets)納米復(fù)合物,結(jié)合核酸外切酶的剪切循環(huán)放大作用,以調(diào)查其在活細胞中的分子探測的能力。結(jié)果證明細胞對適體-FAM/GO-nanosheets納米復(fù)合物的攝取以及細胞內(nèi)靶標(biāo)ATP循環(huán)放大監(jiān)測成功實現(xiàn)。GO-nanosheets在活細胞中的遞送,保護和感測能力表明氧化石墨烯可以是許多生物學(xué)領(lǐng)域的有力候選物,例如DNA和蛋白質(zhì)分析,基因和藥物遞送以及細胞內(nèi)跟蹤等。3.展示了一種DNA納米自組裝結(jié)構(gòu)用以攜帶治療型mi RNA進入并殺死腫瘤細胞,而且在DNA自組裝的初期結(jié)構(gòu)可以用于檢測mi RNA。該體系主要由FAM修飾的三個莖環(huán)結(jié)構(gòu)和一條直鏈DNA組成。當(dāng)遇到靶標(biāo)miRNA時,發(fā)生雜交鏈?zhǔn)椒磻?yīng),三個莖環(huán)結(jié)構(gòu)交替打開形成星狀DNA自組裝結(jié)構(gòu),未反應(yīng)的莖環(huán)結(jié)構(gòu)被MnO_2熒光猝滅,通過檢測星狀DNA自組裝結(jié)構(gòu)的熒光信號可以用以檢測miRNA。以星狀DNA自組裝結(jié)構(gòu)為前體,加入直鏈DNA,在T4連接酶的作用下,直鏈DNA在星狀DNA自組裝結(jié)構(gòu)上連接成環(huán),隨后在T7 RNA聚合酶的作用下,以直鏈DNA為模板發(fā)生滾環(huán)轉(zhuǎn)錄反應(yīng),最終形成基于星狀DNA納米結(jié)構(gòu)的CXCR4莖環(huán)和三螺旋串聯(lián)的DNA自組裝納米結(jié)構(gòu),經(jīng)離心后,形成DNA納米水凝膠,其攜帶CXCR4和三螺旋RNA作為治療型miRNA進入并殺死三陰性乳腺癌細胞。
[Abstract]:MicroRNAs miRNAs, ATP and other important biological molecules are the structural and functional basis of biological and life phenomena. Their contents and activities are directly related to the health of organisms.Among them, miRNA plays an important biological role in regulating gene expression.The establishment of rapid, simple, accurate and sensitive analytical methods for these biomolecules is of great significance for solving many difficult problems and promoting the development of information science, and is the frontier and hot spot of bioanalytical chemistry research at present.In this paper, the detection methods of miRNA and ATP were developed by using DNA self-assembly and cyclic amplification of nucleic acid exonuclease, which is of great significance for the early diagnosis of tumor. The main contents are as follows: 1.The biosensor is based on the cleavage endonuclease (NEase) -assisted cascade template enhancement hybridization (TEHP) and ring-amplification (RCAA) to induce the formation of a G-quadruplex hemin complex.The hairpin probe H1 was immobilized on the surface of the gold electrode by Au-S bond. With the help of DNA, the target RNA let-7d was introduced to form the cleavage site of the incision endonuclease. The hairpin probe was cut open and the target DNA was released.The introduction of hairpin probe H _ 2) triggered two other cascading cycles.The clipped fragments were used as primers for the rca reaction, resulting in a large number of aptamers of hemin. With the help of potassium ions, the aptamer could form the G-quadruplex heme hemin complex, while hemin was the responder of electrochemical detection.The newly designed hypersensitive electrochemical detection method enables the detection limit of let-7d RNA to reach 0.42 fM, and can specifically identify the target from the let-7RNA family.This method has great potential in the early diagnosis of related genetic diseases.The ATP aptamer, carboxyl fluorescein (Fam) / graphene oxide nanocrystalline (GO-nanosheets) nanocomposites were designed and amplified by the shear cycle of nucleic acid exonuclease in order to investigate their ability to detect molecules in living cells.The results showed that cell uptake of aptamer-FAM / GO-nanosheets nanosheets and intracellular target ATP cycle amplification were successful in delivering, protecting and sensing graphene oxide in living cells, suggesting that graphene oxide could be a potent candidate in many biological fields.Examples include DNA and protein analysis, gene and drug delivery, and intracellular tracking.A DNA nano-self-assembly structure was demonstrated to carry therapeutic mi RNA into and kill tumor cells, and it could be used to detect mi rna in the early stages of DNA self-assembly.The system consists of three stem rings modified by FAM and a straight chain DNA.When the target miRNA is encountered, the hybrid chain reaction occurs, and the three stem ring structures open alternately to form the stellate DNA self-assembly structure. The unreacted stem ring structure is quenched by MnO_2 fluorescence. The fluorescence signal of the stellate DNA self-assembly structure can be used to detect the miRNAA by detecting the fluorescence signal of the stellate DNA self-assembly structure.The stellate DNA self-assembly structure was used as the precursor, and the linear DNA was added into the straight strand DNA. Under the action of T4 ligase, the linear DNA was connected to the stellate DNA self-assembly structure to form a ring. Then, under the action of the T7 RNA polymerase, the ring-ring transcription reaction occurred using the straight-stranded DNA as the template.Finally, CXCR4 stem rings based on stellate DNA nanostructures and DNA self-assembled nanostructures in tandem with trihelix were formed. After centrifugation, DNA nanogels were formed, which carried CXCR4 and trihelix RNA as therapeutic miRNA to enter and kill triple-negative breast cancer cells.
【學(xué)位授予單位】：山東師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：TP212.3;R730.4

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本文編號：1744217