本文選題：電化學信號放大 + DNA生物傳感器　； 參考：《青島科技大學》2016年碩士論文

【摘要】：本論文主要基于級聯(lián)信號放大策略構筑了三種電化學DNA生物傳感器,提出了基于靶標循環(huán)和后續(xù)擴增組合信號放大的有效構筑機制,對疾病相關的DNA、小分子等實現(xiàn)了高靈敏、高選擇性分析檢測。主要內容包括:1.基于聚合酶、切口酶等溫擴增技術和酶/金納米顆粒后續(xù)放大策略,構筑了一種高靈敏的DNA生物傳感器。本論文設計了一個發(fā)夾DNA探針(HP),其含有與靶標DNA互補的3'端突出序列,切口酶的識別位點和用來終止聚合反應的烷烴間隔三部分。當靶標DNA存在時,在聚合酶和切口酶作用下,引發(fā)“聚合-切割-鏈置換”反應,打開HP;DNA標記的金納米顆粒可與打開的HP雜交,金納米上的DNA探針標記了生物素,其進一步與標記堿性磷酸酶(ALP)的親和素親和連接,進一步ALP催化1-萘基磷酸鹽(1-NP)的生成1-萘酚,實現(xiàn)對靶標DNA雙重信號放大的檢測,檢測限可低至0.065fM。2.基于Pb~(2+)-DNAzyme誘導切割,末端轉移酶(TdTase)輔助的無模板聚合信號放大策略,發(fā)展了一種簡單靈敏的電化學DNAzyme傳感器,用于特異性檢測Pb~(2+)。首先,將8-17 DNAzyme的發(fā)夾狀底鏈(HP DNA)固定在電極上,它與靶標Pb~(2+)識別后催化切割底鏈,并露出3'-OH端。末端轉移酶(TdTase)催化dUTP-biotin有序地添加到底鏈3'-OH端上,進而實現(xiàn)無模板擴增。利用生物素(biotin)與親和素的親和作用,將親和素-堿性磷酸酶(SA-ALP)連接到電極上,進一步ALP催化1-萘基磷酸鹽(1-NP)的生成1-萘酚,產生電化學信號,實現(xiàn)對靶標Pb~(2+)的高靈敏檢測,檢測限可達到0.043 nM。3.通過結合熵驅動靶標循環(huán)和DNA雜交鏈式反應組合放大策略,構筑了一種等溫、無酶、免標記的高靈敏電化學DNA生物傳感器,用于檢測目標DNA。首先,在金電極表面自組裝上DNA雙鏈探針,該探針的末端突出區(qū)域作為toehold位點,其與靶標DNA(TD)特異性識別雜交,觸發(fā)鏈置換反應,將輔助DNA探針(AP)置換下來并露出第二個toehold位點。這個toehold位點隨后與燃料DNA鏈(FS)識別雜交,進而FS取代了TD和保護鏈(PP),靶標DNA循環(huán)利用。FS的突出序列進一步引發(fā)兩個莖環(huán)DNA(HP-1、HP-2)交替雜交,形成周期性長鏈DNA的聚合體,作為信號載體;利用六氨基合釕配合物(RuHex)作為電化學指示劑,可以實現(xiàn)對靶標DNA的高靈敏檢測。
[Abstract]:In this paper, three kinds of electrochemical DNA biosensors are constructed based on cascade signal amplification strategy, and an effective construction mechanism based on target cycle and subsequent amplification combined signal amplification is proposed, which is highly sensitive to disease-related DNA, small molecules and so on. High selectivity analysis and detection. The main contents include: 1. A highly sensitive DNA biosensor was constructed based on polymerase, notch enzyme isothermal amplification and subsequent amplification strategy of enzyme / gold nanoparticles. In this paper, a hairpin DNA probe (HPN) was designed, which contains 3 'protrusion sequence complementary to target DNA, recognition site of incision enzyme and alkane spacer used to terminate polymerization. In the presence of target DNA, "polymerization-cleaving-chain displacement" reaction was initiated under the action of polymerase and incision enzyme. The gold nanoparticles labeled with HPN DNA could be hybridized with open HP, and DNA probes on gold nanoparticles labeled biotin. It was further linked with the affinity of ALP, and then the 1-naphthol was synthesized by ALP. The detection limit of double signal amplification of target DNA could be as low as 0.065 fM.2. A simple and sensitive electrochemical DNAzyme sensor was developed based on the signal amplification strategy of template free polymeric signal assisted by PBN 2 + DNAzyme induced cleavage and terminal transferase (TdTase). Firstly, the 8-17 DNA zyme was fixed on the electrode, which was recognized with the target PbC-2), and then the base chain was dissected and the 3H-OH end was exposed. The HPS-DNA of 8-17 DNAzyme was immobilized on the electrode, and it was recognized with the target PbC-2). Terminal transferase (TdTase) catalyzes the ordered addition of dUTP-biotin onto the end of the 3- OH chain, which leads to the template free amplification of dUTP-biotin. By using the affinity of biotin) and avidin, SA-ALP) was connected to the electrode to further catalyze the formation of 1-naphthol from 1-naphthophosphate (1-NPP) by ALP, and to produce electrochemical signal. The detection limit can reach 0.043 nM.3. A highly sensitive electrochemical DNA biosensor was constructed by combining entropy driven target cycle and DNA hybridization chain reaction to detect target DNAs. First, a double-stranded probe was self-assembled on the surface of the gold electrode. The protruding region of the probe was used as the toehold site, which was specifically hybridized with the target DNA-TDs, triggered the strand replacement reaction, and replaced the auxiliary toehold probe with the second toehold site. The toehold site was then hybridized with the fuel DNA strand (FSs), and FS replaced the TD and the protective chain. The protruding sequence of the target DNA cycle using .FS further led to the alternant hybridization of the two stem rings, HP-1 and HP-2), forming a periodic long strand DNA polymer. As a signal carrier, RuHexis as an electrochemical indicator can be used to detect target DNA with high sensitivity.
【學位授予單位】：青島科技大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：O657.1

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本文編號：2043674