發(fā)布時(shí)間：2018-03-18 08:37

  本文選題：DNA分子邏輯門　切入點(diǎn)：DNA探針　出處：《寧夏大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：近年來,以半導(dǎo)體材料為基礎(chǔ)的集成電路由于其集成度接近理論的極限,科學(xué)家們正在積極開發(fā)新型計(jì)算機(jī),DNA計(jì)算機(jī)被認(rèn)為是最具有發(fā)展?jié)摿Φ挠?jì)算機(jī)。眾所周知,計(jì)算機(jī)的構(gòu)建以邏輯運(yùn)算為基礎(chǔ),要建立和發(fā)展DNA計(jì)算機(jī),分子邏輯門技術(shù)是一條不可逾越的必經(jīng)之路。由于電化學(xué)檢測方法具有簡單、快速、靈敏、選擇性好等優(yōu)點(diǎn),備受研究者們的青睞。本論文以幾種常見食源性致病菌的特征DNA序列和生物小分子為目標(biāo)物,應(yīng)用電化學(xué)檢測技術(shù),提出了一系列DNA分子邏輯門,并且構(gòu)建了半加器和半減器兩種邏輯運(yùn)算器,為基于DNA分子的邏輯電路提供重要的理論支持,并為核酸和生物小分子的檢測提供了可選擇的方法。(1)在第一章緒論部分介紹了分子邏輯門及邏輯運(yùn)算器的相關(guān)概念并闡述了國內(nèi)外研究進(jìn)展,提出了本輪的研究內(nèi)容和創(chuàng)新點(diǎn)。(2)在第二章提出了基于目標(biāo)DNA與雙標(biāo)記探針競爭結(jié)合模式構(gòu)建的“NOR”型DNA分子邏輯門用于沙門氏菌(Sal)DNA和志賀氏菌(Shi)DNA的檢測。本實(shí)驗(yàn)以二茂鐵(Fc)為電化學(xué)指示劑,首先利用自組裝技術(shù)將巰基標(biāo)記的S1探針修飾在金電極表面,設(shè)計(jì)兩端標(biāo)記Fc的DP探針和S1探針部分互補(bǔ)雜交。當(dāng)目標(biāo)物Sal DNA和Shi DNA任何一個(gè)存在時(shí),由于堿基互補(bǔ)配對原則和臨近表面雜交作用使DP脫離電極表面�；谀繕�(biāo)物存在前后Fc的電化學(xué)信號的變化實(shí)現(xiàn)對Sal DNA和Shi DNA的高選擇性、高靈敏性檢測。由實(shí)驗(yàn)結(jié)果可得,該方法對Sal DNA和Shi DNA的檢測范圍均在1.00 nmol/L～1000.00 nmol/L之間,檢出限分別為0.52 nmol/L和0.72 nmol/L(S/N=3)。該工作以Sal DNA和Shi DNA為輸入,以IFc為輸出,構(gòu)建“NOR”型DNA分子邏輯門。(3)在第三章提出了基于Y構(gòu)型的“OR”型DNA分子邏輯門用于SalDNA和大腸桿菌(E.coli)DNA的檢測。本實(shí)驗(yàn)以Fc為電化學(xué)指示劑,首先將三條相互部分互補(bǔ)雜交的S1、S2和P1探針通過巰基自組裝在金電極表面形成Y構(gòu)型,基于堿基互補(bǔ)配對原則和臨近表面雜交反應(yīng)構(gòu)建了一個(gè)簡單、靈敏、特異性檢測Sal DNA和E.coli DNA的生物傳感器。由實(shí)驗(yàn)結(jié)果可知,該方法對Sal DNA和E.coli DNA的檢測范圍均在10.00 nmol/L～1000.00 nmol/L,檢出限分別為6.42 nmol/L 和 1.58 nmol/L(S/N=3)。以 Sal DNA 和 E.coli DNA 為輸入,以 Fc 信號變化值 △IFc為輸出,構(gòu)建了“OR”型DNA分子邏輯門。(4)在第四章提出了基于發(fā)夾結(jié)構(gòu)探針的用于李斯特桿菌(List)DNA和E.coli DNA分析的半加器的構(gòu)建。本實(shí)驗(yàn)以Fc和亞甲基藍(lán)(MB)為電化學(xué)指示劑,首先將分別標(biāo)記了 Fc和MB的發(fā)夾結(jié)構(gòu)探針S-1、S-2通過巰基自組裝在金電極表面,構(gòu)建了 DNA生物傳感器用于List DNA和E.coli DNA的同時(shí)智能分析。S-1和S-2的環(huán)部分別和List DNA、E.coli DNA完全互補(bǔ)雜交,當(dāng)List DNA或E.coli DNA存在時(shí),只有一個(gè)發(fā)夾結(jié)構(gòu)被打開,List DNA和E.coli DNA都存在時(shí),兩發(fā)夾結(jié)構(gòu)同時(shí)打開。基于目標(biāo)物存在前后Fc和MB在電極表面位置的變化致使相應(yīng)的電流發(fā)生變化,從而實(shí)現(xiàn)對List DNA和E.coliDNA的快速、靈敏、高選擇性分析。分析實(shí)驗(yàn)結(jié)果可得,該方法對List DNA和E.coli DNA的檢測范圍均在20.00 nmol/L～1000.00 nmol/L,檢出限分別為1.98 nmol/L和8.99 nmol/L(S/N=3)。以ListDNA和E.coli DNA作為輸入,以兩條探針電流變化之和∑△I(∑△I=△IMB+△IFc)和信號之比Y(Y=△IFc/△IMB或△IMB/△IFc)作為輸出,同時(shí)構(gòu)建了“AND”型和“XOR”型DNA分子邏輯門。融合這兩種邏輯門,構(gòu)建了具有邏輯運(yùn)算功能的DNA半加器。(5)在第五章提出了用于分析SalDNA和三磷酸腺苷(ATP)的分子半減器的構(gòu)建。本實(shí)驗(yàn)以Fc和MB為電化學(xué)指示劑,首先利用巰基自組裝技術(shù)將分別將標(biāo)有Fc和MB的發(fā)夾結(jié)構(gòu)探針L-p、ATP的適體AP修飾在金電極表面,AP探針結(jié)合了與之完全互補(bǔ)配對C-AP。設(shè)計(jì)L-p和目標(biāo)物Sal DNA特異性雜交,當(dāng)Sal存在時(shí)使L-P的環(huán)部結(jié)構(gòu)形成剛性雙鏈,ATP的存在可以和AP形成復(fù)合物置換出C-AP,拉近MB和電極表面的距離�；谀繕�(biāo)物存在前后,Fc和MB與電極表面距離的變化致使相應(yīng)的電流發(fā)生變化,可以實(shí)現(xiàn)Sal DNA和ATP的高靈敏性和高特異性分析。分析實(shí)驗(yàn)結(jié)果可得,SalDNA檢測范圍為10.00 nmol/L～1000.00 nmol/L,檢出限為2.19nmol/L(S/N=3);ATP 的檢測范圍:10.00nmol/L～1000.00nmol/L,檢出限為 3.88nmol/L(S/N=3)。以List DNA和ATP作為輸入,以兩條探針電流之和ΣI(ΣI=IMB+IFc)、之比Y(Y=△IFc/△IMB或△IMB/△IFc)作為輸出,同時(shí)構(gòu)建了“INHIBIT”型和“XOR”型DNA分子邏輯門。融合這兩種邏輯門,構(gòu)建了具有邏輯運(yùn)算功能的DNA半減器。
[Abstract]:In recent years, based on semiconductor materials and integrated circuits because of its integration is close to the theoretical limit, scientists are actively developing a new type of computer, DNA computer is considered to be the most potential for development of computer. As everyone knows, the computer to construct logic as the basis, to the establishment and development of DNA computer, molecular logic gate technology is the only way which must be passed an impassable. As the electrochemical detection method is simple, rapid, sensitive, good selectivity, has attracted researchers' attention. In this paper, several common foodborne pathogens and biological characteristics of DNA sequence of small molecules as object, application of electrochemical detection technology, put forward a series of DNA molecular logic gate, and construction half adder and half subtractor two logic unit, to provide important theoretical support for logic circuit based on DNA molecules and nucleic acid and biological small Provide alternative method of sub test. (1) in the first chapter introduces the related concepts of molecular logic gate and logic device and describes the study progress at home and abroad, puts forward the research content and innovation points of this round. (2) proposed in the second chapter based on the target of DNA and double labeled probe competition with the model of "NOR" type DNA molecular logic gate (Sal) for Salmonella and Shigella DNA (Shi) DNA detection. In this experiment, two ferrocene (Fc) as the electrochemical indicator, using self assembly technology of S1 probe labeled thiol modified on the electrode surface, both ends of mark design the Fc DP probe and S1 probe complementary hybridization. When the target is Sal DNA and Shi DNA the existence of any one, because of complementary base pairing and near surface hybridization DP from the electrode surface. The electrochemical signal changes before and after the existing object based on Fc To achieve high selectivity for Sal DNA and Shi DNA, high sensitivity detection. From the experimental results, the method of Sal DNA and Shi DNA detection range is between 1 nmol/L and 1000 nmol/L, the detection limits were 0.52 nmol/L and 0.72 nmol/L (S/N=3). The Sal DNA and Shi DNA to work as input in IFc, as output, construction of the "NOR" type DNA molecular logic gate. (3) proposed in the third chapter based on the Y configuration of "OR" type DNA molecular logic gate for SalDNA and Escherichia coli DNA (E.coli) detection. In this experiment, Fc as the electrochemical indicator, the three mutually complementary hybridization S1, S2 and P1 probe Y configuration on the surface of the gold electrode by self-assembled thiol, complementary base pairing and near surface hybridization reaction to construct a simple and sensitive biosensor based on specific detection of Sal, DNA and E.coli DNA. The experimental results show that the method of Sal DNA and E.coli DNA detection range was from 10 nmol/L to 1000 nmol/L, the detection limits were 6.42 nmol/L and 1.58 nmol/L (S/N=3). Sal DNA and E.coli with DNA as input, Fc signal changes in the value of delta IFc as output, the construction of "OR" type DNA molecular logic gate (4) in fourth. Chapter of the hairpin probe for Lester was based on (List) to construct a half adder analysis of DNA and E.coli DNA. In this experiment, Fc and methylene blue (MB) as the electrochemical indicator, first labeled the hairpin probe S-1 Fc and MB S-2, the self-assembled on gold electrode surface. DNA List and E.coli DNA biosensor for DNA and intelligent analysis of ring part.S-1 and S-2 respectively, and List DNA was constructed. E.coli DNA complementary hybridization, List DNA or E.coli DNA when there is only one hairpin structure was opened, List DNA and E.coli DNA There are two, hairpin structure opened at the same time. The target of Fc and MB before and after the change in the position of the electrode surface in the corresponding current changes based on List and E.coliDNA DNA to realize the fast, sensitive, high selective analysis. Analysis of the experimental results, the method of List DNA and E.coli DNA detection range in 20 nmol/L ~ 1000 nmol/L, the detection limits were 1.98 nmol/L and 8.99 nmol/L (S/N=3). The ListDNA and E.coli DNA as input to two probe current changes and I (sigma delta sigma delta I= Delta IMB+ Delta IFc) and the ratio of signal Y (Y= Delta IFc/ delta or delta IMB/ Delta IMB IFc) as the output, and constructs the "AND" and "XOR" type DNA molecular logic gate. The fusion of these two logic gates, with the construction of logic functions DNA half adder. (5) proposed in the fifth chapter for the analysis of SalDNA and adenosine triphosphate (ATP) molecule half subtracter The construction. In this experiment, Fc and MB as electrochemical indicator, hairpin probe L-p first using thiol self-assembled technology will be labeled with Fc and MB, aptamer AP ATP immobilized on the electrode surface, AP probe combined with complementary and matching design of L-p C-AP. and Sal DNA target specific hybridization when the presence of Sal L-P ring structure to form a rigid double chain, the presence of ATP and AP formed a complex replacement of C-AP, between MB and the electrode surface. Based on the distance before and after the object exists, Fc and MB and changes of electrode surface distance in the corresponding current change, analysis can achieve Sal DNA and ATP with high sensitivity and specificity. The analysis of the experimental results, the SalDNA detection range of 10 nmol/L to 1000 nmol/L, the detection limit is 2.19nmol/L (S/N=3); the detection range of ATP: 10.00nmol/L ~ 1000.00nmol/L, the detection limit is 3.88nmol/L (S/N=3). 浠ist DNA鍜孉TP浣滀負(fù)杈撳叆,浠ヤ袱鏉℃帰閽堢數(shù)嫻佷箣鍜屛(危I=IMB+IFc),涔嬫瘮Y(Y=鈻矷Fc/鈻矷MB鎴栤柍IMB/鈻矷Fc)浣滀負(fù)杈撳嚭,鍚屾椂鏋勫緩浜嗏，

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