本文選題：G四股螺旋 + 核酸適體　； 參考：《湖南大學(xué)》2010年碩士論文

【摘要】： 蛋白質(zhì)如免疫球蛋白E及小分子如腺苷等生物分子在維系生物個體的生命活動中起著至關(guān)重要的作用。對這些生命物質(zhì)進(jìn)行快速而有效的監(jiān)測對保持生個體正常的機(jī)能有著直接而現(xiàn)實的意義。生物傳感器在眾多分析方法中獨樹一幟,他不僅具有常規(guī)分析方法的優(yōu)點,而且能模擬生命活動過程,能提供原位、實時分析。核酸適體是一段自核酸文庫中隨機(jī)篩選出的功能化單鏈短DNA或RNA序列,具有化學(xué)穩(wěn)定,尺寸小,價格低廉,高親和力,高特異性以及靶物質(zhì)廣泛等的特點,這使它在構(gòu)建一系列目標(biāo)分子檢測體系中成為人們的首選。本文以核酸適體為目標(biāo)識別探針,主要在以下幾個方面開展工作： (1)基于G四股螺旋和信號增強(qiáng)的熒光寡核苷酸探針用于蛋白質(zhì)的超靈敏檢測【第2章】。本章中,我們報道了一種標(biāo)記芘的富G核酸探針及其在IgE檢測中的應(yīng)用,為探究人類端粒的生物學(xué)功能提供了有益的參考�；诜肿娱gG四股螺旋結(jié)構(gòu),結(jié)合S1核酸酶和目標(biāo)識別核酸適體,我們設(shè)計了一種末端單芘標(biāo)記的寡核酸探針并提出一種新的蛋白質(zhì)檢測方法。該方法不僅有效回避了報告基團(tuán)特異性位點優(yōu)化和芘單體熒光易被核酸堿基猝滅的問題,同時還提出了一種信號增強(qiáng)的響應(yīng)機(jī)理,成功用于均相中IgE超靈敏檢測。該傳感器的線性范圍為4.72×10-12～7.56×10-9M,回歸系數(shù)為0.9941,檢測下限9.45×10-14M。 (2)基于背景消除和信號增強(qiáng)的電化學(xué)核酸適體傳感器用于小分子超靈敏檢測[第3章]。本章中,我們以腺苷為目標(biāo)模型提出了一種多功能的電化學(xué)核酸適體傳感機(jī)理用于小分子的超靈敏檢測。巰基修飾的核酸適體探針上標(biāo)記二茂鐵電活性分子,并將其固定在電極表面,在核酸適體序列的兩端引入大腸桿菌核酸內(nèi)切酶Ⅰ的酶切位點,這不僅能夠扣除背景電流,同時形成一種信號增強(qiáng)的響應(yīng)機(jī)理。沒有目標(biāo)物腺苷存在時,核酸適體折疊成發(fā)夾結(jié)構(gòu),在莖部形成一個酶切雙鏈區(qū),酶切后二茂鐵脫離電極表面,沒有二茂鐵的響應(yīng)電流信號產(chǎn)生。有腺苷存在時,腺苷和核酸適體結(jié)合誘導(dǎo)核酸適體發(fā)生構(gòu)型轉(zhuǎn)變,酶切雙鏈區(qū)域消失,核酸內(nèi)切酶就無法切斷適體探針序列。此時二茂鐵分子仍離電極表面很近,故能夠觀察到二茂鐵的響應(yīng)電流峰。這種傳感檢測體系,在腺苷濃度極低的情況下仍能檢測到電流信號。檢測下限達(dá)到10-13 M,線性響應(yīng)范圍為3.74×10m～3.74×10-8M。我們提出的這種新型電化學(xué)傳感檢測方法極大的拓展了各種基于核酸適體傳感體系用于目標(biāo)檢測的分析范圍。 (3)基于分子信標(biāo)核酸適體熒光生物探針用于腺苷的高選擇性檢測【第4章】。本章中,基于核酸適體結(jié)合目標(biāo)誘導(dǎo)構(gòu)型轉(zhuǎn)換和DNA雜交原理,以腺苷的核酸適體序列為主體設(shè)計分子信標(biāo),提出了一種信號減小的腺苷檢測響應(yīng)機(jī)理。當(dāng)沒有腺苷存在時,互補(bǔ)DNA序列和分子信標(biāo)雜交,形成DNA雙螺旋結(jié)構(gòu),分子信標(biāo)打開,產(chǎn)生很強(qiáng)的熒光。有腺苷存在時,分子信標(biāo)和腺苷結(jié)合后發(fā)生構(gòu)型轉(zhuǎn)換,再向其加入互補(bǔ)DNA序列,熒光響應(yīng)信號變化很小。隨著腺苷分子濃度的降低,體系的熒光響應(yīng)相應(yīng)增強(qiáng),從而實現(xiàn)對腺苷小分子的特異性檢測與精確定量。
[Abstract]:Proteins such as immunoglobulin E and small molecules, such as adenosine, play a vital role in maintaining individual life activities. Rapid and effective monitoring of these living substances has a direct and realistic significance for maintaining the normal functioning of the individual. Biological sensilla is unique in many analytical methods, He not only has the advantages of conventional analytical methods, but also simulates the process of life activity and provides in situ, real-time analysis. The aptamer is a functional single strand short DNA or RNA sequence randomly selected from the nucleic acid library. It has the characteristics of chemical stability, small size, low price, high affinity, high specificity and wide range of target materials. This makes it the first choice in building a series of target molecular detection systems. This paper uses aptamers as the target identification probe, which mainly works in the following aspects:
(1) the use of G four strand helix and signal enhanced fluorescent oligonucleotide probe for protein ultra sensitive detection [chapter second]. In this chapter, we reported a rich G nucleic acid probe for labeling pyrene and its application in IgE detection, which provided useful reference for exploring the biological function of human telomere. Based on intermolecular G four strands spiral structure In combination with S1 nuclease and target identification of aptamers, we designed a terminal single pyrene labeled oligonucleotide probe and proposed a new method for protein detection. This method not only effectively avoids the problem of the optimization of the specific site of the report group and the fluorescence of pyrene monomers easily to be quenched by the nucleic acid base, but also proposes a signal enhancement. The response mechanism is successfully used for IgE ultra sensitive detection in homogeneous phase. The linear range of the sensor is 4.72 x 10-12 to 7.56 x 10-9M, the regression coefficient is 0.9941, and the detection limit is 9.45 x 10-14M..
(2) electrochemical nucleic acid aptamers based on background elimination and signal enhancement are used for small molecular hypersensitive detection [Third]. In this chapter, we use adenosine as the target model to propose a multi-functional electrochemical aptamer sensing mechanism for the ultra sensitive detection of small molecules. A sulfhydryl modified nucleic acid aptamer labeled two metallocene electric probe. The active molecule is immobilized on the surface of the electrode, and the enzyme tangent site of Escherichia coli endonuclease I is introduced at both ends of the nucleotide sequence. This can not only deduct the background current, but also form a signal enhancement response mechanism. When the target adenosine exists, the aptamer is folded into a hairpin structure, and an enzyme is formed in the stem. After the enzyme was cut, two ferrocene was separated from the surface of the electrode, and there was no response current signal produced by the two ferrocene. When adenosine existed, the binding of adenosine and aptamer induced the transformation of the aptamer, the region of the double chain disappears, and the endonuclease could not cut off the sequence of the aptamer probe. At this time, the two ferrocene molecules were still close to the surface of the electrodes, so they could be found to be very close to the surface of the electrode. It is enough to observe the response current peak of two ferrocene. This sensing detection system can still detect the current signal when the concentration of adenosine is very low. The detection limit is 10-13 M, and the linear response range is 3.74 * 10m to 3.74 x 10-8M.. The new electrochemical sensing method proposed by us has greatly expanded a variety of aptamer based sensing. The system is used for the analysis of target detection.
(3) high selectivity detection of adenosine based on molecular beacon aptamer fluorescent biological probe (fourth chapter). In this chapter, a molecular beacon is designed based on aptamer binding target induced transformation and DNA hybridization, and a signal reduction adenosine detection response mechanism is proposed. When adenosine exists, the complementary DNA sequence and molecular beacon hybridize to form a DNA double helix structure, and the molecular beacon opens and produces a strong fluorescence. When the adenosine exists, the molecular beacon and adenosine are combined after the combination of the molecular beacon and the complementary DNA sequence, and the fluorescence response signal becomes small. With the decrease of the adenosine molecular concentration, the fluorescence of the system is fluorescence. In response to the corresponding enhancement, specific detection and accurate quantification of adenosine small molecules were achieved.
【學(xué)位授予單位】：湖南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R341

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 馮德榮;生物傳感器的研究現(xiàn)狀和發(fā)展方向[J];山東科學(xué);1999年04期

，

本文編號：1954610