基于核酸適配體熒光探針的構(gòu)建及應(yīng)用研究
發(fā)布時(shí)間：2018-11-24 15:15
【摘要】：核酸適配體是利用體外篩選技術(shù)得到的一段DNA或RNA序列,它能與靶物質(zhì)高親和力、高特異性結(jié)合,被廣泛應(yīng)用于生物傳感器領(lǐng)域。核酸染料SG是一種結(jié)合于所有雙鏈DNA雙螺旋小溝區(qū)域的具有綠色激發(fā)波長的染料。利用其熒光信號(hào)的變化可以定量檢測(cè)靶物質(zhì)。核酸外切酶III能作用于雙鏈DNA,沿3’→5’方向逐步切去單核苷酸,利用其剪切特性可以提高傳感器的靈敏度。納米金由于其良好的光學(xué)特性,被廣泛應(yīng)用于生物、化學(xué)等領(lǐng)域,近年來已成為科學(xué)家們研究的熱點(diǎn)之一。本論文主要利用納米金和核酸染料的光譜特性來構(gòu)建不同的生物傳感器檢測(cè)四環(huán)素、乙型肝炎病毒DNA、腺苷,主要包括以下三個(gè)部分:(1)基于核酸適配體無標(biāo)記熒光法檢測(cè)四環(huán)素。基于四環(huán)素適配體和信號(hào)轉(zhuǎn)換探針構(gòu)建一個(gè)三螺旋分子開關(guān),加入四環(huán)素后,該三螺旋分子開關(guān)的結(jié)構(gòu)被破壞,四環(huán)素適配體與四環(huán)素特異性結(jié)合,從而釋放出信號(hào)轉(zhuǎn)換探針,然后加入SG,體系熒光降低。SG熒光強(qiáng)度的降低與四環(huán)素的濃度成正比,線性范圍為1.1-23 nM,檢出限是130 pM。本方法能應(yīng)用于牛奶樣品中四環(huán)素的檢測(cè),其加標(biāo)回收率為92.5-96.1%。(2)基于核酸外切酶III和核酸染料SG無標(biāo)記檢測(cè)HBV。首先設(shè)計(jì)兩個(gè)3’-末端突出的發(fā)夾DNA,當(dāng)沒有HBV存在時(shí),ssDNA相繼打開兩個(gè)發(fā)夾DNA,被Exo III剪切后,剩下部分單鏈DNA,加入SG后呈現(xiàn)弱的熒光信號(hào)。當(dāng)HBV存在時(shí),ssDNA與HBV雜交形成部分互補(bǔ)的雙鏈DNA而不與發(fā)夾DNA作用,因此兩個(gè)發(fā)夾DNA能保持其發(fā)夾結(jié)構(gòu)而不被Exo III剪切,加入SG后體系呈現(xiàn)一個(gè)強(qiáng)的熒光信號(hào)。實(shí)驗(yàn)結(jié)果表明,當(dāng)HBV濃度在0.1 10 nM范圍內(nèi),體系熒光增強(qiáng)與HBV濃度呈線性,檢出限為59 pM。該方法簡(jiǎn)單、方便、靈敏度高,且能很好地應(yīng)用于血清樣品中HBV的檢測(cè),其加標(biāo)回收率為93.0-98.0%。(3)基于核酸適配體和納米金無標(biāo)記檢測(cè)腺苷。腺苷不存在時(shí),適配體吸附在納米金表面形成一個(gè)保護(hù)層,保護(hù)納米金在高鹽溶液中依然分散,溶液顏色仍為紅色,加入SG后,SG嵌入適配體中并隨適配體吸附在納米金上,因此拉近了SG與納米金之間的距離,且SG的發(fā)射與納米金的吸收相互重疊,從而發(fā)生熒光共振能量轉(zhuǎn)移,SG的熒光被納米金猝滅。腺苷存在時(shí),腺苷與其適配體特異性結(jié)合,此時(shí)納米金在高鹽溶液中發(fā)生團(tuán)聚,溶液由紅色變?yōu)樗{(lán)色。加入SG后,SG嵌入適配體-腺苷中,熒光恢復(fù)。據(jù)此,可采用比色法、紫外法、熒光法檢測(cè)腺苷。其中,熒光法的靈敏度最高,檢測(cè)范圍為5.5 110 nM,檢出限為0.8nM。在血清樣品中用熒光法檢測(cè)腺苷的加標(biāo)回收率為92.0-103.4%。
[Abstract]:Nucleic acid aptamer is a sequence of DNA or RNA obtained by in vitro screening. It can bind to target material with high affinity and specificity and is widely used in biosensors. Nucleic acid dye SG is a dye with green excitation wavelength that binds to all double stranded DNA double helix grooves. The change of fluorescence signal can be used to quantitatively detect the target material. Nucleic acid exonuclease (III) can incise a single nucleotide in the direction of 3'-5'of double-stranded DNA, and the sensitivity of the sensor can be improved by using its shear characteristics. Nanocrystalline gold has been widely used in biological, chemical and other fields due to its good optical properties. In this paper, different biosensors were constructed to detect tetracycline, DNA, adenosine of hepatitis B virus by using the spectral characteristics of gold nanoparticles and nucleic acid dyes. The main contents are as follows: (1) Detection of tetracycline based on nucleic acid aptamer-free fluorescent assay. A three-helix molecular switch was constructed based on tetracycline aptamer and signal conversion probe. When tetracycline is added, the structure of the three-helix molecular switch is destroyed, and the tetracycline aptamer binds specifically to tetracycline, thus releasing the signal conversion probe. The fluorescence intensity of SG decreased in direct proportion to the concentration of tetracycline. The linear range was 1.1-23 nM, and the detection limit was 130 pM.. The method can be applied to the detection of tetracycline in milk samples. The recoveries of tetracycline in milk samples are 92.5-96.1. (2) HBV. detection is based on nucleic acid exonuclease III and nucleic acid dye SG. At first, two hairpins with protruding ends were designed. When there was no HBV, ssDNA opened two hairpins one after another and DNA, was cut by Exo III. The remaining part of single-stranded DNA, appeared weak fluorescence signal after adding SG. In the presence of HBV, ssDNA hybridized with HBV to form a partially complementary double-stranded DNA without interaction with hairpin DNA. Therefore, the hairpin DNA could maintain its hairpin structure without being cut by Exo III, and the system with SG showed a strong fluorescence signal. The experimental results show that when the concentration of HBV is in the range of 0.1 ~ 10 nM, the fluorescence enhancement of the system is linear with the concentration of HBV, and the detection limit is 59 pM.. The method is simple, convenient and sensitive, and can be applied to the detection of HBV in serum samples. The recovery rate of the method is 93.0-98.0.The (3) adenosine is detected based on aptamer of nucleic acid and gold nanoparticles. When adenosine did not exist, the aptamer adsorbed on the surface of gold nanoparticles to form a protective layer, and the protective nanocrystalline gold was still dispersed in the high salt solution, and the color of the solution was still red. After the addition of SG, SG was embedded in the aptamer and adsorbed on the gold nanoparticles with the aptamer. Therefore, the distance between SG and nanocrystalline gold is narrowed, and the emission of SG overlaps with the absorption of nanocrystalline gold, which results in the fluorescence resonance energy transfer, and the fluorescence of SG is quenched by nanocrystalline gold. In the presence of adenosine, adenosine binds specifically to its aptamer, and the gold nanoparticles reunite in high salt solution, which changes from red to blue. After the addition of SG, SG was embedded in the aptamer-adenosine and the fluorescence recovered. Therefore, colorimetric method, UV method and fluorescence method can be used to detect adenosine. The sensitivity of fluorescence method was the highest, and the detection limit was 0.8 nm in the detection range of 5.5 ~ 110nm 路mol ~ (-1) 路L ~ (-1) 路L ~ (-1). The recoveries of adenosine in serum samples were 92.0-103.4.
【學(xué)位授予單位】：湘潭大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：O657.3
本文編號(hào)：2354208
[Abstract]:Nucleic acid aptamer is a sequence of DNA or RNA obtained by in vitro screening. It can bind to target material with high affinity and specificity and is widely used in biosensors. Nucleic acid dye SG is a dye with green excitation wavelength that binds to all double stranded DNA double helix grooves. The change of fluorescence signal can be used to quantitatively detect the target material. Nucleic acid exonuclease (III) can incise a single nucleotide in the direction of 3'-5'of double-stranded DNA, and the sensitivity of the sensor can be improved by using its shear characteristics. Nanocrystalline gold has been widely used in biological, chemical and other fields due to its good optical properties. In this paper, different biosensors were constructed to detect tetracycline, DNA, adenosine of hepatitis B virus by using the spectral characteristics of gold nanoparticles and nucleic acid dyes. The main contents are as follows: (1) Detection of tetracycline based on nucleic acid aptamer-free fluorescent assay. A three-helix molecular switch was constructed based on tetracycline aptamer and signal conversion probe. When tetracycline is added, the structure of the three-helix molecular switch is destroyed, and the tetracycline aptamer binds specifically to tetracycline, thus releasing the signal conversion probe. The fluorescence intensity of SG decreased in direct proportion to the concentration of tetracycline. The linear range was 1.1-23 nM, and the detection limit was 130 pM.. The method can be applied to the detection of tetracycline in milk samples. The recoveries of tetracycline in milk samples are 92.5-96.1. (2) HBV. detection is based on nucleic acid exonuclease III and nucleic acid dye SG. At first, two hairpins with protruding ends were designed. When there was no HBV, ssDNA opened two hairpins one after another and DNA, was cut by Exo III. The remaining part of single-stranded DNA, appeared weak fluorescence signal after adding SG. In the presence of HBV, ssDNA hybridized with HBV to form a partially complementary double-stranded DNA without interaction with hairpin DNA. Therefore, the hairpin DNA could maintain its hairpin structure without being cut by Exo III, and the system with SG showed a strong fluorescence signal. The experimental results show that when the concentration of HBV is in the range of 0.1 ~ 10 nM, the fluorescence enhancement of the system is linear with the concentration of HBV, and the detection limit is 59 pM.. The method is simple, convenient and sensitive, and can be applied to the detection of HBV in serum samples. The recovery rate of the method is 93.0-98.0.The (3) adenosine is detected based on aptamer of nucleic acid and gold nanoparticles. When adenosine did not exist, the aptamer adsorbed on the surface of gold nanoparticles to form a protective layer, and the protective nanocrystalline gold was still dispersed in the high salt solution, and the color of the solution was still red. After the addition of SG, SG was embedded in the aptamer and adsorbed on the gold nanoparticles with the aptamer. Therefore, the distance between SG and nanocrystalline gold is narrowed, and the emission of SG overlaps with the absorption of nanocrystalline gold, which results in the fluorescence resonance energy transfer, and the fluorescence of SG is quenched by nanocrystalline gold. In the presence of adenosine, adenosine binds specifically to its aptamer, and the gold nanoparticles reunite in high salt solution, which changes from red to blue. After the addition of SG, SG was embedded in the aptamer-adenosine and the fluorescence recovered. Therefore, colorimetric method, UV method and fluorescence method can be used to detect adenosine. The sensitivity of fluorescence method was the highest, and the detection limit was 0.8 nm in the detection range of 5.5 ~ 110nm 路mol ~ (-1) 路L ~ (-1) 路L ~ (-1). The recoveries of adenosine in serum samples were 92.0-103.4.
【學(xué)位授予單位】：湘潭大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：O657.3
【參考文獻(xiàn)】
相關(guān)期刊論文 前9條
1 李蘭英;鄭國金;;紫外-可見分光光度法在藥物分析中的應(yīng)用[J];中國醫(yī)藥指南;2013年30期
2 馮東;李雪梅;王丙蓮;;紫外-可見分光光度法在食品檢測(cè)及食品安全分析中的應(yīng)用[J];食品工業(yè);2013年06期
3 馮彩婷;楊麗霞;李書靜;;紫外-可見分光光度法測(cè)定奶粉中的香蘭素含量[J];河北化工;2012年06期
4 楊季冬;張書然;劉紹璞;;同步-偏振-導(dǎo)數(shù)熒光法同時(shí)測(cè)定三種苯二酚異構(gòu)體的研究[J];化學(xué)學(xué)報(bào);2007年20期
5 徐大光;陰志剛;侯曉強(qiáng);薛冰;;紫外-可見分光光度法在藥物分析中的應(yīng)用及進(jìn)展[J];中國醫(yī)學(xué)物理學(xué)雜志;2006年06期
6 李巧玲;原現(xiàn)瑞;劉景艷;;紫外-可見分光光度法在食品分析中的應(yīng)用進(jìn)展[J];食品工程;2006年01期
7 劉春春;杭海英;;生物大分子相互作用檢測(cè)技術(shù)新進(jìn)展——三色熒光級(jí)聯(lián)熒光共振能量轉(zhuǎn)移技術(shù)[J];生物化學(xué)與生物物理進(jìn)展;2006年03期
8 丁志群;劉文清;張玉鈞;李宏斌;魏慶農(nóng);趙南京;陳東;楊立書;;激光誘導(dǎo)熒光探測(cè)水體中溶解有機(jī)物濃度[J];光子學(xué)報(bào);2006年02期
9 黃賢智;許金鉤;李耀群;;同步熒光法同時(shí)測(cè)定色氨酸、酪氨酸和苯丙氨酸[J];分析化學(xué);1987年03期
相關(guān)博士學(xué)位論文 前1條
1 胡珊;熒光共振能量轉(zhuǎn)移體系的研究及其在均相免疫分析中的應(yīng)用[D];華中科技大學(xué);2010年
，本文編號(hào)：2354208
本文鏈接：http://sikaile.net/kejilunwen/huaxue/2354208.html
最近更新
教材專著
