基于核酸适配体荧光探针的构建及应用研究

发布时间：2018-11-24 15:15

【摘要】：核酸适配体是利用体外筛选技术得到的一段DNA或RNA序列,它能与靶物质高亲和力、高特异性结合,被广泛应用于生物传感器领域。核酸染料SG是一种结合于所有双链DNA双螺旋小沟区域的具有绿色激发波长的染料。利用其荧光信号的变化可以定量检测靶物质。核酸外切酶III能作用于双链DNA,沿3’→5’方向逐步切去单核苷酸,利用其剪切特性可以提高传感器的灵敏度。纳米金由于其良好的光学特性,被广泛应用于生物、化学等领域,近年来已成为科学家们研究的热点之一。本论文主要利用纳米金和核酸染料的光谱特性来构建不同的生物传感器检测四环素、乙型肝炎病毒DNA、腺苷,主要包括以下三个部分:(1)基于核酸适配体无标记荧光法检测四环素。基于四环素适配体和信号转换探针构建一个三螺旋分子开关,加入四环素后,该三螺旋分子开关的结构被破坏,四环素适配体与四环素特异性结合,从而释放出信号转换探针,然后加入SG,体系荧光降低。SG荧光强度的降低与四环素的浓度成正比,线性范围为1.1-23 nM,检出限是130 pM。本方法能应用于牛奶样品中四环素的检测,其加标回收率为92.5-96.1%。(2)基于核酸外切酶III和核酸染料SG无标记检测HBV。首先设计两个3’-末端突出的发夹DNA,当没有HBV存在时,ssDNA相继打开两个发夹DNA,被Exo III剪切后,剩下部分单链DNA,加入SG后呈现弱的荧光信号。当HBV存在时,ssDNA与HBV杂交形成部分互补的双链DNA而不与发夹DNA作用,因此两个发夹DNA能保持其发夹结构而不被Exo III剪切,加入SG后体系呈现一个强的荧光信号。实验结果表明,当HBV浓度在0.1 10 nM范围内,体系荧光增强与HBV浓度呈线性,检出限为59 pM。该方法简单、方便、灵敏度高,且能很好地应用于血清样品中HBV的检测,其加标回收率为93.0-98.0%。(3)基于核酸适配体和纳米金无标记检测腺苷。腺苷不存在时,适配体吸附在纳米金表面形成一个保护层,保护纳米金在高盐溶液中依然分散,溶液颜色仍为红色,加入SG后,SG嵌入适配体中并随适配体吸附在纳米金上,因此拉近了SG与纳米金之间的距离,且SG的发射与纳米金的吸收相互重叠,从而发生荧光共振能量转移,SG的荧光被纳米金猝灭。腺苷存在时,腺苷与其适配体特异性结合,此时纳米金在高盐溶液中发生团聚,溶液由红色变为蓝色。加入SG后,SG嵌入适配体-腺苷中,荧光恢复。据此,可采用比色法、紫外法、荧光法检测腺苷。其中,荧光法的灵敏度最高,检测范围为5.5 110 nM,检出限为0.8nM。在血清样品中用荧光法检测腺苷的加标回收率为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.
【学位授予单位】：湘潭大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657.3

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