基于DNAzyme介导生成金纳米颗粒的比色法快速检测金黄色葡萄球菌肠毒素B

发布时间：2018-05-20 02:30

本文选题：比色法 + 卟啉铁/G-四链体DNA酶　；参考：《重庆医科大学》2017年硕士论文

【摘要】：生物传感技术是由多学科相互交叉渗透形成的高新技术。利用该技术构建的生物传感器作为一种新型的检测装置,可以通过识别元件和换能器将被测物质浓度转化为可检测的光、电等信号。比色传感器因其光信号检测简便快捷,在分析检测领域广泛应用。近年来,纳米材料在生物传感器中的应用研究已经成为热点。尤其是金纳米颗粒(gold nanoparticles,Au NPs),因其独特的光学特性使比色传感器的灵敏度得到大大的提高。金黄色葡萄球菌肠毒素B(staphylococcal enterotoxin B,SEB)作为超抗原和过敏原在变态反应性疾病中起着非常重要的作用,是引起败血症和脓毒症最重要的毒素因子。因此,SEB的检测具有重要的临床意义。本课题是以SEB为检测目标,构建了一种基于金纳米颗粒生成的比色新方法。该方法能够快速简便、低成本、高灵敏地检测SEB,为相关疾病的临床诊断提供了新工具。该方法能够结合临床检测需求使用不同的识别元件,构建通用型的检测方法,为生物传感技术提供了新思路。目的:基于卟啉铁/G-四链体结构的DNAzyme介导金纳米颗粒生成导致的颜色改变,联合适配体作为识别探针构建通用型的比色法检测平台,并以SEB作为待检物的代表,对新构建的比色检测平台进行评价和性能确认。方法:1.金纳米颗粒的生成:在2-(N-吗啉基)乙磺酸(2-(N-morpholino)ethanesulphonic acid,MES)缓冲液中,利用不同浓度的过氧化氢将氯金酸还原成不同分散状态的金纳米颗粒,并对其进行表征。2.具有辣根过氧化物酶活性的DNAzyme的形成:探针G和SEB适配体在反应液中发生杂交反应,加入SEB和卟啉铁,适配体可特异性与SEB结合,探针G与卟啉铁形成具有辣根过氧化物酶活性的DNAzyme。3.比色传感器的构建:利用目标物介导DNAzyme生成,进一步控制金纳米颗粒的合成速度,定量检测样本中的SEB。4.检测条件的优化:对MES缓冲液浓度、氯金酸浓度,过氧化氢浓度,检测时间及适配体和探针G的核酸序列进行优化,以提高信噪比。5.新构建比色法的性能评价:对传感器的最低检测限、线性范围、特异性进行评价。6.实际标本的检测:将已经构建好的比色生物传感器用于血清样本的检测。结果:1.用紫外可见分光光度计、动态光散射、透射电镜对生成的金纳米粒子分别进行光谱学和形态学的表征分析。蓝色和红色金纳米颗粒溶液的紫外可见吸收光谱最大吸收峰分别在604 nm和557 nm处。动态光散射检测得到两种状态的金纳米颗粒的水动力平均直径为282.4 nm和61.9 nm,分布系数为0.347和0.094。透射电镜可见呈均一分散状态和多不规则聚集状态的两种金纳米颗粒。2.用琼脂糖凝胶电泳对探针G和适配体的杂交反应进行表征,结果表明随着SEB浓度的增加,杂交双链逐渐解离。紫外可见分光光度计对DNAzyme的生成进行验证,随着SEB的增加,DNAzyme的生成也增加。3.当目标物加入到构建的比色传感器,DNAzyme生成,降低了金纳米颗粒的生成速率,纳米颗粒溶液呈现蓝色,从而实现目标物的检测。4.优化实验得到的最佳条件为:MES缓冲液的浓度为2 m M,氯金酸的浓度为0.3 m M,过氧化氢的浓度120μM,检测时间反应后15 min,适配体选用APTSEB1,探针G选用G3。5.在最优条件下对新构建的比色传感器进行评价:SEB浓度在0.1pg m L-1-1000 pg m L-1范围内与550 nm处降低的吸光度值呈线性相关,R2=0.9895;SEB肉眼可识别的最低检测限为1 pg m L-1;该传感器能特异识别SEB,在高浓度BSA、凝血酶、Ig G存在的情况下,均只产生微弱的背景信号。6.在血清中加入SEB,配制三组不同浓度的SEB(10 pg m L-1,100pg m L-1,1000 pg m L-1),该法测得的结果与缓冲液配制相同浓度的SEB检测结果无明显差异,表明该传感器可以正确检测血清等复杂基质的临床样本。结论:本研究成功构建了基于卟啉铁/G-四链体结构的DNAzyme介导金纳米颗粒生成的比色法检测平台,该传感器无酶无修饰,操作简单。用于检测SEB时灵敏度高,检测线性范围宽,特异性好,并可用于临床血清样本中SEB的检测。
[Abstract]:Biosensor is a high and new technology which is formed by interpenetration of multidisciplinary. The biosensor constructed by this technology is used as a new type of detection device, and the measured material concentration can be transformed into detectable light, electricity and other signals by identifying elements and transducers. In recent years, the application of nanomaterials in biosensors has become a hot spot. In particular, gold nanoparticles (gold nanoparticles, Au NPs) have greatly improved the sensitivity of colorimetric sensor because of its unique optical properties. Staphylococcus aureus enterotoxin B (staphylococcal enterotoxin B, SEB) As a superantigen and anaphylaxis, it plays a very important role in allergic diseases and is the most important toxin factor in sepsis and sepsis. Therefore, the detection of SEB has important clinical significance. This subject is a new method based on the formation of gold nanoparticles by using SEB as the detection target. This method can be fast. The rapid, simple, low cost, and highly sensitive detection of SEB provides a new tool for the clinical diagnosis of related diseases. This method can use different identification components in combination with clinical detection requirements and construct a general detection method, which provides a new idea for biological sensing technology. Objective: DNAzyme mediated gold nanoparticles based on the structure of porphyrin iron /G- four chain body Color change caused by grain generation, combined aptamers as identification probes to construct a generic colorimetric detection platform, and use SEB as a representative to evaluate and confirm the performance of the newly constructed colorimetric detection platform. Method: the generation of 1. gold nanoparticles: 2- (N-morpholino) ethanesulphonic acid, M at 2- (N- Ma Lanji) ethanesulphonic acid ES) in the buffer, chlorosauric acid was reduced to gold nanoparticles with different dispersing state by hydrogen peroxide with different concentrations, and the formation of DNAzyme was characterized by.2. with horseradish peroxidase activity: hybridization reaction between probe G and SEB aptamers in the reaction liquid, added into SEB and porphyrin iron, the aptamers are specifically associated with SEB The formation of the DNAzyme.3. colorimetric sensor with horseradish peroxidase activity by needle G and porphyrin: using target to mediate DNAzyme formation, further controlling the synthesis speed of gold nanoparticles, and optimizing the SEB.4. detection conditions in the samples: the concentration of MES buffer, the concentration of chloro gold acid, the concentration of hydrogen peroxide, the detection time and the detection time. The nucleic acid sequence of the aptamer and the probe G is optimized to improve the performance evaluation of the signal to noise ratio.5. new construction colorimetric method: the minimum detection limit of the sensor, the linear range, the specificity of the detection of the actual specimens of.6.: a good colorimetric biosensor is used to detect the blood samples. Results: 1. with UV visible spectrophotometry The maximum absorption peaks of the UV visible absorption spectra of blue and red gold nanoparticles were 604 nm and 557 nm respectively. The average hydrodynamic diameter of the gold nanoparticles with two forms was 2, respectively. The average hydrodynamic diameter of the gold nanoparticles with two forms was 2. 82.4 nm and 61.9 nm, the distribution coefficients of 0.347 and 0.094. transmission electron microscopy showed that two gold nanoparticles with homogeneous and irregular aggregation state.2. were characterized by agarose gel electrophoresis on the hybridization reaction of the probe G and the aptamers. The results showed that the hybrid double chains gradually dissociated with the increase of SEB concentration. Ultraviolet visible light was visible. The degree meter validates the generation of DNAzyme. With the increase of SEB, the generation of DNAzyme also increases.3. when the target is added to the constructed colorimetric sensor, DNAzyme is generated, and the formation rate of gold nanoparticles is reduced, and the nano particle solution is blue. Thus the optimum condition for the test of the target detection.4. optimization experiment is the MES buffer solution. The concentration is 2 m M, the concentration of chloro gold acid is 0.3 m M, the concentration of hydrogen peroxide is 120 mu M, the detection time is 15 min, the aptamer is chosen APTSEB1, and the probe G selects G3.5. under the optimal condition to evaluate the newly constructed colorimetric sensor. The SEB concentration is linearly related to the absorbance value which is reduced in 550 places within the 0.1pg m. R2=0.9895; the minimum detection limit for SEB's naked eye is 1 pg m L-1; the sensor can identify SEB specifically. In the presence of high concentration of BSA, thrombin and Ig G, only a weak background signal.6. is added to the serum for SEB, and the results and buffers of the three groups of SEB (10) are prepared. There is no obvious difference between the SEB detection results of the same concentration of liquid, indicating that the sensor can detect the clinical samples of complex matrix of serum and so on. Conclusion: This study successfully constructed a colorimetric detection platform based on the structure of the porphyria iron /G- four chain body structure of DNAzyme mediated gold nanoparticles. The sensor has no enzyme without modification, and the operation is simple. It has high sensitivity, wide linearity and specificity in detecting SEB, and can be used for the detection of SEB in clinical serum samples.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R446.5;TP212.3

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