表面增强拉曼光谱研究DNA碱基与农药相互作用及适配体传感器的应用

发布时间：2018-09-14 19:14

【摘要】：本研究主要利用SERS研究了DNA碱基、碱基与农药的相互作用以及应用核酸配适体传感器检测马拉硫磷。碱基是脱氧核糖核酸(DNA)的主要组成部分,与生物的生长、发育、繁殖等正常生命活动及突变、癌变等异常生命活动密切相关。因此,碱基的性质一直是生物和化学领域的重要研究内容之一。研究DNA碱基与农药相互作用有助于从分子水平上认识农药分子对DNA作用的细节,对了解致毒、致癌、致基因突变产生的机理具有一定的意义。同时可为农药适配体传感器的筛选和构建提供依据,应用农药适配体与表面增强拉曼光谱(SERS)法相结合可以对农药残留分子进行特异性检测,以此为基础建立对农药分子的特异性快速检测方法,是一项具有实际应用价值的工作。首先,通过化学还原法制备出了具有SERS活性的金、银纳米粒子,粒子形貌规整,接近球形。对四种DNA碱基(腺嘌呤、鸟嘌呤、胞嘧啶、胸腺嘧啶)在银上的SERS谱进行研究,运用密度泛函理论(DFT)/B3LYP泛函方法对碱基分子内键振动模拟计算其理论拉曼光谱,并与实验NRS、SERS进行对比,对其峰进行了详细的归属。进一步从理论上研究了银金属团簇对腺嘌呤振动光谱的影响。结果表明,随着银原子数目的增加,腺嘌呤N与Ag距离随Ag原子数的增加而距离变小,模拟光谱和实验SERS能更好地相符合,且其静态极化率值增大,与模拟的拉曼光谱峰强度的变化趋势显现相关性。结合实验SERS谱,说明腺嘌呤环上的N原子与金属银发生了电荷转移。为进一步利用拉曼光谱研究单碱基分子与农药分子的相互作用研究奠定了理论和实验基础。其次,采用SERS方法详细研究了胞嘧啶(Cy)在银上的吸附行为和规律。从理论计算结合实测值探讨了Cy在基底Ag上的增强效应和吸附行为。考察了Cy分子在Ag纳米粒子上的不同吸附时间、浓度、pH等条件对SERS光谱的影响,发现pH影响最大,Cy分子存在两种不同的异构体和三种不同的存在形态,并随酸度变化相互转化而达动态平衡。基于Cy在不同pH时的形态分布和相应的SERS变化规律,结合DFT算得的Cy分子中的电荷分布及在银基底表面的吸附机制,详细探讨了酸碱对Cy分子的SRES光谱影响的内因和吸附机理,在中性和弱碱性时,Cy中的N3和O与Ag形成配位吸附,在pH大于11时,N与Ag形成配位吸附,而O与Ag形成共价吸附。在此基础上,采用UV(紫外光谱)和SERS研究了四种DNA碱基与农药倍硫磷分子在溶液中的相互作用,主要对其作用位点、相互作用强弱进行了研究,结果表明,在腺嘌呤、鸟嘌呤、胞嘧啶含氨基的三种碱基,主要是倍硫磷与这三种碱基的氨基作用是其主要结合位点,而在胸腺嘧啶中,作用对其的影响主要是对其环呼吸振动的影响。根据倍硫磷的分子表面静电势图,11位的S原子是其分子电子密度集中区域,是与碱基作用的主要位点。结合DFT计算表明,以不同位点结合对其环呼吸振动的影响不同,作用位点越靠近碱基环的位置,对环振动拉曼峰的影响越大。最后,利用银溶胶和适配体,发展了一种针对马拉硫磷的特异性检测的SERS方法。通过适配体-马拉硫磷结合靶标物1089、1112 cm-1两处特征峰中1112 cm-1的强度来检测马拉硫磷,结果表明在5×10-7~1×10-5 mol·L-1范围内具有线性,其线性程为I=58.37C(×10-7 mol·L-1)+197.97,R2=0.9920。且通过模拟样品测试和特异性实验验证了该方法的可行性和特异性,该方法测定简单,且具有特异性,有一定的应用价值,可实现样品中马拉硫磷的快速无损检测。
[Abstract]:In this study, SERS was used to study the interaction between DNA bases and pesticides, and the detection of malathion by nucleic acid ligand biosensor. Bases are the main components of DNA, which are closely related to normal life activities such as growth, development, reproduction, mutation, canceration and other abnormal life activities. The study of the interaction between DNA bases and pesticides is helpful to understand the details of the interaction between pesticides and DNA at the molecular level. It is also helpful to understand the mechanism of toxicity, carcinogenesis and gene mutation. It is a valuable work to establish a rapid and specific method for the detection of pesticide residues by combining the aptamer with surface-enhanced Raman spectroscopy (SERS). Firstly, the SERS active pesticide residues were prepared by chemical reduction method. The SERS spectra of four DNA bases (adenine, guanine, cytosine, thymine) on silver were studied. The theoretical Raman spectra of the intramolecular bond vibrations were calculated by density functional theory (DFT) / B3LYP functional method, and compared with experimental NRS and SERS. The results show that the distance between adenine N and Ag decreases with the increase of the number of silver atoms, and the simulated spectra and experimental SERS are in better agreement with each other, and the static polarizability increases with the increase of the number of silver atoms. The results show that the N atoms on the adenine ring have charge transfer with silver. It lays a theoretical and experimental foundation for the further study of the interaction between single base molecule and pesticide molecule by Raman spectroscopy. Secondly, the cytosine is studied in detail by SERS method. The enhancement effect and adsorption behavior of Cy on Ag substrate were discussed by theoretical calculation and measured values. The effects of different adsorption time, concentration and pH on SERS spectra of Cy on Ag nanoparticles were investigated. It was found that pH had the greatest influence on SERS spectra. Cy molecules had two different isomers and three different types. Based on the morphological distribution of Cy molecule at different pH and the corresponding SERS variation law, combined with the charge distribution of Cy molecule calculated by DFT and the adsorption mechanism on the surface of silver substrate, the internal cause and adsorption mechanism of acid-base effect on the SRES spectrum of Cy molecule were discussed in detail. When the pH is higher than 11, N and Ag form coordination adsorption, while O and Ag form covalent adsorption. On this basis, UV (ultraviolet spectroscopy) and SERS were used to study the interaction between four DNA bases and thiophosphorus pesticide molecules in solution, mainly the interaction site and interaction strength were studied. The results showed that the amino-binding sites of the three amino groups in adenine, guanine and cytosine were mainly the amino-binding sites of thiophosphorus and the amino-binding sites of the three amino groups. In thymine, the effect of the amino-binding sites on the cyclic respiratory vibrations was mainly the effect of the amino-binding sites. The results of DFT calculation show that the closer the binding site to the base ring, the greater the influence on the Raman peak of ring vibration. Finally, a special kind of malathion was developed by using silver sol and aptamer. SERS method for anisotropy detection. Malathion was detected by the intensity of 1112 cm-1 in the two characteristic peaks of the aptamer-malathion binding target 1089,1112 cm-1. The results showed that it was linear in the range of 5 *10-7~1 *10-5 mol.L-1, and the linear equation was I=58.37C (*10-7 mol.L-1)+197.97, R2=0.9920. The feasibility and specificity of this method are verified by experiments. The method is simple, specific and has certain application value. It can be used for rapid and nondestructive detection of malathion in samples.
【学位授予单位】：浙江工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657.37;TQ450.1

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