基于固态纳米孔的生物分子辨识实验研究

发布时间：2018-07-23 09:16

【摘要】：现代生命科学的发展,使人类了解到基因是人类的遗传密码,蛋白质是生命活动的执行者。破解基因意味着人类可以掌握自己的生命信息,而知道身体中蛋白质的信息则掌握了身体的全部健康状况。纳米孔技术是未来检测基因及蛋白质最为理想的工具,是目前正在研制的第三代基因测序技术的基础,也将成为未来精准医疗的重要手段。本文对固态纳米孔辨识生物分子的可行性进行实验研究,探索基因分子和蛋白质分子在纳米孔中的易位规律。主要的研究成果和内容如下:1)研究双链λ-DNA(以下简称DNA)在两种条件下(其一,同浓度不同电解质溶液(LiCl、NaCl、KCl);其二,不同浓度同种电解质溶液(LiCl))的过孔行为特征并分析其通过纳米孔的姿态。研究表明,DNA过孔时间与其表面吸附离子的数量和离子吸附能力有关,离子吸附能力越强和离子数量越多,DNA整体净电荷量越少,过孔时间越长。DNA在通过纳米孔时会呈现线性长链拉直过孔和长链折叠过孔两种姿态。2)研究牛血清蛋白(BSA)在纳米孔中过孔行为,探索蛋白质过孔的普遍规律。在施加不同跨膜电压下,研究在纳米孔中强电场下蛋白质的解折叠机理。对不同电解质溶液中,BSA与纳米孔之间相互作用和易位频率进行分析讨论。结果表明,BSA在通过纳米孔时,会出现两次解折叠过程,而且当电场强度到达一定值时,这两次解折叠都是突变而不是传统理念中认为的渐变过程。研究还发现在LiCl、NaCl、KC1三者中,KC1中BSA通过频率最高但阻塞最为严重,LiCl中现象正好相反。3)对比DNA、BSA两种生物分子混合与单独检测的实验结果,探索纳米孔对简单生物分子的辨识能力。实验结果中发现统计数据结果介于二者之间,整体现象和单纯DNA过孔行为类似,同时出现类似蛋白质过孔时的长过孔时间事件,且数量明显少于纯BSA过孔事件,可以从中辨识BSA长过孔时间事件以及DNA正常过孔事件。4)探究固态纳米孔对复杂生物大分子IgG、IgA、IgM的辨识能力。从实验结果中发现,生物大分子在通过纳米孔时,进入纳米孔较为困难却会在孔口附近徘徊产生碰撞信号,而且分子量越大,碰撞信号数量占总信号比率越高。5)探索设置浓度差的方法提高信噪比,以增强固态纳米孔对生物分子辨识能力,优化实验结果。研究发现在纳米孔两侧存在浓度差时,带电生物分子(DNA、BSA)从高浓度往低浓度运动时,其过孔信号能够被放大,浓度差梯度越大,放大效应越明显。而且该方法对生物分子过孔时间没有太大的影响。6)采用多物理场耦合软件(COMSOL)模拟纳米通道内流场情况,对浓度差下的信号放大效应进行模拟仿真。从仿真结果中推断出该效应和生物分子表面电荷、浓度差离子扩散和双电层三者有关。为了验证模拟的结果,改变生物分子表面电荷,观察浓度差下现象是否与猜想一致。实验结果证实了本文推断的合理性。
[Abstract]:With the development of modern life science, human beings understand that genes are human genetic codes and proteins are the executors of life activities. Deciphering genes means that humans have access to their own life information, and that knowledge of proteins in the body holds all the health of the body. Nanopore technology is the most ideal tool for detecting genes and proteins in the future. It is the basis of the third generation gene sequencing technology which is currently being developed. It will also become an important means of accurate medicine in the future. In this paper, the feasibility of identifying biomolecules by solid nanoparticles was studied experimentally, and the translocation of gene molecules and protein molecules in nano-pores was explored. The main research results and contents are as follows: (1) study of double strand 位 -DNA (hereinafter referred to as DNA) under two conditions (one is the same concentration of different electrolyte solution (LiCl-NaCl-KCl), the other is the same concentration of different electrolyte solution (LiCl-NaCl-KCl). The behavior characteristics of the same electrolyte solution (LiCl) in different concentration and the attitude of passing through the nano-pore were analyzed. The results show that the pore time of DNA is related to the amount of ions adsorbed on the surface and the adsorption ability of ions. The stronger the ion adsorption ability and the more the number of ions are, the less the total net charge of DNA is. The longer the pore passage time, the more linear long chain straight and long chain folding pore. 2) the behavior of bovine serum protein (BSA) in the nanometer pore was studied, and the general rule of protein passing through the pore was explored. Under different transmembrane voltages, the unfolding mechanism of protein under strong electric field in nano-pore was studied. The interaction and translocation frequency between BSA and nano-pore in different electrolyte solutions were analyzed and discussed. The results show that there are two unfolded processes when the BSA passes through the nano-pores, and when the electric field intensity reaches a certain value, the two unfolding processes are abrupt rather than the gradual change process thought in the traditional idea. It is also found that in LiCl-NaCl-KC1, the phenomenon of BSA passing through most frequently but blocking most seriously in LiCl is opposite to that in LiCl.) comparing with the experimental results of DNA BSA mixing and individual detection, the ability of identifying simple biomolecules by nano-pore is explored. It was found that the statistical data were somewhere between the two. The whole phenomenon was similar to that of pure DNA, and there was a long time event when the protein passed through the pore, and the number was obviously less than that of pure BSA. It can be used to identify BSA long pore crossing time events and DNA normal pore crossing events .4) to explore the ability of solid nano-pore to identify IgGN IgA + IgM from complex biological macromolecules. From the experimental results, it is found that when the biomolecules pass through the nano-pores, the more difficult it is to enter the nano-pores, but the more the molecular weight of the biomolecules is, the higher the molecular weight is, The higher the ratio of collision signal to total signal is, the higher the ratio of collision signal to total signal is.) the method of setting concentration difference to improve SNR is explored to enhance the recognition ability of solid nano-pore to biomolecules and optimize the experimental results. It is found that when there is a concentration difference between the two sides of the nano-pore, when the charged biomolecules (DNA-BSA) move from high concentration to low concentration, the signal can be amplified. The bigger the gradient of concentration difference is, the more obvious the amplification effect is. Moreover, the method has no significant effect on the pore crossing time of biomolecules. 6) the multi-physical field coupling software (COMSOL) is used to simulate the flow field in nanochannels, and the signal amplification effect under the concentration difference is simulated. From the simulation results, it is inferred that this effect is related to the surface charge of biomolecules, the ion diffusion of concentration difference and the double electric layer. In order to verify the simulation results and change the surface charge of biomolecules, the phenomenon under the concentration difference is observed to be consistent with the conjecture. The experimental results confirm the rationality of the inference in this paper.
【学位授予单位】：东南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q7;TB383.1

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