基于固態(tài)納米孔的生物分子辨識(shí)實(shí)驗(yàn)研究
發(fā)布時(shí)間：2018-07-23 09:16
【摘要】：現(xiàn)代生命科學(xué)的發(fā)展,使人類了解到基因是人類的遺傳密碼,蛋白質(zhì)是生命活動(dòng)的執(zhí)行者。破解基因意味著人類可以掌握自己的生命信息,而知道身體中蛋白質(zhì)的信息則掌握了身體的全部健康狀況。納米孔技術(shù)是未來(lái)檢測(cè)基因及蛋白質(zhì)最為理想的工具,是目前正在研制的第三代基因測(cè)序技術(shù)的基礎(chǔ),也將成為未來(lái)精準(zhǔn)醫(yī)療的重要手段。本文對(duì)固態(tài)納米孔辨識(shí)生物分子的可行性進(jìn)行實(shí)驗(yàn)研究,探索基因分子和蛋白質(zhì)分子在納米孔中的易位規(guī)律。主要的研究成果和內(nèi)容如下:1)研究雙鏈λ-DNA(以下簡(jiǎn)稱DNA)在兩種條件下(其一,同濃度不同電解質(zhì)溶液(LiCl、NaCl、KCl);其二,不同濃度同種電解質(zhì)溶液(LiCl))的過(guò)孔行為特征并分析其通過(guò)納米孔的姿態(tài)。研究表明,DNA過(guò)孔時(shí)間與其表面吸附離子的數(shù)量和離子吸附能力有關(guān),離子吸附能力越強(qiáng)和離子數(shù)量越多,DNA整體凈電荷量越少,過(guò)孔時(shí)間越長(zhǎng)。DNA在通過(guò)納米孔時(shí)會(huì)呈現(xiàn)線性長(zhǎng)鏈拉直過(guò)孔和長(zhǎng)鏈折疊過(guò)孔兩種姿態(tài)。2)研究牛血清蛋白(BSA)在納米孔中過(guò)孔行為,探索蛋白質(zhì)過(guò)孔的普遍規(guī)律。在施加不同跨膜電壓下,研究在納米孔中強(qiáng)電場(chǎng)下蛋白質(zhì)的解折疊機(jī)理。對(duì)不同電解質(zhì)溶液中,BSA與納米孔之間相互作用和易位頻率進(jìn)行分析討論。結(jié)果表明,BSA在通過(guò)納米孔時(shí),會(huì)出現(xiàn)兩次解折疊過(guò)程,而且當(dāng)電場(chǎng)強(qiáng)度到達(dá)一定值時(shí),這兩次解折疊都是突變而不是傳統(tǒng)理念中認(rèn)為的漸變過(guò)程。研究還發(fā)現(xiàn)在LiCl、NaCl、KC1三者中,KC1中BSA通過(guò)頻率最高但阻塞最為嚴(yán)重,LiCl中現(xiàn)象正好相反。3)對(duì)比DNA、BSA兩種生物分子混合與單獨(dú)檢測(cè)的實(shí)驗(yàn)結(jié)果,探索納米孔對(duì)簡(jiǎn)單生物分子的辨識(shí)能力。實(shí)驗(yàn)結(jié)果中發(fā)現(xiàn)統(tǒng)計(jì)數(shù)據(jù)結(jié)果介于二者之間,整體現(xiàn)象和單純DNA過(guò)孔行為類似,同時(shí)出現(xiàn)類似蛋白質(zhì)過(guò)孔時(shí)的長(zhǎng)過(guò)孔時(shí)間事件,且數(shù)量明顯少于純BSA過(guò)孔事件,可以從中辨識(shí)BSA長(zhǎng)過(guò)孔時(shí)間事件以及DNA正常過(guò)孔事件。4)探究固態(tài)納米孔對(duì)復(fù)雜生物大分子IgG、IgA、IgM的辨識(shí)能力。從實(shí)驗(yàn)結(jié)果中發(fā)現(xiàn),生物大分子在通過(guò)納米孔時(shí),進(jìn)入納米孔較為困難卻會(huì)在孔口附近徘徊產(chǎn)生碰撞信號(hào),而且分子量越大,碰撞信號(hào)數(shù)量占總信號(hào)比率越高。5)探索設(shè)置濃度差的方法提高信噪比,以增強(qiáng)固態(tài)納米孔對(duì)生物分子辨識(shí)能力,優(yōu)化實(shí)驗(yàn)結(jié)果。研究發(fā)現(xiàn)在納米孔兩側(cè)存在濃度差時(shí),帶電生物分子(DNA、BSA)從高濃度往低濃度運(yùn)動(dòng)時(shí),其過(guò)孔信號(hào)能夠被放大,濃度差梯度越大,放大效應(yīng)越明顯。而且該方法對(duì)生物分子過(guò)孔時(shí)間沒有太大的影響。6)采用多物理場(chǎng)耦合軟件(COMSOL)模擬納米通道內(nèi)流場(chǎng)情況,對(duì)濃度差下的信號(hào)放大效應(yīng)進(jìn)行模擬仿真。從仿真結(jié)果中推斷出該效應(yīng)和生物分子表面電荷、濃度差離子擴(kuò)散和雙電層三者有關(guān)。為了驗(yàn)證模擬的結(jié)果,改變生物分子表面電荷,觀察濃度差下現(xiàn)象是否與猜想一致。實(shí)驗(yàn)結(jié)果證實(shí)了本文推斷的合理性。
[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.
【學(xué)位授予單位】：東南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q7;TB383.1
本文編號(hào)：2138932
[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.
【學(xué)位授予單位】：東南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q7;TB383.1
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