發(fā)布時(shí)間：2018-05-19 04:30

  本文選題：基因測(cè)序 + 固態(tài)納米孔��； 參考：《吉林大學(xué)》2017年碩士論文

【摘要】：DNA(Deoxyribonucleic Acid,脫氧核糖核酸)蘊(yùn)含了整個(gè)生物體的遺傳信息。能夠快速和準(zhǔn)確獲取DNA序列的信息對(duì)于對(duì)探究生命奧秘,疾病診斷,藥物研發(fā),育種等領(lǐng)域的技術(shù)發(fā)展起到不可忽視的推動(dòng)作用。高通量、低成本的納米孔(直徑為幾個(gè)納米的小孔)測(cè)序技術(shù)由于其能直接檢測(cè)無(wú)標(biāo)記的單分子,是下一代DNA測(cè)序技術(shù)的重要候選者。其原理是通過(guò)檢測(cè)DNA分子易位穿過(guò)納米孔時(shí)引起的特征離子電流來(lái)分辨DNA鏈上的四種不同類(lèi)型的堿基,從而讀取整個(gè)DNA長(zhǎng)鏈的信息。根據(jù)其制作材料的不同,納米孔大致可被分為兩類(lèi):生物納米孔和固態(tài)納米孔。固態(tài)納米孔杰出的熱學(xué)、力學(xué)、化學(xué)穩(wěn)定性和大規(guī)模集成性的優(yōu)勢(shì),使其被廣泛應(yīng)用。然而固態(tài)納米孔的制備受到傳統(tǒng)制備方法復(fù)雜、高成本、低產(chǎn)量的限制。另一個(gè)制約固態(tài)納米孔測(cè)序發(fā)展的難題是:納米孔器件承載膜太厚和DNA穿孔的易位速率太快,導(dǎo)致在空間分辨率和時(shí)間分辨率上都達(dá)不到分辨單堿基的目的。因此,如何有效控制DNA分子在納米孔內(nèi)的傳輸行為,減慢DNA分子的易位速率,對(duì)納米孔測(cè)序技術(shù)突破具有重要意義。首先,本文介紹了一種快速、易操作的制備納米孔方法,在電解質(zhì)溶液中的,通過(guò)在絕緣薄膜上施加可調(diào)電流脈沖,使薄膜發(fā)生電介質(zhì)擊穿形成具有納米精度的單個(gè)固態(tài)納米孔,脈沖參數(shù)可以調(diào)整而適用于不同厚度和材料的薄膜。相比于其他制備納米孔的微納加工工藝,此方法大大降低了操作復(fù)雜性和工藝成本,可實(shí)現(xiàn)批量化生產(chǎn)。我們利用LabVIEW虛擬儀器控制可編程的數(shù)字源表Keithley 2450實(shí)現(xiàn)電介質(zhì)擊穿過(guò)程的測(cè)量與數(shù)據(jù)記錄。此法制備出的氮化硅和石墨烯納米孔穩(wěn)定性良好,在后續(xù)的DNA檢測(cè)應(yīng)用中表現(xiàn)出了優(yōu)異的傳感性能。其次,提出微納米網(wǎng)格和氮化硅納米孔垂直集成的新結(jié)構(gòu),一定電壓下,聚合物納米纖維網(wǎng)格把纏繞的DNA并解開(kāi)成線性狀態(tài),克服納米孔對(duì)較長(zhǎng)DNA分子形成的熵勢(shì)壘,從實(shí)驗(yàn)上觀測(cè)到了減慢長(zhǎng)鏈DNA分子易位速率的現(xiàn)象,DNA易位速率最長(zhǎng)可減緩至136.77/(7。利用MATLAB仿真軟件建立了微納米網(wǎng)格-納米孔結(jié)構(gòu)的物理模型,從理論上計(jì)算了加入納米纖維網(wǎng)格前后,納米孔附近離子濃度和電場(chǎng)分布的變化,解釋了微納米網(wǎng)格影響DNA易位行為的物理機(jī)制。另外,本文還嘗試制備了納米孔集成氧化鋅納米線網(wǎng)格結(jié)構(gòu),為其今后應(yīng)用于DNA測(cè)序領(lǐng)域驗(yàn)證了可行性。值得欣喜的是,本文還利用厚度接近于堿基間距的石墨烯納米孔首次探測(cè)到了單堿基的信號(hào),并采用增加電解質(zhì)粘度的辦法,實(shí)驗(yàn)中利用有機(jī)離子液體BMIMCl將單堿基的易位速率減緩至毫秒量級(jí),并嘗試區(qū)分出不同的堿基種類(lèi)。研究成果將為基于納米孔的DNA測(cè)序技術(shù)研究、蛋白質(zhì)和長(zhǎng)鏈聚合物檢測(cè)等領(lǐng)域的實(shí)際應(yīng)用提供必要的理論依據(jù)、基礎(chǔ)實(shí)驗(yàn)數(shù)據(jù)和技術(shù)儲(chǔ)備。
[Abstract]:DNA(Deoxyribonucleic acid (deoxyribonucleic acid) contains genetic information for the whole organism. The rapid and accurate acquisition of DNA sequences plays an important role in exploring the mystery of life, disease diagnosis, drug research and development, breeding and other fields of technological development. High-throughput and low-cost nano-pore sequencing (several nanometers in diameter) is an important candidate for the next generation of DNA sequencing because of its ability to directly detect unlabeled monolayers. The principle is to detect the characteristic ion currents caused by the translocation of DNA molecules through the nanopores to distinguish four different types of bases in the DNA chain, so as to read the information of the whole DNA long chain. According to the different materials, nano-pores can be divided into two types: biological nano-pores and solid nano-pores. The outstanding thermal, mechanical, chemical stability and large-scale integration advantages of solid-state nano-pore make it widely used. However, the preparation of solid nanoparticles is limited by the traditional preparation methods, such as complex, high cost and low production. Another difficult problem that restricts the development of solid-state nano-pore sequencing is that the carrier film thickness of nano-hole device and the translocation rate of DNA perforation are too fast, resulting in the spatial resolution and time resolution can not achieve the purpose of single base resolution. Therefore, how to effectively control the transport behavior of DNA molecules in nano-pores and slow down the translocation rate of DNA molecules is of great significance for the breakthrough of nano-pore sequencing technology. First of all, this paper introduces a fast and easy to operate method for the preparation of nano-pores in electrolyte solution by applying adjustable current pulses to the insulating film. A single solid nanometer pore with nanometer precision can be formed by dielectric breakdown of the film. The pulse parameters can be adjusted and applied to thin films with different thickness and material. Compared with other micro-nano fabrication processes, this method greatly reduces the operation complexity and process cost, and can realize batch production. We use LabVIEW virtual instrument to control the programmable digital source meter Keithley 2450 to realize the measurement and data recording of dielectric breakdown process. The nano-pore of silicon nitride and graphene prepared by this method has good stability and excellent sensing performance in the subsequent application of DNA. Secondly, a new structure of vertical integration of nanoscale meshes and nano-pores of silicon nitride is proposed. At a certain voltage, polymer nanofilament meshes winding DNA and unwrapping it into a linear state to overcome the entropy barrier formed by nano-pores on longer DNA molecules. The phenomenon of slowing down the rate of translocation of long-stranded DNA molecules has been observed experimentally. The longest rate of DNA translocation can be reduced to 136.77 / 7. The physical model of micro-nano mesh-nano-pore structure was established by using MATLAB simulation software. The changes of ion concentration and electric field distribution near nano-pore before and after the addition of nano-fiber mesh were calculated theoretically. The physical mechanism of the effect of micro-nano-mesh on DNA translocation behavior is explained. In addition, this paper also attempted to prepare nano-pore integrated ZnO nanowire grid structure, which proved the feasibility of its application in the field of DNA sequencing in the future. It is gratifying to note that the signal of a single base is detected for the first time by using graphene nanorods with thickness close to base spacing, and the method of increasing electrolyte viscosity is adopted. In the experiment, the translocation rate of single base was reduced to millisecond by using organic ionic liquid (BMIMCl), and different base species were identified. The research results will provide the necessary theoretical basis, basic experimental data and technical reserve for the research of DNA sequencing technology based on nano-pore, protein and long chain polymer detection and other practical applications.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：Q503

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本文編號(hào)：1908728