本文選題：納米摩擦學(xué) + 粘著�。� 參考：《西南交通大學(xué)》2015年博士論文

【摘要】：作為遺傳信息的載體,DNA是生命在進(jìn)化過程中最為重要的一種物質(zhì)�？焖贉�(zhǔn)確地探測DNA的序列不僅可以幫助人們有效地預(yù)防和治療如糖尿病、血友癥和癌癥等遺傳學(xué)疾病,還能有助于人類解開自身的生命起源之謎。自上世紀(jì)70年代起,人們已先后發(fā)展了三代DNA測序技術(shù)。第一代DNA測序技術(shù)基于Sanger法,耗時15年完成了人類基因組計劃,直接花費約30億美元。第二代DNA測序技術(shù)以高通量為特點,僅需一周、花費不到100萬美元就能完成人類基因組的測序。近年來備受關(guān)注的基于納米孔的第三代DNA測序技術(shù),通過檢測DNA堿基通過納米孔時所產(chǎn)生的特征阻塞電流進(jìn)行測序。憑借超快速、高精度、低成本的優(yōu)勢,納米孔測序技術(shù)有望完成美國國立衛(wèi)生研究院(NIH)設(shè)定的只花費1000美元、在24小時內(nèi)完成個人基因組測序的目標(biāo)。盡管如此,納米孔測序技術(shù)目前仍存在一些關(guān)鍵問題亟需解決,如DNA分子過孔速度過快以至于難以對阻塞電流信號進(jìn)行精確地檢測。研究表明,增大DNA分子與納米孔孔壁間的摩擦力可以在一定程度上降低其過孔速度。然而,以往的研究主要集中于DNA表面自組裝、分子形貌、DNA分子鏈的力學(xué)穩(wěn)定性以及基于DNA分子特定功能器件的研發(fā),有關(guān)DNA分子摩擦學(xué)行為的研究卻鮮見報道。因此,為了全面、深入地理解DNA分子與納米孔材料之間的摩擦作用機(jī)制,亟需系統(tǒng)地開展DNA摩擦學(xué)行為的研究。相關(guān)研究成果不僅可以促進(jìn)基于納米孔的第三代DNA測序技術(shù)的發(fā)展和應(yīng)用,同時也能夠豐富和完善生物納米摩擦學(xué)的理論基礎(chǔ)。本論文借助原子力顯微鏡(AFM),針對DNA分子/納米孔材料摩擦副,重點研究了不同納米孔材料、溶液NaCl濃度、溶液pH值、分子鏈長對DNA分子摩擦學(xué)行為的影響規(guī)律及作用機(jī)制。首先,成功制備出表面吸附有DNA分子的二氧化硅針尖,利用其研究了溶液環(huán)境中DNA與幾種典型納米孔材料之間的摩擦學(xué)行為,進(jìn)而提出了納米孔材料選擇的優(yōu)化建議。在此基礎(chǔ)上,以原子級平整的云母表面為基底成功制備出DNA自組裝薄膜,并使用AFM的氮化硅針尖系統(tǒng)研究了溶液NaCl濃度、溶液pH值以及分子鏈長對DNA摩擦學(xué)行為的影響規(guī)律及作用機(jī)制。同時,在東南大學(xué)陳云飛教授課題組的幫助下,對不同pH值溶液中DNA分子通過氮化硅納米孔的過孔情況進(jìn)行了測試,以驗證本論文有關(guān)溶液pH值優(yōu)化的研究結(jié)果。論文的主要結(jié)論及創(chuàng)新點如下：(1) 成功制備出表面吸附有DNA分子的二氧化硅針尖,揭示出不同納米孔材料與DNA分子之間摩擦學(xué)行為的變化規(guī)律。使用3-氨丙基三乙氧基硅烷偶聯(lián)劑修飾二氧化硅針尖,將DNA分子自組裝于其表面,成功地制備出表面吸附有DNA分子的二氧化硅針尖。在此基礎(chǔ)上,使用該針尖系統(tǒng)研究了DNA分子與5種典型的納米孔材料(氮化硅、石英、APS-石英、石墨烯、石墨)之間的摩擦學(xué)行為。結(jié)果表明,在相同載荷下5種納米孔材料與DNA分子之間的摩擦力從大到小依次為：APS-石英氮化硅石英石墨烯石墨。其中石墨烯和石墨由于擁有特殊的微觀層狀結(jié)構(gòu),其與DNA之間的摩擦力明顯小于其余3種納米孔材料；通過不同納米孔材料表面的接觸角測試,發(fā)現(xiàn)液下環(huán)境中疏水表面有可能在一定程度上增大DNA分子與材料間的摩擦力；通過APS-石英、氮化硅和石英表面力曲線的測試,發(fā)現(xiàn)帶正電的APS-石英表面與DNA分子之間會產(chǎn)生靜電引力,有助于增大DNA-SiO2針尖與基底間的相互作用力,進(jìn)一步導(dǎo)致摩擦力的增大。該部分研究結(jié)果針對納米孔材料的選擇提出相關(guān)建議,也為后續(xù)DNA摩擦行為研究中針尖材料的選擇提供了參考。(2) 闡明了NaCl溶液濃度對DNA分子構(gòu)象及摩擦行為的影響規(guī)律。在云母表面成功制備出DNA自組裝薄膜,使用氮化硅針尖測試了溶液的NaCl濃度對DNA分子構(gòu)象及摩擦行為的影響規(guī)律。結(jié)果顯示,隨著溶液中帶正電的Na離子濃度的增大,越來越多的DNA分子骨架外側(cè)磷酸基團(tuán)所帶的負(fù)電荷被屏蔽,DNA分子從較為伸展的構(gòu)象逐漸轉(zhuǎn)變?yōu)椤熬�圈盤繞”的構(gòu)象。此外,在相同載荷下,當(dāng)溶液NaCl濃度較低時,由于DNA分子的剛度較高,針尖與DNA分子間的摩擦力較大�？梢�,較低的NaCl濃度能使DNA分子更為伸展,有利于DNA分子在良好的姿態(tài)下進(jìn)入并通過納米孔,同時,較大的摩擦力利于降低DNA分子的過孔速度。因此,較低的NaCl濃度更利于DNA分子的納米孔測序。(3) 揭示了溶液pH值對DNA分子構(gòu)象及摩擦行為的作用機(jī)制。在不同pH值的溶液中,使用氮化硅針尖在DNA薄膜表面進(jìn)行了粘著與摩擦行為的測試,揭示了溶液pH值對DNA分子構(gòu)象及摩擦力的影響機(jī)制。研究表明,溶液pH值的變化將對DNA分子堿基之間,以及DNA分子與氮化硅針尖之間的相互作用力產(chǎn)生影響,從而會進(jìn)一步影響DNA分子的構(gòu)象及其與氮化硅針尖的摩擦行為。當(dāng)pH較低時,DNA分子與氮化硅針尖之間存在靜電引力,在一定程度上增大了兩者之間的相互作用力,導(dǎo)致摩擦力增大；當(dāng)pH值較高時,DNA分子與針尖之間存在靜電斥力,在一定程度上平衡了部分外加載荷,導(dǎo)致摩擦力降低。此外,不同pH值下DNA構(gòu)象的變化也能影響摩擦力的大小。低pH值下DNA分子的吸附構(gòu)象主要為“平躺型”,針尖在剪切時會受到較大的阻力；高pH下DNA分子的吸附構(gòu)象主要為“鏈圈型”,并且部分分子鏈伸展至溶液中,針尖在剪切時受到的阻力較小。在低pH值溶液中,平躺的構(gòu)象有利于DNA分子的一端進(jìn)入并保持良好的姿態(tài)通過納米孔；同時,低pH值溶液中DNA分子與氮化硅納米孔材料的摩擦力較大,利于降低DNA分子的過孔速度。最后,不同pH值溶液中DNA穿越納米孔的實驗也同樣驗證了較低的溶液pH值能降低DNA分子的過孔速度,因此,較低的溶液pH值更利于DNA分子的納米孔測序。(4) 研究表明鏈長對DNA分子摩擦學(xué)行為的影響不大,納米孔測序?qū)NA分子的讀取長度不受摩擦限制。使用不同鏈長的DNA分子在云母表面制備出自組裝薄膜,采用氮化硅針尖研究了鏈長對DNA分子構(gòu)象及摩擦行為的影響規(guī)律。結(jié)果表明,DNA分子以鏈圈和平躺共存的構(gòu)象吸附于APS修飾的云母基底表面；隨著鏈長的增大,DNA分子更為伸展,利于DNA分子以良好的姿態(tài)進(jìn)入并通過納米孔。在較低的載荷范圍內(nèi),鏈長對DNA分子與氮化硅針尖之間的摩擦力影響不大,DNA分子的過孔速度受鏈長的影響較小。因此,從摩擦學(xué)的角度來看,納米孔測序的讀取長度不受限制。綜上所述,本論文系統(tǒng)研究了納米孔材料、溶液NaCl濃度、溶液pH值以及分子鏈長對DNA分子摩擦學(xué)行為的影響規(guī)律和作用機(jī)制,研究結(jié)果不僅豐富了生物納米摩擦學(xué)的理論基礎(chǔ),而且為基于納米孔的第三代DNA測序技術(shù)的優(yōu)化提供了理論依據(jù)。
[Abstract]:As a carrier of genetic information, DNA is the most important substance in the evolution of life. Rapid and accurate detection of DNA sequences can not only help people effectively prevent and treat genetic diseases such as diabetes, hemophilia and cancer, but also help to unravel the mystery of the origin of life. Since the 70s of last century, people The three generation of DNA sequencing technology has been developed successively. The first generation of DNA sequencing technology based on the Sanger method has completed the human genome project for 15 years and costs about $3 billion. The second generation DNA sequencing technology is characterized by high throughput, requiring only one week and less than 1 million dollars to complete the sequencing of the human genome. The third generation DNA sequencing technology in nanoscale has been sequenced by detecting the characteristic blocking current produced by the DNA base through the nanopore. With the advantage of super fast, high precision and low cost, the nanoscale sequencing technology is expected to complete the US National Institutes of Health (NIH) for only $1000 and complete the individual genome within 24 hours. In spite of this, there are still some key problems to be solved in nanoscale sequencing technology. For example, the speed of the DNA molecules is too fast to accurately detect the blocking current signal. The study shows that the increase of the friction force between the DNA and the pore wall of the nanopore can be reduced to a certain extent. The research focused on the surface self-assembly of DNA, the molecular morphology, the mechanical stability of the DNA molecular chain and the development of the specific functional devices based on the DNA molecules. The research on the tribological behavior of the DNA molecules is rarely reported. Therefore, in order to understand the mechanism of the friction between the DNA molecules and the nanomorp materials, it is urgent to systematically understand the mechanism of the friction between the DNA molecules and the nanomorp materials. The research on the tribological behavior of DNA can not only promote the development and application of the third generation DNA sequencing technology based on nanoscale, but also enrich and improve the theoretical basis of the biological nano tribology. This paper focuses on the study of the friction pairs of DNA molecules / nanoscale materials with the help of atomic force microscopy (AFM). The influence law and mechanism of nano pore material, solution NaCl concentration, solution pH value and molecular chain length on the tribological behavior of DNA molecules. First, the silicon dioxide tip with DNA molecules adsorbed on the surface was successfully prepared, and the tribological behavior between DNA and several typical nanoscale materials in the solution environment was studied, and then the nanopore was proposed. On this basis, DNA self assembled film was successfully prepared on the basis of atomic level smooth mica surface, and the effect of NaCl concentration of solution, pH value of solution and molecular chain length on the tribological behavior of DNA and the mechanism of action were studied by AFM silicon nitride needle point system. At the same time, Chen Yunfei teaching at Southeast University was used. In order to verify the results of the study on the optimization of the pH value of the solution, the main conclusions and innovation points of this paper are as follows: (1) the successful preparation of the silicon dioxide tip with DNA molecules on the surface was successfully prepared, and the different nanometers were revealed. The change law of tribological behavior between the pore material and the DNA molecule. Using the 3- ammonia propyl triethoxy silane coupling agent to modify the silica pinpoint, the DNA molecules are self assembled on the surface of the silicon dioxide, and the silica pinpoints with DNA molecules adsorbed on the surface have been successfully prepared. On this basis, the DNA molecules and 5 typical kinds of nanofilms are studied using this needle point system. The tribological behavior between the mesoporous materials (silicon nitride, quartz, APS- quartz, graphene, graphite). The results show that the friction between the 5 nanoscale materials and the DNA molecules under the same load is in turn: APS- quartz silicon nitride quartz graphene graphite. Among them, graphene and graphite have special microstructure, and they are with D The friction between NA is significantly less than the other 3 kinds of nanoscale materials. Through the contact angle test of the surface of different nanoscale materials, it is found that the hydrophobic surface in the underwater environment may increase the friction between the DNA and the material to a certain extent. Through the test of the surface force curve of APS- quartz, silicon nitride and quartz, the positive APS- stone is found. The electrostatic attraction between the DNA and the surface of the UK will help to increase the interaction between the tip and the substrate of the DNA-SiO2, and further lead to the increase of the friction force. The results of this study provide some suggestions for the selection of nanoscale materials, and provide a reference for the selection of the tip materials in the subsequent DNA friction study. (2) clarified The influence of the concentration of NaCl solution on the conformation and friction behavior of DNA molecules. The DNA self assembled film was successfully prepared on the mica surface. The influence of NaCl concentration on the conformation and friction of DNA was measured with the NaCl concentration of the silicon nitride. The results showed that as the concentration of positive Na ions in the solution increased, more and more DNA fractions were found. The negative charge of the phosphoric group outside the subframework is shielded, and the DNA molecules gradually change from the more extensional conformation to the "coil coiled" conformation. In addition, when the concentration of the solution NaCl is low, the friction force between the needle tip and the DNA molecule is larger when the concentration of the DNA molecule is high. The lower NaCl concentration can make the DNA molecule More extensional, it is beneficial for DNA molecules to enter and pass through nanoscale in good posture, and the larger friction force is beneficial to reducing the pore velocity of DNA molecules. Therefore, the lower NaCl concentration is more beneficial to the nanoscale sequencing of DNA molecules. (3) the mechanism of the pH value of the solution on the conformation and friction of DNA molecules is revealed. In the solution of different pH values. On the surface of the DNA film, the adhesion and friction behavior of the silicon nitride needle was tested. The influence mechanism of the solution pH value on the conformation and friction of the DNA molecule was revealed. The study shows that the change of the pH value of the solution will affect the interaction between the DNA molecular base and the interaction between the DNA molecule and the silicon nitride needle. The conformation of DNA molecules and the friction behavior of the silicon nitride needle are affected. When the pH is low, there is an electrostatic force between the DNA and the tip of the silicon nitride needle, which, to a certain extent, increases the interaction between the two and causes the friction force to increase. When the pH value is high, there is an electrostatic repulsion between the DNA molecule and the needle tip, and the equilibrium is balanced to a certain extent. The friction force is reduced by partial loading. In addition, the change of DNA conformation under different pH values can also affect the size of the friction force. The Adsorption Conformation of DNA molecules at low pH values is mainly "flat", and the tip of the needle will be subjected to greater resistance in shear; the adsorption structure of DNA molecules under high pH is mainly "chain ring", and some molecular chains are used. In the solution, the tip of the needle is less resistant to shear. In a low pH value solution, the flat conformation is beneficial to the entry of the DNA molecule and maintain a good posture through the nanopore; at the same time, the friction force of the DNA molecule in the low pH solution is larger than that of the silicon nitride nanomaterials, which is beneficial to the reduction of the pore velocity of the DNA molecule. Finally, the difference in the pore velocity of the DNA molecule is different. The experiment of DNA through the nanoport in pH value solution also verified that the lower pH value of the lower solution can reduce the pore velocity of the DNA molecule. Therefore, the lower pH value is beneficial to the nanoscale sequencing of the DNA molecules. (4) the study shows that the chain length has little effect on the tribological behavior of the DNA molecules, and the reading length of DNA molecules by nanomorp sequencing is not limited by the friction limit. The self assembled film was prepared on the mica surface using DNA molecules with different length of chain. The effect of chain length on the conformation and friction of DNA was studied by the tip of silicon nitride. The results showed that the conformation of DNA molecules coexisted peacefully on the surface of the APS modified mica surface. With the increase of the chain length, the DNA molecules were more extended. In a relatively low load range, the chain length has little effect on the friction between the DNA molecules and the silicon nitride pinpoint, and the pore velocity of the DNA molecule is less affected by the chain length. Therefore, from the tribological point of view, the reading length of the nanoscale sequencing is not limited. In summary, this is the case. The influence rule and mechanism of the nano pore material, the concentration of solution NaCl, the pH value of the solution and the length of the molecular chain on the tribological behavior of DNA molecules are systematically studied in this paper. The results not only enrich the theoretical basis of the biological nano tribology, but also provide a theoretical basis for the optimization of the third generation DNA sequencing technology based on the nanopore.
【學(xué)位授予單位】：西南交通大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：Q523

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9 李經(jīng)建;冀中華;蔡生民;;小溝結(jié)合方式中的DNA媒介電荷轉(zhuǎn)移[A];第十三次全國電化學(xué)會議論文摘要集（下集）[C];2005年

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