【作者】 刘航；

【导师】 刘全俊；

【作者基本信息】 东南大学 ， 生物医学工程， 2018， 博士

【摘要】 纳米孔传感器作为一种廉价、无需标记、高灵敏度且通量较高的单分子传感系统,近二十年来已经受到越来越多的关注。目前,使用最多的仍然是生物纳米孔,由于其较好的可重复性和稳定性,以及较高的检测精度,生物纳米孔被广泛地应用在各种有机无机分析物的检测和分析中,以及它们之间单分子水平的反应机制研究中。最常用做生物纳米孔的有α-溶血素(α-hemolysin)蛋白和耻垢分枝杆菌孔蛋白(Msp A),但它们的野生型蛋白用于纳米孔分析时往往会存在自发阻塞和过孔速率较快等缺陷,必须经过蛋白质工程的相应突变改造后才可以达到特异性识别目标分析物以及降低过孔速率提高检测精度等目的。本课题为了着眼于更高精度纳米孔系统构建的探索,首先摸索了集成孔内纳米电极的固态纳米孔的制备方法和工艺参数,然后选择了常用的α-溶血素构建了生物纳米孔单分子检测系统,并对其稳定性影响因素进行了研究。另外,利用α-溶血素纳米孔研究了部分双链结构(duplex)DNA的解链过程,实现了对单链DNA分子间单个碱基差异性的区分。然后,使用蛋白质工程相关技术对野生型Msp A蛋白进行相应改造,并将新型的突变Msp A蛋白(Mut-Msp A)应用于不同碱基单链DNA均聚物的检测和区分,实现了高分辨率生物纳米孔检测系统的构建。本论文的研究内容主要包括以下几个方面:1.集成孔内纳米电极纳米孔的制备本课题采用了一种跨尺度纳米电极的制备方法,从5μm宽的金(Au)线上制备了一个10 nm宽的纳米电极。制作过程主要为四部分:(1)通过低压化学气相沉积(LPCVD)沉积Au-Si3N4结构;(2)通过电子显微镜初步刻蚀电极;(3)沉积Si3N4膜封装刻蚀好的电极芯片;(4)通过氦离子显微镜(HIM)在三明治结构的纳米孔上刻蚀出纳米孔。与其它方法相比,该方法只用到了电子显微镜,更简单、普遍且有效。制作中以10 nm的纳米孔和10 nm电极间隙为例进行实践,证实了该方法可适用于大批量生产的纳米孔,且纳米孔的间隙可以随纳米孔的孔径而改变。本研究为后续更高精度的固态-生物复合纳米孔构建奠定了基础。2.生物纳米孔单分子检测系统的构建本课题通过考量不同的磷脂分子、流体装置、脂双层制备方法和孔蛋白等生物纳米孔系统构建过程中的关键影响影响因素,并通过一系列的电化学检测手段,对构建系统的稳定性进行了综合评估。最终确定DPh PC能在较宽的温度范围和氧化环境中依旧保持稳定,WARNER公司的流体池装置最便于实验操作,通过刷涂法制备磷脂双分子层效率最高且不需要其它仪器辅助。制备出的α-溶血素孔在1M KCl溶液20°C时单通道电导约为1 n S,而突变Msp A纳米孔的单通道电导约为2.8 n S,构建出的生物纳米孔系统不仅有较好的可重复性,也有较高的信噪比和分辨率,可达到对DNA进行单分子水平检测的需求。3.α-溶血素孔对DNA分子间单碱基差异的区分通过将一条设计好的17-mer的短链探针分别与两条只有单碱基差异的57-mer目标长链退火,形成了完全互补配对的和含有一对错配碱基的部分双链(duplex)结构DNA。分析它们各自在一系列偏置电压驱动下穿过α-溶血素纳米孔通道颈缩区时的解链过程,发现典型的易位事件分为了五种类型,提取真正发生解链行为的目标事件中的对应解链阶段的电流阻塞幅值和持续时间等相关信息,经统计分析发现,杂交产物的易位时间普遍在百毫秒级以上,比单纯的探针或模板链百微秒级的易位时间明显增长。在140 m V电压下,两种只有一个碱基对差异的杂交产物的解链时间有着显著差异,完美互补的为430±34 ms,而有一对碱基错配的为15±1 ms,相差近30倍。另外,可以观察到双链结构的解链时间与电压成线性关系,随着施加的偏置电压的逐渐升高,解链时间反而逐渐减少。由此可见,使用α-溶血素纳米孔可以在单分子水平上区分DNA双链间的单碱基差异,这种简单的生物传感策略可广泛地应用于SNP位点的检测,疾病分类、医疗诊断中。4.新型Msp A突变体蛋白表达和纯化及其对DNA均聚物的检测研究Msp A蛋白突变的位点及相应效果,确定使用蛋白质工程相关技术将野生型Msp A蛋白颈缩区带负电的氨基酸残基突变为不带电的氨基酸残基,和邻近颈缩区区域的部分不带电的氨基酸残基突变为带正电的氨基酸残基的突变方案,确定并合成目的基因序列,在大肠杆菌BL21(DE3)中原核表达,并将目标蛋白质分离纯化及分析鉴定。最终表达的突变型Msp A蛋白的浓度达到0.86 mg/ml,且纯度达到了80%以上。使用该突变体蛋白制备的纳米孔检测体系,基线平稳,基础噪声低于5 p A,且可以反复组装,相同电压下的开孔电流水平偏差在±3 p A左右将得到的新型Msp A突变体蛋白用于单链DNA均聚物的检测,结果显示易位速度显著降低。且在同等实验条件下,相同DNA均聚物过突变的速率比通过野生型Msp A纳米孔的速率降低了近10倍。综上所述,本课题通过研究固态纳米孔内纳米电极的制备,确定了批量制造集成孔内纳米电极的固态纳米孔的制备工艺,后续通过构建α-溶血素和Msp A突变体生物纳米孔,系统地研究了DNA分子的易位和解链行为机制,为下一步将集成孔内纳米电极的固态纳米孔和突变Msp A纳米孔结合构建固态-生物复合纳米孔提供了理论参考。更多还原

【Abstract】 As a low-cost,high sensitivity and high throughput single molecule sensing system,the nanopore sensor has received more and more attention in the past twenty years.At present,the commonly used nanopores are still biological nanopores.Due to its good repeatability and stability,and higher detection accuracy,biological nanopores was widely applied on various organic and inorganic molecular detection and analysis,as well as study of their reaction mechanism at single molecular level.Most commonly used biological nanopore are alpha hemolysin（α-HL）protein and mycobacterium smegmatis porin（MspA）.However,the wild type proteins can’t directly be used for nanopore analysis because their spontaneous blockage and faster translocation rate.Mutant proteins can be formed by using some protein engineering technology,which can finally achieve specificity for the identification of the target analytes,lower the translocation speed,and to improve the detection accuracy.From this point,we firstly constructed the single molecule detection system based on biological nanopores,and studied the factors which could affect its stability.In addition,the alpha hemolysin nanopore was utilized to study the unzipping kinetic of duplex DNA,and finally achieved discrimination of single nucleotide differences between duplex DNA structures.Protein engineering technology was used to form a novel MspA protein（Mut-MspA）mutation based on wild type MspA protein,which was then applied to detect and discriminate four types of DNA homopolymer,and finally used on construction of higher resolution nanopore detection system.The contents of this thesis including:1.Construction of biological nanopore system for single molecule detectionIn this work,a cross-scale method of fabricating nano-electrodes was proposed,and a 10 nm-width nano-electrode was got from 5μm-width aurum（Au）line.Fabrication procedures involves four steps:（i）Depositing of Au-Si₃N₄ structure by Low Pressure Chemical Vapor Deposition（LPCVD）and Electron Beam Evaporation,（ii）Etching a suitable width of nano-electrode by microscopes,（iii）Depositing 100nm thickness Si₃N₄ on the Au-Si₃N₄ by LPCVD,（iv）Drilling a suitable diameter hole on the sandwich structure by Helium-ion microscopes（HIM）.Compared with the traditional methods,only electron microscopes were involved in this process,it was a simple and effective method to fabricate electrode-embedded nanopore.The feasibility of the method has been demonstrated by practice and a 10 nm nanopore with 10 nm-width nano-electrodes was fabricated by this method.This method will be a new way to fabricate transverse electrode-embedded nanopore in fabricating smaller than 2 nm gap of nano-electrodes in the future.2.Construction of biological nanopore system for single molecule detectionWe had studied the key influencing factors within the nanopore construction processes by comparing of different types of phospholipid molecule,fluid device,lipid bilayer preparation methods and porins,Through a series of electrochemical detection means,a comprehensive assessment was made on the stability of nanopore system,which ultimately helped to determine a efficient and stable preparation method.The constructed nanopore platform had not only good repeatability,but also high signal-to-noise ratio and resolution,and could achieve single molecule detection level for DNA.3.Single Nucleotide Discrimination byα-Hemolysin NanoporeA well designed 17-mer short probe was respectively annealed with two 57-mer target DNA and forming a complete complementary duplex DNA and a duplex DNA contained a pair of mismatched bases.By analysing of their unzipping processes through the constriction zone ofα-HL nanopore channel under a bias voltage,we found the translocation time of the hybridization products was obvious increased than the probe or the template strand.The duplexes only with single base-pair difference have significant differences on unzipping duration.The unzipping duration time of perfect complementary duplex was 430±34 ms,but the duration of single base pair mismatch duplex was only 15±1 ms,which nearly increased by 30 times.Therefore,α-HL nanopore can be used to discriminate single base difference within ssDNA at single molecule level.This simple biosensing strategy can be widely used on detection of SNP sites,disease classification and medical diagnosis.4.Expression and purification of a novel MspA mutant and detection of DNA polymers.By using protein engineering technology,we had replaced three negative charged amino acid residues within the construction zone with uncharged amino acid residues,and three uncharged amino acid residues in the adjacent area of construction zone to positively charged amino acid residues based on the wild-type MspA protein in Escherichia coli.Finally,the new MspA mutant protein was used to detect the DNA polymer of the same chain length,and the results showed that the new type of mutant MspA porin was able to distinguish four different kinds of DNA polymer.However,under the same experimental conditions,the translocation rate of the same DNA homopolymer through the Mut-MspA nanopore decreased nearly 10 times compared with the alpha hemolysin nanopore.In summary,we studied the technical parameter for preparation of nano-electrodes in solid-state nanopores,and subsequently constructsα-hemolysin and MspA mutant biological nanopore systems.The mechanism of translocation and melting behavior of DNA molecules was systematically studied,which provided a theoretical reference for the next step to integrate solid-state nanopores and mutated MspA nanopore to construct solid-biocomposite nanopores.更多还原