Aerolysin纳米孔道对寡聚核苷酸的高灵敏单分子检测

自纳米孔道单分子电化学技术提出以来,为了构建性能良好的纳米孔道,研究人员一直在寻找不同的孔道材料. 本研究探索了Aerolysin生物纳米孔道在寡聚核苷酸检测方面的可能性. 实验结果表明,与常用的α-溶血素纳米孔道相比,Aerolysin纳米孔道在寡聚核苷酸检测方面表现出更强的空间和时间分辨能力. 三个碱基长度的寡聚核苷酸可对Aerolysin纳米孔道造成约为40%的电流阻断. 阻断时间表现出电压相关性,随电压的升高而减小. 与其他生物纳米孔道相比,Aerolysin纳米孔道无需任何基因突变、化学修饰即可实现对单个寡聚核苷酸的超灵敏分析. 未来,Aerolysin纳米孔道将有可能应用于DNA损伤检测、microRNA分析以及其他基于纳米孔道的单分子分析检测.     

电化学限域SiN_X纳米孔道检测核酸分子的研究

固体纳米孔道因其机械强度高、尺寸可控、易于表面修饰及集成化设计等优点被广泛应用于DNA、RNA和蛋白质等生物分子的检测研究.为了检测单个单链核酸分子,本研究采用电化学刻蚀法可控制备了单个SiN_X固体纳米孔道,通过SiN_X固体纳米孔道限域空间效应增强了纳米孔道与短链核酸分子之间的弱相互作用,从而实现了核酸分子的单分子水平检测.通过研究不同孔径(3.1和8.5 nm)纳米孔道与核酸分子间的弱相互作用差异,有效区分了核酸分子在限域空间内产生的过孔和碰撞两种个体行为,加深了对固体纳米孔道限域空间内核酸分子电化学行为的理解.     

单分子电泳:纳米孔道电化学的新认识

该文旨在从电泳分离技术的角度认识纳米孔道电化学单分子分析技术,这种技术可以作为"单分子电泳"来理解和研究。纳米孔道电化学单分子分析技术与电泳的本质都是采用外加电场使待测分子产生电迁移。待测分子性质不同,且与介质材料孔道外露基团相互作用不同,使得分子移动速度具有差异,据此实现分离识别。气单胞菌溶素（Aerolysin）纳米孔道,由于其孔径与待测分子尺寸相匹配,其孔道内壁可以看作是由氨基酸组成的具有调控单个分子电迁移能力的特异性孔道界面。每一个氨基酸残基都相当于一个探测单元,在电场力的作用下,待测分子逐一进入孔道时与每一个探测单元相互作用方式、程度与时长不同,从而形成了单个待测分子特征的迁移速度和迁移运动轨迹。在纳米孔道实验中,每秒可以有上千个待测分子穿过孔道,产生特征阻断电流信号。通过对这些信号的阻断电流、阻断时间、阻断频率、信号特征等进行统计分析,可以从"单分子电泳"水平对单个待测物实现高通量的分辨和识别。该文以Aerolysin纳米孔道分辨仅有一个核苷酸差异的寡聚核苷酸（5'-CAA-3'、5'-CAAA-3'、5'-CAAAA-3'）为例,详细阐述了纳米孔道"单分子电泳"的单核苷酸分辨能力,展现了电化学限域空间在电泳单分子水平分离技术上的应用。     

纳米孔道单分子电化学测量仪器的稳定性研究

纳米孔道分析技术是一种基于电化学空间限域效应的单分子检测技术。测量纳米孔道产生的单分子皮安级微弱电流信号对电化学测量仪器的电流分辨、时间分辨和抗噪音能力提出了挑战。Cube纳米孔道电化学测量仪器通过设计频率补偿电路、前置电流放大器测量系统和基于现场可编程逻辑门阵列(FPGA)的高速数字处理电路,实现了便携式超灵敏电化学测量仪器对微弱电流信号的高时间分辨、高电流分辨,以及低噪音的放大、采集和快速处理。稳定性是仪器能够应用于实际单分子测量分析的重要衡量指标之一。该文通过高阻值电阻对该仪器进行稳定性测试,在截止滤波频率为5、10、100 kHz条件下,Cube纳米孔道仪器获取的电流基线的噪音均方根(RMS)值分别比商品化仪器减小了80.0%、87.5%、48.2%,证明Cube纳米孔道仪器抑制噪音能力更强,电流分辨能力更好,仪器测量稳定性更佳。进一步通过统计比较施加电压值的实际值和标准偏差,结果显示该仪器施加电压误差小,其仪器施加电压标准偏差仅为施加电压变化量(10 mV)的0.14%。同时,通过Aerolysin纳米孔道检测Poly(dA)4的实验,对比Cube仪器和商品化仪器在不同施加电压下获取的单分子信号残余电流程度,得到两者误差均小于0.01,结果具有可重复性。因此,Cube纳米孔道仪器具有稳定性好、灵敏度高、便携性强的特点,可应用于纳米孔道单分子分析。     

用于纳米孔道分析的四通道电化学传感器设计与仿真

纳米孔道技术是一种基于空间限域的超灵敏的单分子分析技术.通过研究单个分子限域于纳米孔道中所产生的离子电流的变化,可在单分子尺度上获取其结构、尺寸、电性及与孔道间弱相互作用的信息.目前主要应用的纳米孔道测量仪器单次实验仅能测量单个纳米孔道,其检测通量较低.本文基于实验室前期自主设计研制的单通道纳米孔道测量仪器Cube-D2上,比较研究了两种互阻放大器的测量特性,从而选择了合适的测量电路设计了四通道电化学传感器放大电路.进一步通过仿真验证了四通道电化学传感器设计方案的可行性,为阵列化高通量纳米孔道单分子电化学测量仪器的设计提供了理论基础.     

Aerolysin纳米孔核酸检测灵敏区域的协同相互作用探索研究

纳米孔单分子检测技术以其简便、快速、高通量及无需标记等特点, 应用于DNA及蛋白质测序, 更有望实现单分子动态构象变化的研究. Aerolysin(气单胞菌溶素)纳米孔道由于其特有的较长的β-桶限域区(β-barrel)及孔内壁丰富的带电荷氨基酸残基, 在单个寡聚核苷酸分子分析中展现出极高的灵敏性. 本设计利用dA₁₄-4-X, dA₁₄-11-X, dA₁₄-4-X-11-X (X=C, T, G)等单个寡聚核苷酸探针分子, 研究了Aerolysin的两个灵敏区域R1和R2, 探索了R1灵敏区域对单个碱基弱相互作用的差异, 实现区分单个碱基差异. 进一步实验证明, R1灵敏区域对单个碱基类型差异的灵敏区分不受R2灵敏区域被碱基A、C、T占位所影响. 然而, 当R2区域被碱基G占位时, 会使R1区域丧失对整个孔道电流的主导性. 本研究有助于理解Aerolysin对单个寡聚核苷酸分子的超灵敏测量机制.     

基于Aerolysin纳米孔道对单个c-di-AMP分子的检测研究

环二腺苷酸(c-di-AMP)是原核细胞中普遍存在的第二信使, 不仅能够有效调控细胞生长、离子转运、细胞壁代谢平衡等多种生理过程, 还能引发I型干扰素应答, 激发机体天然免疫反应. 本实验使用单个气单胞菌溶素(Aerolysin)纳米孔道蛋白构建的单分子界面, 对c-di-AMP进行单分子测量研究. 为提高Aerolysin纳米孔对带负电小分子化合物的测量灵敏度, 本实验利用LiCl为支持电解质, 有效屏蔽Aerolysin孔口表面负电荷, 减小c-di-AMP与Aerolysin纳米孔之间的静电排斥, 从而显著增强了Aerolysin纳米孔道对单个c-di-AMP分子的检测能力. 实验结果显示, 在90 mV电压下, 每分钟在LiCl中获得的有效穿孔事件的数量最高可达同条件KCl支持电解质的30倍, 且有效穿孔事件占总体事件的比例在不同电压下提升了7~11倍. 进一步表明, 使用LiCl支持电解质, 可有效增强Aerolysin孔道对带负电小分子化合物的测量灵敏度. 因此, 本研究实现了Aerolysin纳米孔道对单个环二核苷酸的高灵敏免标记检测, 有望为单分子水平上阐明新型免疫干扰机制提供新的分析方法.     

纳米孔道单分子电化学测量的低噪音控温系统研究

纳米孔道检测技术是一种利用单个分子测量界面实现在单分子水平上测量DNA、RNA、蛋白、多肽等生物分子的高灵敏的单分子检测技术. 由于单个分子与纳米孔道的相互作用受热力学控制,亟需精准控制纳米孔道单分子分析的实验温度. 因此,本文研制了一种低噪音控温系统用于具有皮安级电流分辨的纳米孔道单分子实验,以实现精确调控测量时的环境温度. 该系统利用半导体制冷片的热电效应对检测池环境加热/制冷,通过对高精度热敏电阻进行电磁屏蔽以实现在温度反馈的同时避免噪音的引入. 利用比例-积分-微分算法进行控制,达到高精度快速控温的要求. 该系统控温精度为±1 °C,无额外噪音引入至超灵敏纳米孔道单分子测量,获得了25 °C到5 °C下Poly(dA)5与单个气单胞菌溶素（Aerolysin）分子界面间作用产生信号的差异,应用于研究单分子与纳米孔道相互作用的热力学行为.     

生物纳米孔道技术在非基因测序方面的研究与应用

纳米孔道分析技术是一种低成本、快速、无需标记的单分子检测技术,仅有20多年的发展历史,在DNA单分子测序领域展示出较好的应用前景,现已有商业化的产品面世且趋于成熟.越来越多的研究表明,纳米孔可作为一个通用的单分子传感器.本文综述了生物纳米孔道分析技术对蛋白质、多肽和核酸等单个分子与孔道间相互作用、动力学和热力学过程的实时监测以及多种生物大分子和金属离子的定量检测等方面的研究进展.在纳米孔技术中,电化学检测系统也十分重要,本文还特别介绍了高带宽及超低电流分辨仪器和相关软件的相关进展.     

基于石英纳米孔道的单颗粒尺寸分布分析

发展了一种基于石英纳米孔道的单颗粒电化学动态分析方法, 用于单个CdSe/ZnS量子点纳米颗粒的尺寸分布分析. 其机制是向石英纳米孔道两端施加电压, 表面带有正电荷的单个CdSe/ZnS量子点纳米颗粒在电场力驱动下由管内向管外运动, 当量子点纳米颗粒穿过纳米孔道尖端狭小的限域空间时, 其表面正电荷使石英纳米孔道内电荷密度增加, 孔道内的电化学限域效应进一步将电荷密度增加的信息放大并转变为可读的离子流增强信号. 通过对动态离子流信号解析可实时获取具有2种不同尺寸的量子点纳米颗粒所导致的2类过孔事件信息, 从而对在限域空间内运动的纳米颗粒进行尺寸分布分析.     

Monitoring disulfide bonds making and breaking in biological nanopore at single molecule level

Monitoring subtle changes in ionic current flow through a nanopore could be applied to observe single molecule reaction. Here,we introduced cysteine to substitute for lysine at position 238 constructing a mutant aerolysin K238 C. It could be regarded as a nanoreactor to efficiently visualize chemical bonds making and breaking. The compound 5,5′-dithiobis-(2-nitrobenzoic acid)(DTNB) was selected as a reactant coming into collisions on the interface of the pore to occur a reversible reaction. Our results showed that the mutant aerolysin could respond to three molecules of DTNB simultaneously and reflect corresponding levels with distinguishable current signals. Therefore, this method constitutes a simple, generic tool for monitoring single molecule reaction, which evokes a guidance for the mutant aerolysin towards the application of tracking other more reactions at single molecule level.     

Real-time Event Recognition and Analysis System for Nanopore Study

To achieve fast and accurate analysis of weak current signal of nanopore-based single molecule detection, an online data process based on adaptive threshold algorithm with data buffering technique and finite impulse response filtering was designed. A software system based on the data process was developed for online recognition and analysis of nanopore events during nanopore experiment. To testify the performance of the algorithm and software system, ideal signals with different noise level (20–100 pA) were generated at bandwidth ranging from 3 kHz to 100 kHz. The result showed that this software system was stable at different bandwidths and sampling rates and could be used in analyzing the signals at high noise. The proposed software system was further applied to aerolysin nanopore experiment for detection of poly(dA)₄ molecules. The results showed that the data process system could be applied in real nanopore recording experiment with high accuracy and speed.     

Dynamics of unfolded protein transport through an aerolysin pore

Protein export is an essential mechanism in living cells and exported proteins are usually translocated through a protein-conducting channel in an unfolded state. Here we analyze, by electrical detection, the entry and transport of unfolded proteins, at the single molecule level, with different stabilities through an aerolysin pore, as a function of the applied voltage and protein concentration. The frequency of ionic current blockades varies exponentially as a function of the applied voltage and linearly as a function of protein concentration. The transport time of unfolded proteins decreases exponentially when the applied voltage increases. We prove that the ionic current blockade duration of a double-sized protein is longer than that assessed for a single protein supporting the transport phenomenon. Our results fit with the theory of confined polyelectrolyte and with some experimental results about DNA or synthetic polyelectrolyte translocation through protein channels as a function of applied voltage. We discuss the potential of the aerolysin nanopore as a tool for protein folding studies as it has already been done for α-hemolysin.     

Aerolysin单分子界面的构建及高选择性单分子检测

提出了一种基于Aerolysin膜蛋白质分子构建单分子界面的方法, 运用蛋白质工程技术对单分子界面进行定点修饰, 所建立方法灵活、 可控且重复性好. 采用Poly(dA)₄为探针分子对修饰后的单分子界面进行了表征, 结果表明, 在孔口处的Arginine修饰影响了寡聚核苷酸的选择性. 为进一步理解Aerolysin单分子界面及合理设计功能性单分子界面提供了参考.     

Discrimination of single‐stranded DNA homopolymers by sieving out G‐quadruplex using tiny solid‐state nanopores

Nanopore sensor has been developed as a promising technology for DNA sequencing at the single‐base resolution. However, the discrimination of homopolymers composed of guanines from other nucleotides has not been clearly revealed due to the easily formed G‐quadruplex in aqueous buffers. In this work, we report that a tiny silicon nitride nanopore was used to sieve out G tetramers to make sure only homopolymers composed of guanines could translocate through the nanopore, then the 20‐nucleotide long ssDNA homopolymers could be identified and differentiated. It is found that the size of the nucleotide plays a major role in affecting the current blockade as well as the dwell time while DNA is translocating through the nanopore. By the comparison of translocation behavior of ssDNA homopolymers composed of nucleotides with different volumes, it is found that smaller nucleotides can lead to higher translocation speed and lower current blockage, which is also found and validated for the 105‐nucleotide long homopolymers. The studies performed in this work will improve our understanding of nanopore‐based DNA sequencing at single‐base level.     

Protein detection by nanopores equipped with aptamers

Protein nanopores have been used as stochastic sensors for the detection of analytes that range from small molecules to proteins. In this approach, individual analyte molecules modulate the ionic current flowing through a single nanopore. Here, a new type of stochastic sensor based on an αHL pore modified with an aptamer is described. The aptamer is bound to the pore by hybridization to an oligonucleotide that is attached covalently through a disulfide bond to a single cysteine residue near a mouth of the pore. We show that the binding of thrombin to a 15-mer DNA aptamer, which forms a cation-stabilized quadruplex, alters the ionic current through the pore. The approach allows the quantification of nanomolar concentrations of thrombin, and provides association and dissociation rate constants and equilibrium dissociation constants for thrombin·aptamer interactions. Aptamer-based nanopores have the potential to be integrated into arrays for the parallel detection of multiple analytes.     

Detection of structured single‐strand DNA via solid‐state nanopore

Nanopore is a single‐molecule analysis method which also employed electrophoresis has achieved promising single‐molecule detections. In this study, we designed two kinds of confined spaces by fabricating solid‐state nanopores with desirable diameters to study the structured single‐strand DNA of C‐rich quadruplex. For the nanopore whose diameter is larger than the quadruplex size, the DNA molecule could directly translocate through the nanopore with extremely high speed. For the nanopore whose diameter is smaller than the quadruplex size, DNA molecule which is captured by nanopore could return to the solution without translocation or unzip the quadruplex structure into single‐strand and then pass the nanopore. This study certifies that choosing a suitable sensing interface is the vital importance of observing detailed single‐molecule information. The solid‐state nanopores hold the great potential to study the structural dynamics of quadruplex DNA molecule.     

Electrokinetic particle translocation through a nanopore

Nanoparticle electrophoretic translocation through a single nanopore induces a detectable change in the ionic current, which enables the nanopore-based sensing for various bio-analytical applications. In this study, a transient continuum-based model is developed for the first time to investigate the electrokinetic particle translocation through a nanopore by solving the Nernst-Planck equations for the ionic concentrations, the Poisson equation for the electric potential and the Navier-Stokes equations for the flow field using an arbitrary Lagrangian-Eulerian (ALE) method. When the applied electric field is relatively low, a current blockade is expected. In addition, the particle could be trapped at the entrance of the nanopore when the electrical double layer (EDL) adjacent to the charged particle is relatively thick. When the electric field imposed is relatively high, the particle can always pass through the nanopore by electrophoresis. However, a current enhancement is predicted if the EDL of the particle is relatively thick. The obtained numerical results qualitatively agree with the existing experimental results. It is also found that the initial orientation of the particle could significantly affect the particle translocation and the ionic current through a nanopore. Furthermore, a relatively high electric field tends to align the particle with its longest axis parallel to the local electric field. However, the particle's initial lateral offset from the centerline of the nanopore acts as a minor effect.