DNA sequencing technology based on nanopore sensors by theoretical calculations and simulations

DNA sequencing based on nanopore sensors is a promising tool for third-generation sequencing technol- ogy because of its special properties, such as revolutionized speed and low cost. With about two decades of nanopore technology development, the pioneering work has dem- onstrated the ability of nanopores to perform single-mole- cule detection and DNA sequencing. However, the microscopic mechanisms of DNA transport dynamics through nanopores remain largely unknown. Currently, DNA microscopic transport in a nanopore is difficult to characterize and several unexpected experimental obser- vations are equivocal. This limitation can be resolved using theoretical calculations and simulations. These computa- tional methods can monitor the entire dynamic process that DNA undergoes in solution at a single-atom resolution that can accurately unveil the mystery of DNA transport dynamics and predict certain unexpected phenomena. This paper mainly reports the recent applications of computa- tional and simulation methods applied to the study of DNA transport through both biological and synthetic nanopores. We hope the theoretical calculations and simulations of DNA transport through nanopores can benefit the design of DNA sequencing devices.     

Gold nanorod translocation through a solid-state nanopore

Recently, nanopores have been used in an essential technique for detecting single molecule with high sensitivity. The initial application of nanopores to DNA and RNA sequencing has been expanded to sensing pro- teins and nanoparticles, including Bovine serum albumin, silica nanoparticles, polystyrene beads, and others. In our study, for the first time, a positively charged gold nanorod was investigated using a solid-state nanopore device. Various gold nanorods passed through the nanopore with different current blockages and duration times, providing a measurement of the nanorod diameter, length, and charge. Our findings indicate that nanopore sensing might be a new method for characterizing the size, shape, and charge of nanoparticles.     

Detection and analysis of DNA recapture through a solid-state nanopore

Motion control of a single molecule through a solid-state nanopore offers a new perspective on detecting and analyzing single biomolecules. Repeat recapture of a single DNA molecule reveals the dynamics in DNA translocation through a nanopore and may significantly increase the signal-to-noise ratio for DNA base distin- guishing. However, the transient current at the moment of voltage reversal prevents the observation of instantly recaptured molecules and invalidates the continuous DNA ping-pong control. We performed and analyzed the DNA translocation and recapture experiment in a silicon nitride solid-state nanopore. Numerical calculation of molecular motion clearly shows the recapture dynamics with different delay times. The prohibited time when the data acquisition system is saturated by the transient current is derived by equivalent circuit analysis and finite element simulation. The COMSOL simulation reveals that the membrane capacitance plays an important role in determining the electric field distribution during the charging process. As a result of the transient charging process, a non-constant driving force pulls the DNA back to nanopores faster than theoretically predicted. The observed long time constant in the transient current trace is explained by the dielectric absorption of the membrane capacitor.     

Nanopore-based sensing devices and applications to genome sequencing: a brief history and the missing pieces

A nanopore on an impermeable membrane, which separates two chambers containing electrolytic solu- tion, can be used as a nanometre-sized Coulter counter for single-molecule biological sensing. With an applied poten- tial, charged molecules are electrically dragged through the pore, and the analytical information is sequentially read out from the current blockades. Nucleic acid, which is an elec- trically charged polymer, is an ideal analyte for nanopore analysis and nanopore sequencing. With the advantages of high-speed, label-free and single-molecule resolution, a nanopore sequencer is considered to be the most promising candidate for the third-generation DNA sequencing. In this review, a brief history of nanopore sequencing to date is summarized and discussed along with future prospects. Although successfully demonstrated for known viral gen- ome sequences, the nanopore sequencing technique still requires missing pieces like improved accuracy, automation and throughput for clinical diagnosis-level applications.     

Ion-beam sculpting at nanometre length scales.

Manipulating matter at the nanometre scale is important for many electronic, chemical and biological advances, but present solid-state fabrication methods do not reproducibly achieve dimensional control at the nanometre scale. Here we report a means of fashioning matter at these dimensions that uses low-energy ion beams and reveals surprising atomic transport phenomena that occur in a variety of materials and geometries. The method is implemented in a feedback-controlled sputtering system that provides fine control over ion beam exposure and sample temperature. We call the method "ion-beam sculpting", and apply it to the problem of fabricating a molecular-scale hole, or nanopore, in a thin insulating solid-state membrane. Such pores can serve to localize molecular-scale electrical junctions and switches and function as masks to create other small-scale structures. Nanopores also function as membrane channels in all living systems, where they serve as extremely sensitive electro-mechanical devices that regulate electric potential, ionic flow, and molecular transport across cellular membranes. We show that ion-beam sculpting can be used to fashion an analogous solid-state device: a robust electronic detector consisting of a single nanopore in a Si3N4 membrane, capable of registering single DNA molecules in aqueous solution.     

An integrated current measurement system for nanopore analysis

Nanopore-based techniques have attracted increasing attention as a unique tool for single-molecule analysis. To accurately detect individual motions of each single molecule, nanopore techniques are used to develop an ultrasensitive current measurement system. This work proposes an integrated current measurement system con- taining an amplifier system and a current signal acquisition system with a high current resolution and a high temporal resolution for nanopore analysis. The exploration and achievements in instrument and signal processing endow nanopore techniques with reliability, affordability, and portability, which make a great leap toward its real applications.     

An integrated semiconductor device enabling non-optical genome sequencing

The seminal importance of DNA sequencing to the life sciences, biotechnology and medicine has driven the search for more scalable and lower-cost solutions. Here we describe a DNA sequencing technology in which scalable, low-cost semiconductor manufacturing techniques are used to make an integrated circuit able to directly perform non-optical DNA sequencing of genomes. Sequence data are obtained by directly sensing the ions produced by template-directed DNA polymerase synthesis using all-natural nucleotides on this massively parallel semiconductor-sensing device or ion chip. The ion chip contains ion-sensitive, field-effect transistor-based sensors in perfect register with 1.2 million wells, which provide confinement and allow parallel, simultaneous detection of independent sequencing reactions. Use of the most widely used technology for constructing integrated circuits, the complementary metal-oxide semiconductor (CMOS) process, allows for low-cost, large-scale production and scaling of the device to higher densities and larger array sizes. We show the performance of the system by sequencing three bacterial genomes, its robustness and scalability by producing ion chips with up to 10 times as many sensors and sequencing a human genome.     

An atomic force microscopy study of coal nanopore structure

Coal nanopore structure is an important factor in understanding the storage and migration of absorbed gas in coal. A new method for studying coal nanopore structures is proposed. This idea is based on the nano-level resolution of atomic force microscopy, which can be employed to observe the structural features of coal nanopores clearly, conduct quantitative three-dimensional measurements and obtain structural parameters. Analysis results show that coal nanopores are mainly metamorphic pores and intermolecul...     

Investigation of self-assembled protein dimers through an artificial ion channel for DNA sensing

Artificial nanopores have become promising tools for sensing DNA. Here, we report a new technique for sensing DNA through a conical-shaped nanopore embed- ded in track-etched polyethylene terephthalate （PET） membrane. Two different streptavidin-conjugated mono- valent DNA probes were prepared that can bind to two distinct segments （at either end） of the target DNA. The size of target DNA-linked to the two streptavidin-conju- gated monovalent DNA probes is double that of the indi- vidual probes. By precisely controlling the tip diameter of the conical nanopore embedded in the PET polymer, events due to the translocation of the streptavidin-conjugated monovalent DNA probes through the nanopore can be fil- tered and purposely undetected, whereas the current pulses due to the translocation of the target DNA-induced self- assembled complexes can be detected. The two streptavi- din-conjugated DNA probes cannot be linked by multi- mismatched DNA. Therefore, multi-mismatched （non- specific） DNA will not induce any current pulse signatures. The current pulse signatures for the self-assembled com- plex can be used to confirm the presence of the target DNA. The size-dependent detection of self-assembled complexes on the molecular level shows strong promise for the detection of biomolecules without interference from the probes.     

基因芯片的应用

基因芯片技术是建立在杂交序列基本理论上的分子生物学技术，具有高度平行性、多样性、微型化和自动化的特点．目前，该技术已用于DNA测序、基因表达分析、新基因的发现、基因单核苷酸多态性(SNPs)研究、基因诊断、药物筛选等领域，而且其应用范围还在不断扩展．本丈概述了基因芯片的原理及应用。     

Development of novel and sensitive sensors based on microcantilever of atomic force microscope

Recently, the development of sensors based on microfabricated cantilevers of atomic force microscope (AFM) has attracted considerable attention from the designers of novel physical, chemical, and biological sensors. Many kinds of sensors have been developed taking the advantages of its high-resolution imaging, force measurement and force sensitivity, such as immunosensor and DNA biosensor and the sensors for detection of intermolecular interaction. This paper reviews the progress made in this field and discusses the signal transfer principles by which the design of the sensors is achieved.     

基于微加速度传感器的高速动态车辆超载检测

针对动态车辆超载检测,基于微加速度传感器建立了一种新型的高速动态超载检测方法.桥梁采用简支梁模型,车/桥相互作用模型化为两个具有固定间距点力匀速通过桥面,系统分析了两点力情况的桥梁振动,建立了两点力情况下桥梁振动与力的函数方程,从而奠定了基于微加速度传感器的实际车辆超载检测的理论基础.同时利用先进微传感器搭建了高速超载车检测试验平台,进行了传感器安装位置选择的研究.理论仿真和试验结果证明,该方法可以较准确地进行高速动态超载车辆的检测.     

从噬菌体展示七肽肽库筛选α-淀粉酶特异性配体

为了建立蛋白质亲和配基的高效筛选方法，以淀粉酶为靶分子，利用交替洗脱法从七肽噬茵体展示库中筛选具有高亲和力的噬茵体配体．结果表明，交替洗脱法筛选出的特异性配体的回收率和ELISA信号值均优于酸洗脱法；采用交替洗脱法能够更加有效和迅速地筛选到淀粉酶的高亲和力配体．分析筛选得到的不同噬茵体克隆DNA插入片断的氨基酸序列，发现了可能与配体高亲和性有关的氨基酸残基．     

Single-molecule analysis of DNA uncoiling by a type II topoisomerase

Type II DNA topoisomerases are ubiquitous ATP-dependent enzymes capable of transporting a DNA through a transient double-strand break in a second DNA segment. This enables them to untangle DNA and relax the interwound supercoils (plectonemes) that arise in twisted DNA. In vivo, they are responsible for untangling replicated chromosomes and their absence at mitosis or meiosis ultimately causes cell death. Here we describe a micromanipulation experiment in which we follow in real time a single Drosophila melanogaster topoisomerase II acting on a linear DNA molecule which is mechanically stretched and supercoiled. By monitoring the DNA's extension in the presence of ATP, we directly observe the relaxation of two supercoils during a single catalytic turnover. By controlling the force pulling on the molecule, we determine the variation of the reaction rate with the applied stress. Finally, in the absence of ATP, we observe the damping of a DNA crossover by a single topoisomerase on at least two different timescales (configurations). These results show that single molecule experiments are a powerful new tool for the study of topoisomerases.     

基于多传感器融合的抓取控制研究

针对现有抓取系统中双目视觉定位精度较差,易受环境影响,使得抓取的成功率较低,鲁棒性不强的现象,本文提出采用视觉、红外测距传感器、触觉传感器和编码器等多传感器数据融合的方法,设计并实现了一种可靠的、鲁棒性强的、能自动调整抓取力的抓取系统.通过双目视觉辅以单目相机和红外测距传感器来精确定位,改善抓空情况;通过集成触觉传感器和编码器,对抓取过程中的力-位进行实时监测,减少目标物体破碎和滑落的现象,并通过实验证明了相对于单传感器,多传感器数据融合能大大改善抓取的成功率,提高系统的性能.     

Study of the interaction of DNA and histones by spin-stretching and droplet evaporation

Molecular combing is a powerful and simple method for aligning DNA molecules onto a surface. Using this technique combined with fluorescence microscopy, DNA-histone complexes are stretched on a hydrophobic polymethyl methacrylate (PMMA) surface and observed directly. We have developed a new method to stretch single DNA-histone complexes, termed spin-stretching. The results show that the histones markedly enhance DNA binding to the PMMA surface. DNA winds around the histones and therefore decreases in length. The number of histones that bind to each DNA molecule is found to correlate with the histone concentration. The combed DNA-histone complexes are found to depend on two factors: the binding force on the surface and the centrifugal force at its local position. Na+ ions should compete with histones for binding to DNA; however, the observed competitive binding effect of Na+ ions at low concentrations was negligible.     

Towards molecular electronics with large-area molecular junctions

Electronic transport through single molecules has been studied extensively by academic and industrial research groups. Discrete tunnel junctions, or molecular diodes, have been reported using scanning probes, break junctions, metallic crossbars and nanopores. For technological applications, molecular tunnel junctions must be reliable, stable and reproducible. The conductance per molecule, however, typically varies by many orders of magnitude. Self-assembled monolayers (SAMs) may offer a promising route to the fabrication of reliable devices, and charge transport through SAMs of alkanethiols within nanopores is well understood, with non-resonant tunnelling dominating the transport mechanism. Unfortunately, electrical shorts in SAMs are often formed upon vapour deposition of the top electrode, which limits the diameter of the nanopore diodes to about 45 nm. Here we demonstrate a method to manufacture molecular junctions with diameters up to 100 microm with high yields (> 95 per cent). The junctions show excellent stability and reproducibility, and the conductance per unit area is similar to that obtained for benchmark nanopore diodes. Our technique involves processing the molecular junctions in the holes of a lithographically patterned photoresist, and then inserting a conducting polymer interlayer between the SAM and the metal top electrode. This simple approach is potentially low-cost and could pave the way for practical molecular electronics.     

骨外固定横向力测量传感器的研制

针对骨外固定的临床要求,研制了一种可与骨针和固定、调节装置方便连接的骨外固定横向力测量传感器,该传感器具有体积小、灵敏度高等优点,适用于各种骨外固定器。     

Atomic-scale imaging of DNA using scanning tunnelling microscopy

The scanning tunnelling microscope (STM) has been used to visualize DNA under water, under oil and in air. Images of single-stranded DNA have shown that submolecular resolution is possible. Here we describe atomic-resolution imaging of duplex DNA. Topographic STM images of uncoated duplex DNA on a graphite substrate obtained in ultra-high vacuum are presented that show double-helical structure, base pairs, and atomic-scale substructure. Experimental STM profiles show excellent correlation with atomic contours of the van der Waals surface of A-form DNA derived from X-ray crystallography. A comparison of variations in the barrier to quantum mechanical tunnelling (barrier-height) with atomic-scale topography shows correlation over the phosphate-sugar backbone but anticorrelation over the base pairs. This relationship may be due to the different chemical characteristics of parts of the molecule. Further investigation of this phenomenon should lead to a better understanding of the physics of imaging adsorbates with the STM and may prove useful in sequencing DNA. The improved resolution compared with previously published STM images of DNA may be attributable to ultra-high vacuum, high data-pixel density, slow scan rate, a fortuitously clean and sharp tip and/or a relatively dilute and extremely clean sample solution. This work demonstrates the potential of the STM for characterization of large biomolecular structures, but additional development will be required to make such high resolution imaging of DNA and other large molecules routine.     

原子力显微镜观测DNA分子的表面沉积与扩展

原子力显微镜具有原子级分辨率,能够对生物样品进行观察.用原子力显微镜观察了DNA分子在Si、云母片及修饰过的云母片表面的沉积与扩展,对3种情况作了比较,用超声振荡方法可以有效地切断DNA分子链.