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.     

纳米金在DNA电化学传感器中的应用研究

利用自组装单分子膜原理,通过层层组装的方法将金纳米粒子(AuNPs)和DNA探针分别固定到金电极表面制备成探针电极。结果表明纳米金可以使响应电流大大增强,有利于提高检测的灵敏度;同时DNA自组装到纳米金修饰的金电极上,形成一层致密的分子膜,可使响应电流下降。     

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.     

基于DNA探针和FISH技术对两种有害浮游植物的检测研究

针对两种典型的有害浮游植物微小原甲藻和Takayama pulchellum(T.pulchellum)各设计了4条特异性探针,并采用基于PCR管载体的荧光原位杂交检测方法检测了这些探针的标记效果。实验结果表明,针对微小原甲藻和T.pulchellum核糖体大、小亚基DNA(LSU和SSU rDNA)的序列信息所设计的8条荧光标记探针均能够特异性地识别这些目标藻。各探针的标记效果有一定差异,经过标记的目标藻细胞在单种和自然水样混合样品中均可以通过荧光显微镜进行识别和区分。针对微小原甲藻和T.pulchellum的荧光原位杂交检测方法的建立将有助于对样品中这些目标藻进行快速准确地检测和监测。     

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.     

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.     

Retarding and manipulating of DNA molecules translocation through nanopores

Nanopores are emerging sensitive sensors that can detect and analyze single charged molecule. Nanopores present a promising approach for sequencing human gen- ome below US$1,000 because of its superior performance, such as high throughput and low cost. However, a dominant bottleneck, that is, the high translocation speed of DNA molecules, has to be overcome. This property decreases accuracy of nanopore sensors to the single-base level. In this review, we mainly introduce the recent research works of retarding and manipulating of DNA motion through nanopores by actively control of three forces, which are the driving force, interaction force between nanopore and molecule, and exterior drag force. Lastly, conclusion and further outlook are presented pore-based DNA sequencing on future directions of nano- technology.     

基于改进YOLOv5s和传感器融合的目标检测定位

针对无人车环境感知过程中相机无法提供道路目标的位置信息,激光雷达点云稀疏以致检测方面难以达到很好效果的问题,提出一种通过融合两者信息进行目标检测和定位的方法。采用深度学习中YOLOv5s算法进行目标检测,通过联合标定进行相机与激光雷达外参的获取以转换传感器之间的坐标,使雷达点云数据能投影到相机图像数据中,得到检测目标的位置信息,最后进行实车验证。结果表明,所提算法能在搭载TX2嵌入式计算平台的无人车自动驾驶平台上拥有27.2 Hz的检测速度,并且在一段时间的检测环境中保持12.50%的漏检率和35.32 m的最远识别距离以及0.18 m的平均定位精度。将激光雷达和相机融合,可实现嵌入式系统下的道路目标检测定位,为嵌入式平台下环境感知系统的搭建提供了参考。     

深度卷积神经网络的遥感植被检测方法

【目的】植被检测是城市生态研究的重要手段,然而由于遥感图像中植被存在阴影区域、遮挡区域以及色彩上的畸变等,导致当前的植被检测精度较低。基于遥感卫星影像,采用深度学习技术快速有效地检测出城市中的植被区域,为植被资源统计等相关研究提供依据。【方法】选用深度卷积神经网络模型,对高分辨率遥感影像中的植被区域进行检测。对不同的优化器,通过设置不同的卷积核大小,对精度进行对比分析。最后对网络层数进行研究,对设置合适网络层数进行分析,用构造的深度卷积神经网络在实验数据上进行植被区域检测。【结果】利用卷积神经网络处理二维图像时,无需手动提取特征,进行简单少量的预处理后,直接把图像输入到CNN模型中进行训练,即可实现图片的识别分类功能。降低了预处理的难度,同时局部感知和权值共享大幅度地减少了参数量,加快了计算速度。次抽样还能保证图像处理后的平移、旋转、缩放和拉伸的不变性。解决了传统方法计算量和样本量大、结构复杂以及费时的缺点。在采集到的高分辨率紫金山区域的遥感图像中,通过设计的多层卷积神经网络模型对区域中的植被资源进行分析,对比和研究不同的优化器、卷积核和网络层数,植被检测精度达到95.4%,明显高于当前众多植被检测算法。【结论】在深度学习中,目标检测的精度依赖于网络的结构设置,通过对优化器、卷积核以及网络层数进行设定,可以明显提高目标检测效率和精度。     

一种玻璃载片共价键固定DNA的新方法

目的：建立了一种简单、快速、高效共价固定DNA到玻璃其质上的方法。方法：玻片用β-（对、间氯甲基苯基）三氯硅乙烷处理在表面自组装形成带有苄氯功能基的单层硅烷膜，然后再用NaI丙酮溶液处理将苄氯基转化为苄碘基。巯基修饰的DNA通过巯基与苄碘基反应形成硫醚键共价偶联到这一自组装的硅烷膜上。结果：放射性分析证实玻璃基质表面共价固定DNA的密度为3.2nmol/m^2。用放射性标记的目标DNA进行杂交试验表明最大杂交密度的0.77nmol/m^2。结论；本方法只需一步偶联反应，反应时间短、偶联效率高、操作简单、不需要严格的试剂，而且制得的DNA芯片有较高的温度稳定性，可方便地用于核酸杂交和探测研究。     

基于改进RFBNet算法的遥感图像目标检测

针对遥感图像中的小目标存在信息少、易受背景干扰、特征表达较弱等缺陷,导致目前通用目标检测算法在对这类小目标进行检测时效果不理想的问题,为提高对遥感图像中小目标的检测能力,提出一种基于RFBNet的改进算法.该算法以RFBNet为框架,首先利用自校正卷积取代特征提取网络中的常规卷积,以扩展感受野丰富输出,进而强化对弱特征...     

Monitoring the binding of metal cations and histones to DNA in real time using fluorescence assays

The binding of cations (Na +,K +,Mg 2+,Ca 2+,Mn 2+) and histones to DNA can be studied using fluorescence assays.Here,we measured the fluorescence intensity and fluorescence anisotropy of DNA and DNA-histone complexes in the presence of cations.We demonstrate that when different cations are added into a DNA solution,the fluorescence intensities of the stained DNA are reduced by different amounts.Compared with divalent cations,monovalent cations had a weaker effect on fluorescence intensity and fluorescence anisotropy.Divalent (Mn 2+,Mg 2+,Ca 2+) cations markedly enhanced the fluorescence anisotropy of DNA.The binding modes of monovalent and divalent cations to DNA may be different.Divalent cations can change the structure of DNA molecules,or promote the assembly of DNA strands.The addition of histones causes DNA condensation,which mostly occurs during the first few seconds.Cation binding to DNA is abrupt,and is much faster than that of histones.     

分子动力学模拟烷烃链条数对其在氧修饰石墨烯纳米孔中迁移的影响

为了分析高分子在纳米孔中迁移问题,更好地为纳米器件的设计提供理论支持,采用分子动力学方法模拟了一系列链条数不同的烷烃分子在氧修饰石墨烯纳米孔中的钻孔,研究了链条数N对钻孔条数的影响,给出了均方位移MSD和扩散系数D随N变化的规律.研究结果表明:穿孔条数随着链条数的增加上升明显,但当链条数多到一定程度后,穿孔烷烃数较为缓慢地下降.     

基于中空PtCu纳米复合材料的电化学传感界面构建及其对2-羟基蒽醌的测定

中空PtCu纳米复合材料因其内部的空心结构和粗糙的多孔表面,具有较大的比表面积和良好的渗透性,可以有效提高2-羟基蒽醌的电子传递速率和氧化峰电流.基于此,构建了一种高灵敏度、高选择性、可快速测定2-羟基蒽醌的电化学传感平台.利用循环伏安法和微分脉冲伏安法探讨了2-羟基蒽醌在中空PtCu纳米复合材料修饰电极上的电化学氧化...     

配合物与G-四链体DNA相互作用的研究

端粒在维持基因组稳定、癌症和衰老相关的生理过程中发挥着重要作用,鸟嘌呤通过形成G-四分体平面堆积成G-四链体结构,DNA二级结构参与一些重要的生物调控过程。人们已经在生物体内发现G-四分体结构的存在,它们大量存在于基因的启动区域表明G-四链体可能参与调节基因表达。以G-四链体为新靶标设计能与G-四链体DNA相互作用的小分子,为开发抗癌药物提供了新途径。最近的许多研究证明金属配合物与G-四链体DNA具有有效的相互作用,有望开发成新的抗癌药物。     

The Escherichia coli O157:H7 DNA detection on a gold nanoparticle-enhanced piezoelectric biosensor

This paper presents development of a quartz crystal microbalance （QCM） biosensor for real-time detection of E. coil O157：H7 DNA based on nanogold particles amplification. Many inner Au nanoparticles were immobilized onto the thioled surface of the Au electrode, then more specific thiolated sin- gle-stranded DNA （ssDNA） probes could be fixed through Au-SH bonding. The hybridization was induced by exposing the ssDNA probe to the complementary target DNA of E. coli O157：H7 gene eaeA, then resulted in a mass change and corresponding frequency shifts （ △f ） of the QCM. The outer avidin-coated Au nanoparticles could combine with the target DNA to increase the mass. The electrochemical techniques, cyclic voltammetry （CV） and electrochemical impedance spectroscopy （EIS） were adopted to manifest and character each step. The target DNA corresponding to 2.0×10^3 colony forming unit （CFU）/mL E. coil O157：H7 cells can be detected by this biosensor, so it is practical to develop a sensitive and effective QCM biosensor for pathogenic bacteria detection based on specific DNA analysis. The piezoelectric biosensing system has potential for further applications, such as food safety and environment monitoring, and this approach lays the groundwork for incorporating the method into an integrated system for in-field bacteria detection.     

2,3-二巯基乙二酸自组装膜电极对多巴胺和尿酸的同时测定及其应用

利用电化学方法研究了2,3-二巯基乙二酸(DMSA)自组装膜的电化学行为,发现DMSA自组装膜对于多巴胺和尿酸的氧化能够起到明显的电催化作用.在优化条件下,多巴胺与尿酸的氧化峰电流分别与其浓度在1.0×10-4～1.0×10-3和8.0×10-4～9.0×10-3 mol/L内呈良好的线性关系,检出限分别为3.0 ×10-5和8.0×10-5 mol/L.该电极用于实际样品的测定,结果满意.     

多PIR动态扫描下的区域目标定位方法

在研究现有PIR的目标定位技术的基础上,提出了一种动态下使用红外热释电传感器(PIR)的新型目标区域定位方法;该方法使用多PIR探测节点构成感知单元,该探测节点在电机的带动下作往复式匀速旋转运动;根据多次试验下红外信号的特点,采用峰峰值时间差法和帧差法,确定目标坐标信息。根据PIR在某一时间内只能扫描到某一区域的特性将感知单元分为不同区域,进行区域目标定位;该方法使用多PIR构成探测节点,实现动态探测区域扫描;并能够在目标信息较少的情况下进行快速定位,提高定位精度;并且利用多时段多PIR的扫描探测,可以判别目标的运动速度和方向。实验结果表明,在给定实验条件下,使用该方法可实现对大范围区域进行快速定位,同时给出目标的运动轨迹。     

可见光遥感图像中舰船目标检测的多阶阈值分割方法

针对遥感图像中海洋复杂背景下的海上舰船目标检测,提出了基于自适应多阶阈值分割舰船目标检测方法.使用该方法对Spot,Quickbird,Ikonos,Landsat等不同卫星数据来源、不同分辨率、不同内容的12 704个样本进行了试验,成功检测了1 104个目标中的1 098个,检测率达到99.5%,同时虚警率保持在较低水平.实验证明,该方法对遥感图像中的海上舰船目标检测适应性强、检测率高、稳定性好.