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化学进展 2020, Vol. 32 Issue (5): 562-580 DOI: 10.7536/PC190914 前一篇   后一篇

• 综述 •

纳米孔生物分子检测研究

林子涵1,3, 陈煌1, 董嘉伟1, 赵道辉2,**(), 李理波1,**()   

  1. 1. 华南理工大学化学化工学院 广州 510640
    2. 湖北大学化学与化工学院 武汉 430062
    3. 湖北大学楚才学院 武汉 430062
  • 收稿日期:2019-09-09 修回日期:2019-12-12 出版日期:2020-05-15 发布日期:2020-02-20
  • 通讯作者: 赵道辉, 李理波
  • 基金资助:
    国家自然科学基金项目(21908046); 制浆造纸工程国家重点实验室开放基金(华南理工大学201828)(SCUT201828); 湖北省自然科学基金项目(2019CFB293); 广东省自然科学基金项目(2019A1515011121); 广州市科技计划项目(201804010219); 中央高校基本科研业务经费()

Nanopore-Based Biomolecular Detection

Zihan Lin1,3, Huang Chen1, Jiawei Dong1, Daohui Zhao2,**(), Libo Li1,**()   

  1. 1. School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou 510640, China
    2. College of Chemistry and Chemical Engineering, Hubei University, Wuhan 430062, China
    3. Chucai College, Hubei University, Wuhan 430062, China
  • Received:2019-09-09 Revised:2019-12-12 Online:2020-05-15 Published:2020-02-20
  • Contact: Daohui Zhao, Libo Li
  • About author:
    ** e-mail: (Daohui Zhao);
  • Supported by:
    National Natural Science Foundation of China(21908046); State Key Laboratory of Pulp and Paper Engineering(SCUT201828); Hubei Natural Science Foundation(2019CFB293); Guangdong Natural Science Foundation(2019A1515011121); Guangzhou Technology Project(201804010219); Fundamental Research Funds for the Central Universities()

纳米孔单分子检测技术是一种集操作简单、灵敏度高、检测速度快、无需标记等优点的传感检测技术,广泛应用于蛋白质检测、基因测序和标志物检测等领域。基因测序的费用、灵敏度和精度是该检测技术的发展中亟待解决的主要问题,而开发新型的纳米孔材料则是解决这些问题的关键手段。本文从纳米孔材料的选择和设计角度出发,综述了三种不同的纳米孔,即蛋白质等生物纳米孔、固态纳米孔和新型二维材料纳米孔在生物分子检测方面的应用现状,并比较了生物纳米孔与固态纳米孔的差别。本文也重点阐述了二维材料纳米孔在生物分子检测中的实验和模拟研究进展。最后,对纳米孔检测技术的发展前景进行了展望。

Nanopore-based single molecular detection technology is a sensing technology that combines the advantages of simple operation, high sensitivity, fast speed, and no labeling. It is widely used in protein detection, gene sequencing, and marker detection. The cost, sensitivity and accuracy are the main challenges in developing such technology, and to develop new nanopore materials is the key to solve them. From the perspective of selecting and designing nanopore materials, the application status of three different nanopores: biological nanopore, solid-state nanopore and novel two-dimensional(2D) material nanopore in biomolecule detection are reviewed. Meanwhile, the differences between biological nanopores and solid-state nanopores are compared in details. The article also emphasizes the experimental and simulation research progress of 2D material nanopores in biomolecule detection. Finally, the future development of nanopore-based detection technology is also discussed.

Contents

1 Introduction

2 Basic principles and molecular simulation methods of nanopore-based single molecule detection technology

2.1 The basic principle of nanopore detection

2.2 Molecular dynamics simulation

3 Application of nanopores in biomolecular detection

3.1 Application of biological nanopore in biomolecule detection

3.2 Application of solid-state nanopores in biomolecule detection

3.3 Comparison of biological nanopores and solid-state nanopores

4 Biomolecular detection with two-dimensional(2D) material nanopore

4.1 Introduction to 2D materials

4.2 Experimental study of 2D nanopore in biomolecule detection

4.3 Simulation study of 2D nanopore in biomolecule detection

5 Conclusion and outlook

()
图1 2004~2015年下一代测序技术的发展历程[5]
Fig. 1 Development of next-generation sequencing technologies during 2004~2015[5]. Reproduced with permission from copyright(2017) Nature Publishing Group
表1 商业化公司的测序平台的对比[5]
Table 1 Comparison of available sequencing platforms[5]
图2 离子阻塞电流检测方法的检测原理示意图[26]
Fig. 2 Schematic diagram of ionic current blockage method[26]
表2 三种电子传感检测技术的检测原理[19]
Table 2 The sensing principles of three electronic sensing technologies[19]. Reproduced with permission from copyright(2014), Royal Society of Chemistry
图3 纳米孔单分子检测技术的发展历程[42]
Fig. 3 Development of nanopore-based single-molecule detection technologies[42]. Reproduced with permission from copyright(2016) Nature Publishing Group
表3 常用的生物纳米孔
Table 3 Details of commonly utilized biological nanopores
图4 生物纳米孔在核苷酸检测中的应用。(a~c)Aerolysin纳米孔检测短的寡核苷酸[49](dAn, n=2, 3, 4, 5, 10)。 (a)短的寡核苷酸穿过Aerolysin纳米孔的示意图;(b)在无dAn和加入dAn情况下的代表性电流轨迹,红色三角形表示典型的阻塞事件,其放大图在电流轨迹的右边;(c)高斯拟合得到的dAn的阻塞事件的直方图。(d~f)用Phi29纳米孔检测dsDNA[68]。(d)用Phi29纳米孔检测dsDNA的装置示意图;(e)35 bp dsDNA的结构序列图及其穿孔电流轨迹;(f)双交叉DNA的结构序列图及其穿孔电流轨迹
Fig. 4 Applications of biological nanopores for the detection of nucleotides.(a~c) Detection of oligonucleotides(polydeoxyadenines(dAn), n=2, 3, 4, 5, 10) with an aerolysin pore[49]. (a) Schematic illustration of a short oligonucleotide passing through an aerolysin nanopore.(b) Representative current traces recorded without dAn and with the addition of dAn. The red triangles denote typical blockades shown in the insets.(c) Histogram for the blockade events associated with dAn with Gaussian fits.(d~f) Detection of dsDNA with phi29 nanopore[68].(d) Illustration of dsDNA translocating through a phi29 nanopore.(e) Schematic and 2D sequence of 35 bp dsDNA. Typical current trace showing the translocation of 35 bp dsDNA showing single-level events.(f) Schematic and 2D sequences of double crossover DNA(DX-DNA). Typical current trace showing the translocation of DX-DNA showing double-level events. Reproduced with permission from copyright(2016) Nature Publishing Group
图5 生物纳米孔在检测蛋白中的应用。 用α-HL纳米孔间接检测蛋白质分子,通过比较初始肽底物及其剪切产物产生的电流调制来确定胰蛋白酶Trypsin活性[80]
Fig. 5 Applications of biological nanopores in detecting proteins. Indirect detection of protein molecules with α-HL. Comparison of current modulations measured for the initial peptide substrate and its cleaved products can be performed to determine trypsin activities[80]. Reproduced with permission from copyright(2016) American Chemical Society
表4 常见的固态纳米孔的特点
Table 4 Characteristics of solid-state nanopores
图6 利用2.5 nm的SiNx纳米孔检测dsDNA结构[111]。图为DNA产物和错配的DNA产物的系统示意图(左),典型的电流轨迹(中),停留时间对数直方图(右)
Fig. 6 Detection of various dsDNA structures with a 2.5 nm SiNx nanopore[111]. The picture shows the system diagram of DNA products and mismatched DNA products(left), typical current trace(middle), semi-logarithmic plot of residence time(right). Reproduced with permission from copyright(2015) American Chemical Society
图7 不同类型的超薄2D材料示意图[120]
Fig. 7 Schematic illustration of different kinds of typical ultrathin 2D nanomaterials[120]. Copyright 2015, American Chemical Society
图8 长通道纳米孔与2D纳米孔识别精度比较[125]
Fig. 8 Schematic representation of DNA translocation through long channel nanopores and 2D nanopores[125]. Copyright 2016, Royal Society of Chemistry
表5 利用2D材料进行生物分子检测的实验研究
Table 5 Experiments of biomolecule detection by 2D materials
图9 利用石墨烯纳米孔进行DNA测序的实验研究 (a)Dekker实验室[97],通过阻塞电流可识别非折叠、部分折叠、全折叠的dsDNA。(b)Golovchenko实验室[12],可识别非折叠、折叠的dsDNA。(c)Drndic实验室[99],可识别非折叠、折叠的dsDNA。(d)Bashir实验室[98]
Fig. 9 Experimental studies on DNA sequencing using graphene nanopores. (a) Dekker lab[97], the non-folded, partially folded and folded dsDNA could be identified by blockade current. Copyright(2010) American Chemical Society.(b) Golovchenko lab[12], the non-folded and folded dsDNA could be identified. Copyright(2010) Nature Publishing Group.(c) Drndic lab[99], the non-folded and folded dsDNA could be identified. Copyright(2010) American Chemical Society.(d) Bashir lab[98]. Copyright(2012) American Chemical Society
图10 (a)MoS2 纳米孔DNA测序的实验装置示意图[101]。(b)剥落的单层MoS2薄片的光学图。(c)转移后的MoS2片层的光学图。(d) λ-DNA穿过20 nm MoS2 纳米孔的电流轨迹(蓝线);黑线是λ-DNA穿过SiN x 纳米孔的电流轨迹。(e)选取4个独立的电流阻塞事件来表明λ-DNA在孔中的结构:非折叠(1),部分折叠(2),折叠(3),碰撞事件(4)
Fig. 10 (a) Schematic illustration of DNA sequencing using a MoS2 nanopore[101].(b) Optical image of a freshly exfoliated monolayer MoS2 flake.(c) Optical image after the chosen flake has been transferred to the desired location.(d) A typical trace(in blue) of λ-DNA translocation through a 20 nm MoS2 nanopore. The upper trace(in black) is an example of λ-DNA translocation through a SiN x nanopore.(e) Selected individual events with quantized current drops implying multiple conformations of λ-DNA within the nanopore, i.e., unfolded(1), partially folded(2), folded(3), and bumping event(4). Reproduced with permission from copyright(2014) American Chemical Society
表6 利用2D材料进行生物分子检测的模拟研究
Table 6 Theoretical simulations f biomolecule detection by 2D materials
Material Pore size
(nm)
Simulation method Signal characteristics Molecular size Ion concentration ref
graphene 2.4 MD poly(G-C) > poly(A-T) 45 bases 1 M KCl 142
graphene 1.1~1.5 MD+BD poly(A)≈poly(G)< poly(T) < poly(C) 20 bases 1.7 M KCl 143
graphene 1.6~5.0 MD The SNR increases with the decrease of aperture 45 bases 1.0 M KCl 144
graphene 2.0~3.0 MD Conductivity is related to pore thickness 12 bases 1.0 M NaCl 129
graphene 2.4 MD The fluctuation of signal is related to the DNA structure 48 bases 1.0 M NaCl 145
graphene 1.5 MD Increasing the number of graphene layers can improve the accuracy 45 bases 1.0 M KCl 146
graphene 1.6 MD The charge regulates DNA perforation 20 bases 1.0 M KCl 147
graphene 2.1 MD Hybrid hole can improve the detection accuracy 20 bases 1.0 M NaCl 148
graphene 1.6 MD Ion current + perforation time 15 bases 1.0 M KCl 149
graphene 2.2 MD The perforation rate is related to the type of polypeptide and the number of graphene layers 48 residues 1.0 M KCl 150
graphene 10 MD IgG3 > IgG2 IgG 0.5 M NaCl 151
MoS2 2.3 MD+DFT SNR > 15 16 bases 1.0 M NaCl 130
MoS2 2.5 MD+DFT Pull force + ionic current 6 bases 5 mM KCl 152
h-BN 2.4 MD poly(A-T)40 <poly(G-C)40 40 bases 1.0 M KCl 153
graphene 1.5 SMD Pull force + ionic current Thioredoxin 2 M KCl 154
graphene 1.0 SMD G > A > T > C 11 bases 155
graphene 1.0 SMD G > A > T > C 12 bases 156
graphene 1.1 SMD Dependent on the structure of the hole 6 bases 157
graphene 1.6, 2.4 SMD Independent of pore size and shape 14 bases 1 M KCl 158
图11 (a)DNA穿过MoS2 纳米孔。(b)不同碱基和碱基对(CG或AT)穿过MoS2纳米孔时产生的离子电流。(c)不同电压和温度下MoS2纳米孔的信噪比[130]
Fig. 11 (a) DNA translocation through a MoS2 nanopore.(b) Ionic current for different bases and combination of CG or AT bases through the MoS2 nanopore.(c) Signal-to-noise ratio of MoS2 nanopore for different biases and temperatures[130]. Copyright 2014 American Chemical Society
图12 利用2D材料纳米孔检测DNA甲基化的模拟系统[161]。(a)DNA甲基化位点。(b)模拟的纳米孔装置示意图
Fig. 12 Simulation system of DNA methylation detection with 2D material nanopores[161].(a) Schematic showing a CpG dinucleotide site in an mDNA molecule in complex with a MBD1 protein.(b) Schematic of the simulated nanopore device. Reproduced with permission from copyright(2017) Nature Publishing Group
图13 采用SMD模拟拉DNA穿过石墨烯纳米孔的模拟示意图及拉伸过程产生的不同碱基的特征力峰[155]
Fig. 13 Schematic diagram of DNA translocation through graphene nanopore by SMD simulations, and the characteristic force peaks for different bases[155]. Reproduced with permission from copyright(2014) American Chemical Society
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纳米孔生物分子检测研究