The confined geometry of nanopores enables a wealth of chemistry and analysis to be conducted at the single-molecule scale. Yi-Lun Ying, Aleksandar P. Ivanov and Vincent Tabard-Cossa report on recent developments discussed at the 2020 Nanopore Electrochemistry Meeting.纳米孔的受限几何形状使得能够在单分子规模上进行大量的化学和分析。 应义伦，Aleksandar P.Ivanov和Vincent Tabard-Cossa报告了在2020年纳米孔电化学会议上讨论的最新进展。

One of the most significant challenges in modern analytical sciences is the ever-growing need to characterize single-molecules and molecular assemblies with high spatial and temporal resolution. Nanopores are nanoscale-sized channels that address this challenge by providing a confined space for detecting single entities such as small molecules, nucleic acid polymers, proteins, viruses and nanoparticles using an electrical signal1,2,3. When a voltage is applied across the nanopore, its confined geometry forms a 3D sensing interface that is probed continuously by an ionic current. Molecules that are temporarily confined or transported through the nanopore can be identified by their current modulation. Various methods could be used to controllably construct nanopore structures in a range of soft and solid-state materials. These may include the self-assembly of proteins in lipid bilayers or block copolymer membranes, the drilling or dielectric breakdown of solid-state membranes, or even by simple pulling of glass capillaries down to nanoscale dimensions3,4.现代分析科学中最重大的挑战之一是对以高时空分辨率表征单分子和分子组装体的需求不断增长。纳米孔是纳米级通道，通过提供一个狭窄的空间来利用电信号1,2,3检测单个实体，例如小分子，核酸聚合物，蛋白质，病毒和纳米颗粒，从而解决了这一难题。当在纳米孔上施加电压时，其有限的几何形状会形成3D感应界面，该界面会被离子电流连续探测。可以通过电流调制来识别暂时限制或转运通过纳米孔的分子。可以使用各种方法来可控地构建一系列软和固态材料中的纳米孔结构。这些可能包括蛋白质在脂质双层或嵌段共聚物膜中的自组装，固态膜的钻孔或介电击穿，甚至可以通过简单地将玻璃毛细管拉至纳米尺寸3,4来完成。

By confining single molecules, nanopore sensors could access information on the chemical and physical properties of molecular entities, which is remarkably wealthier than only looking at ensemble-average measurements. The approach has already proven its prowess for long-read nucleic acid sequencing and is making its first strides towards the formidable goal of achieving protein sequencing. Beyond sequencing, it offers an elegant solution to a diverse array of contemporary challenges, for example, by providing novel insights into outstanding ionic and molecular transport problems, controllable chemical synthesis, precision diagnostics and advances in green energy conversion.通过限制单个分子，纳米孔传感器可以访问有关分子实体的化学和物理性质的信息，这比仅查看集合平均测量值要丰富得多。该方法已经证明了其在长时间阅读核酸测序中的能力，并且正在朝着实现蛋白质测序的强大目标迈出第一步。除测序之外，它还为各种当代挑战提供了一个优雅的解决方案，例如，通过提供对未解决的离子和分子传输问题，可控化学合成，精确诊断和绿色能源转换方面的新见解。

The 2020 Nanopore Electrochemistry Meeting (9–14th October) provided an opportunity for scientists worldwide to share recent advances and exchange ideas on the growing number of potential applications that continuously drive the rapid development of nanopore analysis (Fig. 1). The virtual meeting was hosted and organized by Yi-Tao Long and Hong-Yuan Chen from Nanjing University, China and Mathias Winterhalter from Jacobs University, Germany. Over 800 participants from across the globe came together to attend over 60 presentations and discuss more than 1000 questions online. These discussions covered an extensive range of nanopore-related topics spanning across physical and life sciences, medicine and engineering. The speakers shared new insights on biological suprabiomolecular assemblies, bionics, functional (bio)materials, chemical reactions and unconventional transport in nano-confined environments. There was significant focus on emerging nanopore methods for controllable chemical synthesis, protein analysis, future precision diagnostics using molecular barcodes and carriers, biosensing and molecular information storage using DNA nanostructures, single-cell analysis, and for providing new insights into the dynamics of biological processes.2020年纳米孔电化学会议（10月9日至14日）为全世界的科学家提供了一个分享最新进展并就不断推动纳米孔分析快速发展的潜在应用交流想法的机会（图1）。虚拟会议由中国南京大学的龙逸涛和陈洪元以及德国雅各布斯大学的Mathias Winterhalter主持和组织。来自全球的800多名与会者聚集在一起，参加了60多个演讲，并在线讨论了1000多个问题。这些讨论涵盖了与纳米孔相关的广泛主题，涉及物理和生命科学，医学和工程学。演讲者分享了有关生物超分子组装，仿生学，功能性（生物）材料，化学反应和纳米受限环境中非常规运输的新见解。人们非常关注新兴的纳米孔方法，这些方法可用于可控制的化学合成，蛋白质分析，使用分子条形码和载体的未来精确诊断，使用DNA纳米结构的生物传感和分子信息存储，单细胞分析以及提供对生物过程动力学的新见解。 

A new emerging domain in measurement sciences, nanopore electrochemistry, uses a nanopore to characterize single entities with spatial resolution down to sub-nanometre scale and temporal resolution reaching sub-microseconds. The electrochemical confinement of protons, ions, charges, analytes, reactions, interactions and fluidics inside nanopore channels offers a powerful toolkit for the analytical sciences and provides new insights into outstanding scientific questions in protein science, controllable chemical synthesis, and precision diagnostics.

测量科学中一个新兴领域是纳米孔电化学，它使用纳米孔来表征单个实体，其空间分辨率低至亚纳米级，时间分辨率达到亚微秒。纳米孔通道内质子，离子，电荷，分析物，反应，相互作用和流体的电化学限制为分析科学提供了强大的工具包，并提供了对蛋白质科学，可控化学合成和精确诊断中杰出科学问题的新见解。

One exciting trend was the growing versatility of biological nanopores. The hallmark of the protein nanopore is the atomically defined interior, which offers a designable bioreaction interface and excellent control of the local environment. Hagan Bayley’s team (University of Oxford, UK) shared their latest results using an α-hemolysin nanopore as a “nanoreactor” for single-molecule covalent chemistry. When an analyte molecule covalently attaches to the reactive site on the pore interior wall, the chemical reaction can be inferred by a change in ionic current. The intermediates in a reaction can be registered and their lifetimes recorded, yielding the rate constant for all steps. In an organic chemistry context, Scott L. Cockroft (University of Edinburgh, UK) showed that nanopores could resolve rapid click reaction equilibria. Henry S. White (University of Utah, USA) discussed acid–base chemistry in base-flipping dynamics. Learning from the quantum-confined superfluid (QSF) effect in biological ion channels, Lei Jiang’s group (Technical Institute of Physics and Chemistry, China) aimed to arrange single molecules in artificial nanopores to improve the efficiency of chemical reactions. Yunfei Chen (Southeast University, China) discussed dehydrated ion interactions in a highly confined nanopore solution. Chemistry under nanopore confinement thus permits many notable applications, including in situ site-specific modification of biopolymers, selective transmembrane signalling in synthetic cells and enantioselective catalysis. Nanopore confinement also enables the control of electrochemical phenomena such as permselective ion transport (when an ion is preferentially transported), gated transport and redox cycling. To this end, Paul W. Bohn (University of Notre Dame, USA) showed hierarchically organized nanopore electrode arrays to control the transport and reactivity of redox molecules.

一种令人兴奋的趋势是生物纳米孔的多功能性不断增长。蛋白质纳米孔的标志是原子定义的内部，提供了可设计的生物反应界面和对本地环境的出色控制。 Hagan Bayley的小组（英国牛津大学）分享了他们的最新结果，他们使用α-溶血素纳米孔作为单分子共价化学的“纳米反应器”。当分析物分子共价附于孔内壁上的反应位点时，可以通过离子电流的变化来推断化学反应。可以记录反应中的中间体并记录其寿命，从而获得所有步骤的速率常数。在有机化学背景下，Scott L. Cockroft（英国爱丁堡大学）表明，纳米孔可以解决快速点击反应的平衡问题。亨利·怀特（Henry S. White）（美国犹他大学）在碱翻转动力学中讨论了酸碱化学。蒋磊的小组从生物离子通道中的量子限制超流体（QSF）效应中学到了东西，旨在将单个分子排列在人造纳米孔中，以提高化学反应的效率。陈云飞（中国东南大学）讨论了在高度受限的纳米孔溶液中的脱水离子相互作用。因此，在纳米孔限制下的化学方法具有许多显着的应用，包括生物聚合物的原位定点修饰，合成细胞中的选择性跨膜信号传导和对映选择性催化。纳米孔的限制还能够控制电化学现象，例如全选择性离子迁移（优先迁移离子时），门控迁移和氧化还原循环。为此，Paul W. Bohn（美国圣母大学）展示了分层组织的纳米孔电极阵列，以控制氧化还原分子的转运和反应性。

Typical biological nanopores also provide some of the most advanced detectors for proteomic analysis at the single-molecule level. Juan Pelta (University of Paris-Saclay, France) described steps toward nanopore sequencing of proteins using a wild-type aerolysin nanopore, a powerful sensor for size discrimination of peptides. Together with Jan C. Behrends (University of Freiburg, Germany), they revealed that the aerolysin nanopore could identify single amino acid differences within a polyarginine carrier. By tuning the two sensing regions inside a mutant aerolysin nanopore, Yi-Tao Long’s group (Nanjing University, China) demonstrated enhanced sensitivity of nanopores for identifying peptide post-translation modifications and DNA lesions. Giovanni Maglia (University of Groningen, the Netherlands) introduced a sequencing concept based on assembling a nanopore protein sequencer with a proteasome that unfolds the protein structures. Biological nanopores are naturally suitable to study protein–protein interactions (PPI) and protein conformational states. Liviu Movileanu (Syracuse University, USA) showed a sensor platform based on a tether, a peptide adaptor and a protein receptor that could identify low- and high-affinity PPI in a complex mixture. Proteins are folded into 3D shapes of different sizes and solid-state nanopores have diameters that can be tuned easily to study a wide range of protein structures and biomolecular complexes. Moreover, solid-state nanopores allow current recordings in harsh conditions such as high voltage (force), high temperature, extreme pH and strong denaturants. Cees Dekker (TU Delft, the Netherlands) presented “NEOtrap”, a technique for long-term trapping (minutes to hours) of individual proteins inside a solid-state nanopore. A DNA-origami nanosphere is docked to a lipid-coated nanopore, forming a confined region and a highly charged environment where proteins can be trapped via electro-osmotic flow. Michael Mayer (Université de Fribourg, Switzerland) shared his vision of using polymer and lipid-coated solid-state nanopores for high-resolution characterization of single protein amyloid particles. Sébastien Balme (Université de Montpellier, France) described how conical track-etched nanopores, with their high aspect ratio, are well suited to characterize protein protofibril growth or degradation.典型的生物纳米孔还为单分子水平的蛋白质组学分析提供了一些最先进的检测器。 Juan Pelta（法国巴黎萨克莱大学）描述了使用野生型气溶素纳米孔对蛋白质进行纳米孔测序的步骤，这是一种用于区分多肽大小的强大传感器。他们与德国弗莱堡大学的Jan C. Behrends一起发现，溶血素纳米孔可以识别聚精氨酸载体内的单个氨基酸差异。通过调节突变的溶菌素纳米孔内部的两个感应区域，Yi-Tao Long的小组（中国南京大学）证明了纳米孔对识别肽段翻译后修饰和DNA损伤的敏感性增强。 Giovanni Maglia（荷兰格罗宁根大学）介绍了一种测序概念，该概念的基础是将纳米孔蛋白质测序仪与可折叠蛋白质结构的蛋白酶体组装在一起。生物纳米孔自然适合研究蛋白质-蛋白质相互作用（PPI）和蛋白质构象状态。 Liviu Movileanu（美国雪城大学）展示了一种基于系链，肽接头和蛋白质受体的传感器平台，该平台可识别复杂混合物中的低亲和力和高亲和力PPI。蛋白质被折叠成不同大小的3D形状，并且固态纳米孔的直径可以轻松调整，以研究各种蛋白质结构和生物分子复合物。而且，固态纳米孔可以在苛刻的条件下记录电流，例如高压（力），高温，极端的pH值和强变性剂。 Cees Dekker（荷兰TU Delft）提出了“ NEOtrap”技术，该技术可将固态纳米孔中的单个蛋白质长期捕获（数分钟至数小时）。 DNA折纸纳米球停靠在脂质包裹的纳米孔上，形成一个狭窄的区域和一个高度带电的环境，可以通过电渗流捕获蛋白质。 Michael Mayer（瑞士弗里堡大学）分享了他的观点，即使用聚合物和脂质包裹的固态纳米孔对单个蛋白质淀粉样蛋白颗粒进行高分辨率表征。 SébastienBalme（法国蒙彼利埃大学）描述了具有高纵横比的圆锥形轨迹蚀刻纳米孔如何非常适合表征蛋白质原纤维的生长或降解。

In terms of emerging healthcare applications, it was exciting to see several innovative applications employing solid-state nanopores. Amit Meller (Technion, Israel) shared the latest results from his lab’s work towards using portable nanopore devices for rapid analysis of clinical samples. The technology allows for quantification of RNA expression based on the synthesis and single-molecule counting of gene-specific cDNAs without the need for amplification. Joshua Edel’s and Aleksandar Ivanov’s groups (Imperial College London, UK) demonstrated multiplexed microRNA detection directly in native clinical samples without the need for sample processing using electro-optical nanopore readouts of molecular carriers grafted with molecular beacons. Justin Gooding (University of New South Wales, Australia) adopted a different approach by using magnetic nanoparticle carriers to separate analytes selectively and performed nanopore detection of bioanalytes in whole blood. Vincent Tabard-Cossa (University of Ottawa, Canada) reported on a digital scheme capable of quantifying the concentration of a protein biomarker from serum using DNA nanostructures as proxies for the presence (“1”) or absence (“0”) of the target captured via a magnetic bead-based sandwich immunoassay.

在新兴的医疗保健应用方面，令人兴奋的是，看到了一些采用固态纳米孔的创新应用。以色列理工学院的阿米特·梅勒（Amit Meller）分享了他实验室在使用便携式纳米孔设备快速分析临床样品方面所做工作的最新结果。该技术可以基于基因特异性cDNA的合成和单分子计数对RNA表达进行定量，而无需进行扩增。约书亚·埃德尔（Joshua Edel）和Aleksandar Ivanov（英国伦敦帝国学院）的研究小组直接在天然临床样品中演示了多重microRNA检测，而无需使用通过光电信标技术对接枝有分子信标的分子载体进行电光纳米孔读数处理。 Justin Gooding（澳大利亚新南威尔士大学）采用了另一种方法，即使用磁性纳米颗粒载体选择性地分离分析物，并对全血中的生物分析物进行纳米孔检测。 Vincent Tabard-Cossa（加拿大渥太华大学）报告了一种数字方案，该方案能够使用DNA纳米结构作为目标存在（“ 1”）或不存在（“ 0”）的代表来量化血清中蛋白质生物标志物的浓度。通过基于磁珠的夹心免疫分析法捕获。

The majority of these new solid-state nanopore technologies largely rely on molecular probes or carriers based on DNA nanostructures functionalized with recognition sites that enable selective detection of specific analytes. The carrier usually enables more-efficient transport and detection of analytes with a heterogeneous charge that may otherwise be challenging to sense. There are, however, other applications. Ulrich Keyser’s group (University of Cambridge, UK) demonstrated how DNA nanotechnology could be used for information storage and data processing using nanopores. They showed how the density of information could be expanded by increasing the number and the structure of barcodes grafted to a long linear DNA carrier, including 4-way, 6-way and 12-way DNA junctions, that would correspond to logical bits with depths beyond 0 and 1.

这些新的固态纳米孔技术大部分都依赖于基于具有识别位点功能的DNA纳米结构的分子探针或载体，从而能够选择性检测特定的分析物。载体通常可以更有效地运输和检测带有异质电荷的分析物，否则这些异质电荷很难检测。但是，还有其他应用程序。 Ulrich Keyser的小组（英国剑桥大学）展示了如何利用纳米孔将DNA纳米技术用于信息存储和数据处理。他们展示了如何通过增加嫁接到长线性DNA载体（包括4向，6向和12向DNA连接点）上的条形码的数量和结构来扩展信息密度，这些条形码对应于具有深度的逻辑位超过0和1。

While the COVID-19 pandemic forced many of us to distance physically, this virtual meeting’s format can serve as a model for bringing the scientific community closer. A forum for early-career scientists and a training workshop for newcomers took place within the meeting to encourage the next generation of researchers in the nanopore field. The 2020 Nanopore Electrochemistry Meeting was just the start of many more international interactions within this diverse community: following the conference’s success, the Nanopore Weekly Meetings were launched in late October and take place online every Monday. These meetings provide an open platform for researchers worldwide to share new results and exchange ideas on a variety of nanopore-related topics.

尽管COVID-19大流行迫使我们中的许多人物理距离，但这种虚拟会议的形式可以作为使科学界更紧密联系的典范。会议期间举行了一个针对早期职业科学家的论坛和一个针对新移民的培训讲习班，以鼓励纳米孔领域的下一代研究人员。 2020年纳米孔电化学会议只是这个多元化社区中更多国际交流的开始：会议成功举办后，10月下旬启动了纳米孔每周会议，并在每个星期一在线举行。这些会议为世界各地的研究人员提供了一个开放的平台，以分享新的结果并就各种与纳米孔有关的话题交换意见。