Electrophoresis of poly(dT)_(20) through α-hemolysin nanopore in high concentration potassium chloride solution

Experiments of poly（dT）20 electrophoresis throughα-hemolysin nanopores were performed to unveil the electrophoretic transport mechanism of DNA through nanopores in high concentration potassium chloride solution. It is found that there are two obvious current blockades induced by poly（dT）20 translocation and collision events. Both blockade currents increase linearly with the applied bias voltage. However, the normalized blockade currents are almost kept the same although variable bias voltages are applied. The collision time of poly（dT）20 in the luminal site of the pore remains constant for different voltages. The translocation speed of poly（dT）20through the nanopore decreases with the increase of bias voltage. It is because as the potential increases, the drag force on the homopolymer helps it to crumple into a cluster much easier due to the poor stacking of thymine residues compared with homopolymers consisting of other nucleotides. Molecular dynamics simulations further confirm the experimental results. Increasing the applied bias voltage can slowdown the translocation velocity of the flexible poly（dT）20, which favors increasing the precision of single molecule detection by using nanopores.

基于Nanopore平台的测序试剂QzTGS HLA MX9在HLA基因分型中的评价研究

目的 探讨基于Nanopore平台的测序试剂QzTGS HLA MX9(TGS测序)在人类白细胞抗原(human leukocyte antigen, HLA)基因分型中应用的性能。方法 选取北京市红十字血液中心实验室常规HLA分型检测样本48份,采用PCR-SBT和TGS两种测序法分别对全部样本进行HLA基因分型,比较两种方法的基因分型结果及性能。结果 TGS法和PCR-SBT法对48份样本的HLA-A、-B、-C、-DRB1和-DQB1的基因检测结果,在高分辨水平上完全一致,TGS法可以对全部样本进行直接指定新基因及单一组合分型结果。TGS法在耗时、新基因判定、读长、机器维护、模棱两可结果和实时数据等性能方面优势更明显。结论 与PCR-SBT测序技术相比,基于Nanopore平台的测序试剂QzTGS HLA MX9在指定HLA基因结果方面准确性更高、耗时更短。

基于Nanopore测序技术的非洲猪瘟病毒全基因组测序方法建立

非洲猪瘟(ASF)是由非洲猪瘟病毒(ASFV)引起的一种高度传染性和致死性疫病,近年来给我国生猪产业的健康发展造成了沉重打击。ASFV庞大的基因组导致人们难以及时掌握流行毒株的全基因组序列。本研究旨在利用Nanopore三代测序技术建立一种简便可靠的ASFV全基因组测序方法。设计覆盖ASFV全基因组的31对引物并分为4个引物池对样本进行扩增,通过Nanopore测序技术对扩增产物进行测序,进一步优化相关生物信息学分析方法,最终成功建立了ASFV全基因组测序方法。应用该方法成功从某环境拭子样本中获取一株全长为189 416 bp的ASFV全基因组测序。经一代测序验证表明,在B646L、EP402R、E183L、MGF_360-12L、MGF_505-3R和I177L等关键基因及部分变异位点上,本方法结果与一代测序结果一致性100%;在全基因组水平上,本方法结果与二代测序结果一致性为99.94%。此外,在这项研究中,采用Nanopore测序技术发现了NP1450L基因与NP419L基因间区存在56 bp的重复序列插入(通过一代测序技术进行了验证),但是二代测序未能发现这一显著的变异特征。本研究成功建立了基于Nanopore技术的ASFV全基因组测序方法,该方法具有良好的简便性和可靠性,为当前ASF的防控和分子流行病学研究提供了一个重要手段。

基于Nanopore测序技术分析广东地区1株PRRSV-2的基因组特征

为促进Nanopore测序技术在美洲型猪繁殖与呼吸综合征病毒(PRRSV-2)全基因组测序中的应用并进一步了解当前广东地区PRRSV-2全基因组特征,本试验从广东地区某屠宰场采集1份PRRSV-2阳性样本,提取核酸进行靶向扩增,对扩增产物进行文库构建和测序,使用Samtools和Medaka等软件对测序数据进行生物信息学分析和拼接,得到样本的全基因组序列。选取5′端1~4 500核苷酸位点和开放阅读框5(ORF5)基因进行一代测序验证,评估本试验的准确性。进一步使用MEGA 11软件对所得的序列进行全基因组遗传进化分析、核苷酸相似性分析和NSP2蛋白氨基酸变异分析;使用RDP4软件和SimPlot软件进行重组分析。结果显示,本试验完成1份PRRSV-2阳性样本的全基因组测序,序列命名为GDYJ0718-7。2Samtools软件分析结果显示,本试验的平均测序深度为1 838×,覆盖PRRSV-2所有编码区。本试验Nanopore测序结果与一代测序结果一致性为100%。全基因组遗传进化分析结果显示,GDYJ0718-7属于QYYZ-like毒株,与参考株QYYZ(JQ308798)的核苷酸相似性仅为86.3%。NSP2蛋白氨基酸变异分析结果显示,GDYJ0718-7呈现1 aa+36 aa+29 aa的独特缺失模式。重组分析发现,GDYJ0718-7可能由QYYZ-like毒株和JXA1-like毒株重组形成。本试验通过Nanopore测序技术发现了广东地区QYYZ-like毒株变异的复杂性和新特点,为PRRSV-2的防控提供了技术和分子信息支持。

Voltage-modulated polymer nanopore field-effect transistor for multi-sized nanoparticle detection

Solid-state nanopores offer a range of distinct advantages over biological nanopores,such as structural diversity and greater stability and durability;this makes them highly promising for high-resolution nanoparticle sensing.Biological nanopores can exhibit gating characteristics with stress-responsive switches and can demonstrate specificity toward particular molecules.Drawing inspiration from biological nanopores,this paper introduces a novel polymer nanopore with field-effect characteristics,leveraging a conductive polymer in its construction to showcase intriguing gating behavior.Notably,in this device,the polymer layer serves as the gate,enabling precise control over the source–drain current response inside and outside the pore by simply adjusting the gate voltage.This unique feature allows fine-tuning of the nanopore’s sensitivity to nanoparticles of varying sizes and facilitates its operation in multiple modes.Experimental results reveal that the developed polymer nanopore field-effect transistor demonstrates remarkable selectivity in detecting nanoparticles of various sizes under different applied voltages.The proposed single device demonstrates the exceptional ability to detect multiple types of nanoparticle,showcasing its immense potential for a wide range of applications in biological-particle analysis and medical diagnostics.

Formation mechanism of nanopores in dense films of anodic alumina

Constant-current anodization of pure aluminum was carried out in non-corrosive capacitor working electrolytes to study the formation mechanism of nanopores in the anodic oxide films.Through comparative experiments,nanopores are found in the anodic films formed in the electrolytes after high-temperature storage(HTS)at 130°C for 240 h.A comparison of the voltage-time curves suggests that the formation of nanopores results from the decrease in formation efficiency of anodic oxide films rather than the corrosion of the electrolytes.FT-IR and UV spectra analysis shows that carboxylate and ethylene glycol in electrolytes can easily react by esterification at high temperatures.Combining the electronic current theory and oxygen bubble mold effect,the change in electrolyte composition could increase the electronic current in the anodizing process.The electronic current decreases the formation efficiency of anodic oxide films,and oxygen bubbles accompanying electronic current lead to the formation of nanopores in the dense films.The continuous electronic current and oxygen bubbles are the prerequisites for the formation of porous anodic oxides rather than the traditional field-assisted dissolution model.

Diagnosis and treatment of refractory infectious diseases using nanopore sequencing technology:Three case reports

BACKGROUND Infectious diseases are still one of the greatest threats to human health,and the etiology of 20%of cases of clinical fever is unknown;therefore,rapid identification of pathogens is highly important.Traditional culture methods are only able to detect a limited number of pathogens and are time-consuming;serologic detection has window periods,false-positive and false-negative problems;and nucleic acid molecular detection methods can detect several known pathogens only once.Three-generation nanopore sequencing technology provides new options for identifying pathogens.CASE SUMMARY Case 1:The patient was admitted to the hospital with abdominal pain for three days and cessation of defecation for five days,accompanied by cough and sputum.Nanopore sequencing of the drainage fluid revealed the presence of orallike bacteria,leading to a clinical diagnosis of bronchopleural fistula.Cefoperazone sodium sulbactam treatment was effective.Case 2:The patient was admitted to the hospital with fever and headache,and CT revealed lung inflammation.Antibiotic treatment for Streptococcus pneumoniae,identified through nanopore sequencing of cerebrospinal fluid,was effective.Case 3:The patient was admitted to our hospital with intermittent fever and an enlarged neck mass that had persisted for more than six months.Despite antibacterial treatment,her symptoms worsened.The nanopore sequencing results indicate that voriconazole treatment is effective for Aspergillus brookii.The patient was diagnosed with mixed cell type classical Hodgkin's lymphoma with infection.CONCLUSION Three-generation nanopore sequencing technology allows for rapid and accurate detection of pathogens in human infectious diseases.

Adsorption behavior of CO_(2)/H_(2)S mixtures in calcite slit nanopores for CO_(2) storage:An insight from molecular perspective

It is acknowledged that injecting CO_(2) into oil reservoirs and saline aquifers for storage is a practical and affordable method for CO_(2) sequestration.Most CO_(2) produced from industrial exhaust contains impurity gases such as H_(2)S that might impact CO_(2) sequestration due to competitive adsorption.This study makes a commendable effort to explore the adsorption behavior of CO_(2)/H_(2)S mixtures in calcite slit nanopores.Grand Canonical Monte Carlo(GCMC)simulation is employed to reveal the adsorption of CO_(2),H_(2)S as well as their binary mixtures in calcite nanopores.Results show that the increase in pressure and temperature can promote and inhibit the adsorption capacity of CO_(2) and H_(2)S in calcite nanopores,respectively.CO_(2)exhibits stronger adsorption on calcite surface than H_(2)S.Electrostatic energy plays the dominating role in the adsorption behavior.Electrostatic energy accounts for 97.11%of the CO_(2)-calcite interaction energy and 56.33%of the H_(2)S-calcite interaction energy at 10 MPa and 323.15 K.The presence of H_(2)S inhibits the CO_(2) adsorption in calcite nanopores due to competitive adsorption,and a higher mole fraction of H_(2)S leads to less CO_(2) adsorption.The quantity of CO_(2) adsorbed is lessened by approximately 33%when the mole fraction of H_(2)S reaches 0.25.CO_(2) molecules preferentially occupy the regions near the po re wall and H_(2)S molecules tend to reside at the center of nanopore even when the molar ratio of CO_(2) is low,indicating that CO_(2) has an adsorption priority on the calcite surface over H_(2)S.In addition,moisture can weaken the adsorption of both CO_(2) and H_(2)S,while CO_(2) is more affected.More interestingly,we find that pure CO_(2) is more suitable to be sequestrated in the shallower formations,i.e.,500-1500 m,whereas CO_(2)with H_(2)S impurity should be settled in the deeper reservoirs.

Nanopore测序揭示m6A修饰铁死亡相关基因在中晚期头颈部鳞状细胞癌中的表达及临床意义

目的研究N6-甲基腺苷(m6A)修饰在中晚期头颈部鳞状细胞癌(HNSCC)的作用和基因相关通路,为头颈部HNSCC患者的个体化精准治疗提出新的观点及研究参考。方法选取3对临床中晚期HNSCC患者的癌及其癌旁组织(6个样本)进行测序,从癌和癌旁组织的转录组、m6A相关差异基因中筛选出两者共同的差异基因,通过基因本体(GO)分析、pathway分析及蛋白相互作用(PPI)分析,找出其中的枢纽(hub)基因,随后对网络中的基因进行基因集变异分析(GSVA),从而得到m6A修饰影响的基因及通路。结果从癌与癌旁组织的差异基因中筛选出96个转录组m6A相关的上调基因、159个m6A相关下调基因;信号通路分析发现PPAR通路、免疫相关通路下调,代谢通路上调;构建PPI后发现,CDK1呈现上调趋势,而DCN及ARG2均呈现下调趋势;GSVA分析发现PI3K/AKT信号通路在癌组织中上调;PPI中的hub基因共表达分析发现AGR2和CDK1在m6A调控下,与铁死亡相关基因联系很密切。结论m6A修饰与FOXA1、RPL8、DNC、CDK1、ApoE、POSTN、YWHAZ、MMP13等铁死亡相关基因紧密相关,m6A修饰可能通过铁死亡相关基因影响免疫、代谢相关通路,从而在中晚期HNSCC中起重要作用。

宏基因组Nanopore测序在儿童呼吸道病原微生物快速检测中的应用价值

目的探索宏基因组Nanopore(三代)测序在儿童呼吸道病原微生物快速检测中的应用价值。方法对收集的1例重症肺炎患儿的肺泡灌洗液标本,传统方法检测为腺病毒抗原阳性,肺炎支原体和EB病毒PCR检测阳性,患儿经治疗痊愈出院。以此肺泡灌洗液标本提取DNA后,分别行宏基因组三代和二代测序。建立微生物参考基因组数据库,建立三代测序数据实时分析流程,分别将三代和二代测序数据和参考数据库比对,检测并鉴定病原微生物。结果三代、二代测序和传统方法检测从收到样本至检出腺病毒用时分别为3.2 h、55.5 h和48 h,检测费用人民币4200、3600和1790元。三代测序在开始后的12 min即检出1093条腺病毒序列,测序完成后,共得到4920000条序列,检出腺病毒99284条序列,占总序列数的2.02%;检出肺炎支原体和EB病毒序列各1条。二代测序检出的微生物组成和三代类似。结论宏基因组三代测序能实现对所有潜在儿童呼吸道病原微生物的快速实时检测。

A Combination of N2 and CO2 Adsorption to Characterize Nanopore Structure of Organic-Rich Lower Silurian Shale in the Upper Yangtze Platform, South China： Implications for Shale Gas Sorption Capacity

The pores in shales are mainly of nanometer-scale, and their pore size distribution is very important for the preservation and exploitation of shale gas. This study focused on the organic-rich Lower Silurian black shale from four wells in the Upper Yangtze Platform, and their TOC, mineralogical composition and pore characterization were investigated. Low pressure N2 and CO2 adsorption were conducted at 77.35 K and 273.15 K, respectively, and the pore structures were characterized by modified Brunauer-Emmett-Teller （BET）, Dubinin-Radushkevich （DR）, t-plot, Barrett- Joyner-Halenda （BJH） and density functional theory （DFT） methods and then the relationship between pore structure and shale gas sorption capacity was discussed. The results indicate that （1） The Lower Silurian shale has high TOC content of 0.92%~96%, high quartz content of 30.6%-69.5%, and high clays content of 24.1%-51.2%. The total specific surface area varies from 7.56 m^2/g to 25.86 m^2/g. Both the total specific surface area and quartz content are positively associated with the TOC content. （2） Shale samples with higher TOC content have more micropores, which results in more complex nanopore structure. Micropore volumes/surface areas and non-micropore surface areas all increase with the increasing TOC content. （3） A combination of N2 and CO2 adsorption provides the most suitable detection range （~0.3-60 nm） and has high reliability and accuracy for nanopore structure characterization. （4） The TOC content is the key factor to control the gas sorption capacity of the Lower Silurian shale in the Upper Yangtze Platform.

Application of Nanopore Sequencing Technology in the Clinical Diagnosis of Infectious Diseases

Infectious diseases are an enormous public health burden and a growing threat to human health worldwide.Emerging or classic recurrent pathogens,or pathogens with resistant traits,challenge our ability to diagnose and control infectious diseases.Nanopore sequencing technology has the potential to enhance our ability to diagnose,interrogate,and track infectious diseases due to the unrestricted read length and system portability.This review focuses on the application of nanopore sequencing technology in the clinical diagnosis of infectious diseases and includes the following:(i)a brief introduction to nanopore sequencing technology and Oxford Nanopore Technologies(ONT)sequencing platforms;(ii)strategies for nanopore-based sequencing technologies;and(iii)applications of nanopore sequencing technology in monitoring emerging pathogenic microorganisms,molecular detection of clinically relevant drug-resistance genes,and characterization of disease-related microbial communities.Finally,we discuss the current challenges,potential opportunities,and future outlook for applying nanopore sequencing technology in the diagnosis of infectious diseases.

A biosensor based on graphene nanoribbon with nanopores:a first-principles devices-design

A biosensor device, built from graphene nanoribbons （GNRs） with nanopores, was designed and studied by first- principles quantum transport simulation. We have demonstrated the intrinsic transport properties of the device and the effect of different nucleobases on device properties when they are located in the nanopores of GNRs. It was found that the device＇s current changes remarkably with the species of nucleobases, which originates from their different chemical compositions and coupling strengths with GNRs. In addition, our first-principles results clearly reveal that the distinguished ability of a device＇s current depends on the position of the pore to some extent. These results may present a new way to read off the nucleobases sequence of a single-stranded DNA （ssDNA） molecule by such GNRs-based device with designed nanopores

Current blockade mechanism for DNA translocation through solid-state nanopore with different membrane thickness

The current blockade mechanism for λ -DNA translocation under electrical field is investigated through solid-state nanopores with different pore thicknesses. The conductance of a nanopore system mainly consists of the contribution of the pore and access region, and the latter becomes dominant when the nanopore thickness gradually decreases to atomic layer thickness. Based on the existing model of nanopore resistance, a simplified model which describes the relative current blockade during the X-DNA translocation through the nanopores is deduced to quantitatively present the relationship between nanopore thickness and relative current blockade. Results show that the relative current blockade is effectively increased by reducing the nanopore diameter but it decreases with the decreasing nanopore thickness. A two-stage schematic is proposed to increase the relative current blockade by setting a much smaller resistance region. Experimental results show a 21. 9% increase in the relative current blockade with the proposed schematic.

Rapid and Accurate Sequencing of Enterovirus Genomes Using MinION Nanopore Sequencer

Objective Knowledge of an enterovirus genome sequence is very important in epidemiological investigation to identify transmission patterns and ascertain the extent of an outbreak. The MinION sequencer is increasingly used to sequence various viral pathogens in many clinical situations because of its long reads, portability, real-time accessibility of sequenced data, and very low initial costs. However, information is lacking on MinION sequencing of enterovirus genomes. Methods In this proof-of-concept study using Enterovirus 71 （EV71） and Coxsackievirus A16 （CA16） strains as examples, we established an amplicon-based whole genome sequencing method using MinION. We explored the accuracy, minimum sequencing time, discrimination and high-throughput sequencing ability of MinION, and compared its performance with Sanger sequencing. Results Within the first minute （min） of sequencing, the accuracy of MinION was 98.5% for the single EV71 strain and 94.12%-97.33% for 10 genetically-related CA16 strains. In as little as 14 min, 99% identity was reached for the single EV71 strain, and in 17 min （on average）, 99% identity was achieved for 10 CA16 strains in a single run. Conclusion MinION is suitable for whole genome sequencing of enteroviruses with sufficient accuracy and fine discrimination and has the potential as a fast, reliable and convenient method for routine use.

A glass nanopore electrode for single molecule detection

We have developed a simple method for fabricating robust and low noise glass nanopore electrodes with pore size 10±5 nm to detect single molecules.β-Cyclodextrin was used as model compound for characterization.In 1.0 mol/L NaCl solution,the molecules generated current pulses of 2-5 pA with noise level less than 0.8 pA.A slide mode and a plug mode were suggested for the way ofβ-cyclodextrin single molecule moving into the glass nanopores.

Effect of salt gradients on DNA translocation through solid-state nanopores

Aiming at the issues of controlling the translocation speed of DNA through a solid-state nanopore and enlarging the signal-to-noise ratio of ionic current modulation, which are challenges for the application of nanopore technology in DNA detection, salt concentration gradients are applied across the nanopore to investigate their influence on the DNA translocation time and signal-to-noise ratio. Experimental data demonstrates that, in symmetric concentration conditions, both the current blockade and dwell time for A-DNA translocation through a solid-state nanopore increase along with potassium chloride concentration. When the concentration in the trans chamber is decreased from 1 to 0.1 mol/L, keeping the concentration of the cis chamber at 1 mol/L, the normalized current blockade is found to be increased by one order. The increased dwell time and enhanced signal-to-noise ratio are achieved with salt gradients across the nanopore, which can improve the sensitivity when detecting DNA samples.

An analytical model for water-oil two-phase flow in inorganic nanopores in shale oil reservoirs

The existence of water phase occupies oil flow area and impacts the confined oil flow behavior at the solid substrate in inorganic nanopores of shale oil reservoirs,resulting in a completely different flow pattern when compared with the single oil phase flow.This study proposes an analytical model to describe the water-oil two-phase flow.In this model,water slippage at the solid substrate is considered while oil slip is introduced to calculate the oil movement at the solid-oil boundary in dry conditions.It is proven that the oil flow profiles of both the two-phase model and single-phase model show parabolic shapes,but the oil flow capacity drops when water takes up the flow space and the impact of water is more significant when the pore dimension is smaller than 30 nm.Also,the oil flow velocity at a pore center is found to drop linearly given a larger water saturation in wet conditions.The effects of surface wettability and oil properties on water-oil flow are also discussed.Compared with the existing singlephase models,this model describes oil flow pattern in the wet condition with the incorporation of the influence of nanopore properties,which better predicts the oil transport in actual reservoir conditions.Water-oil relative permeability curves are also obtained to improve oil yield.

Molecular dynamics simulation of water transport through graphene-based nanopores: Flow behavior and structure characteristics

The flow behavior of pressure-driven water infiltration through graphene-based slit nanopores has been studied by molecular simulation.The simulated flow rate is close to the experimental values,which demonstrates the reasonability of simulation results.Water molecules can spontaneously infiltrate into the nanopores,but an external driving force is generally required to pass through the whole pores.The exit of nanopore has a large obstruction on the water effusion.The flow velocity within the graphene nanochannels does not display monotonous dependence upon the pore width,indicating that the flow is related to the microscopic structures of water confined in the nanopores.Extensive structures of confined water are characterized in order to understand the flow behavior.This simulation improves the understanding of graphene-based nanofluidics,which helps in developing a new type of membrane separation technique.

Manipulation and optimization of electron transport by nanopore array targets

The transport of sub-picosecond laser-driven fast electrons in nanopore array targets is studied.Attributed to the generation of micro-structured magnetic fields,most fast electron beams are proven to be effectively guided and restricted during the propagation.Different transport patterns of fast electrons in the targets are observed in experiments and reproduced by particle-in-cell simulations,representing two components:initially collimated low-energy electrons in the center and high-energy scattering electrons turning into surrounding annular beams.The critical energy for confined electrons is deduced theoretically.The electron guidance and confinement by the nano-structured targets offer a technological approach to manipulate and optimize the fast electron transport by properly modulating pulse parameters and target design,showing great potential in many applications including ion acceleration,microfocus x-ray sources and inertial confinement fusion.