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.

Direct detection of C9orf72 hexanucleotide repeat expansions by nanopore biosensor

Diagnostic C9orf72 hexanucleotide repeat expansions(C9-HRE)is essential for the early and accurate diagnosis of amyotrophic lateral sclerosis(ALS)and will provide support for the prognosis and gene therapy of ALS.In the present study,by combining catalytic hairpin assembly(CHA)with Mycobacterium smegmatis porin A(MspA)nanopore,a new nanopore-based strategy for the detection of C9-HRE was reported.Less than 30 repeats of C9-HRE could be detected via this method,and the results have the potential to help distinguish between patients and healthy individuals.Moreover,the method demonstrated its great specificity for C9-HRE by identifying other repeat expansions.Given the high selectivity,this approach had been successfully used to detect C9-HRE in cell and blood samples with high accuracy.This detection strategy is user-friendly and has a strong anti-interference ability,thus providing a powerful tool for clinical diagnosis.

Determination of Gossypol in Trace Level by Flow Injection Analysis with Chemiluminescence Detection

This work reports the single-molecule detection of gossypol by flow injection analysis with chemiluminescence method. The method is based on the reaction of luminol with ferricyanid in sodium hydroxide medium sensitized by gossypol. Under the optimum conditions, the CL intensity is proportional to the concentration of gossypol over the range of 1.11×10^-17-2.78×10^-16 mol/L in acid solution and 8.00×10^-11-7.39×10^-8mol/L in neutral solution with correlation coefficients 0.9983 and 0.9905, respectively. The detection limits is 1.60×10^-18 mol/L （S/N=3）. The proposed method has been applied for the determination of the gossypol in cottonseeds and pharmaceutical formulations with satisfactory results.

Single-molecule biotechnology for protein researches

Cells employ proteins to perform metabolic functions and maintain active physiological state through charge transfer and energy conversion.These processes are carried out in a narrow space precisely and rapidly,which,no doubt,bring great difficulty for their detection and dissection.Fortunately,in recent years,the development and expansion of single-molecule technique in protein research make monitoring the dynamical changes of protein at single-molecule level a reality,which also provides a powerful tool for the further exploration of new phenomena and new mechanisms of life activities.This paper aims to summarize the working principle and essential achievements of single-molecule technique in protein research in recent five years.We focus on not only dissecting the difference of nanopores,atomic force microscope,scanning tunneling microscope,and optical tweezers technique,but also discussing the great significance of these single-molecule techniques in investigating intramolecular and intermolecular interactions,electron transport,and conformational changes.Finally,the opportunities and challenges of the single-molecule technique in protein research are discussed,which provide a new door for single-molecule protein research.

Nanopore-Based Metal Ion Detection and Metal Ion-Mediated Nanopore Sensing

With the continuous development of nanotechnology,single-molecule nanopore detection has become a popular research topic.In this review,we summarize the application of biological nanopores for metal ions detection as well as overview the function of metal ions in the ion-mediated nanopore detection of different analytes in recent decades.According to the previous reports,biological nanopores utilize two strategies to detect metal ions.First,the specific binding sites are engineered in the nanopore to slow down the translocation rate of metal ions,resulting in the diverse specific current blockage signals.Secondly,the external molecule probes are added in the detection system to interact with metal ions,leading to the characteristic changes in the signals.At the same time,the external addition of metal ions into the nanopore detection systems enhances the sensitivity and selectivity through the changes of pore charges,the coordination with analytes or indirect detection.This review provides a summary on the role of metal ions in the application of nanopore detection technology.

Wavelength-dependent three-dimensional single-molecule superlocalization imaging for yoctomole detection of thyroid-stimulating hormone on a quantum dot nanobiosensor

A wavelength-dependent three-dimensional(3D)superlocalization imaging method on gold nanoislands(GNIs)chip was developed as a supersensitive single-molecule thyroid-stimulating hormone(TSH)nanobiosensor.Scattered and fluorescent signals from gold nanoislands on the substrate and quantum dots(QDs)nanoprobes were simultaneously isolated and acquired within an evanescent field layer generated by total internal reflection(TIR)of incident light using a dual-view device.The 3D TIR fluorescence images of TSH-bound QDs on the GNIs were obtained using z-axis optical sectioning at 10nm intervals before/after immunoreaction to identify the optimal conditions for detection.The localized centroid position of QD nanoprobes and GNI were distinguished at a subdiffraction limit resolution using 3D Gaussian fitting to the point spread function.The QD TSH nanobiosensor using wavelength-dependent 3D TIR fluorescence-based single-molecule localization microscopy(3D TIRF-SLM)imaging technique showed an excellent detection limit of 90 yoctomoles(~54 molecules)and a wide linear dynamic range of 1.14 zmol/L-100 pmol/L for TSH.The detection sensitivity was about 4.4×10^(9)times higher than conventional enzyme-linked immunosorbent assay and could successfully quantify TSH in human serum.The wavelength-dependent 3D TIRF-SLM technique may emerge as a reliable platform for ultrahigh-sensitive nanobiosensors at the single-molecule level and early diagnosis with quantification of disease-related ultra-tracebiomolecules.

双层纳米孔的制造与应用

由于具有孔径可调节、物理化学性质稳定、极端环境适应性强、集成度高等优点,固态纳米孔逐渐成为最具潜力的单分子测序工具。在固态纳米孔发展过程中,提高其单分子检测精度一直是研究人员关注的重点。近年来,双层纳米孔受到了广泛关注。与传统的单层纳米孔相比,双层纳米孔具有的孔-腔-孔结构提供了2个分子识别位点和纳米受限空间。双孔提供的2个分子识别位点可以在单次过孔事件中获得2次目标信号,所获得的双重检测信号不仅丰富了检测信息,也为信号分析提供了最为直接的对比信息源。此外,双层孔中的空腔还可作为单分子化学反应器。因此,双层纳米孔的出现拓宽了纳米孔传感器的应用范围,在单分子检测方面具有广阔的应用前景。本文概述了纳米孔的发展历程,并重点介绍了双层纳米孔的制造方法及其在单分子检测领域的应用。

Real-time identification of multiple nanoclusters with a protein nanopore in single-cluster level

It is important and challenging to analyze nanocluster structure with atomic precision.Herein,α-hemolysin nanopore was used to identify nanoclusters at the single molecule level by providing two-dimensional(2D)dwell time–current blockage spectra and translocation event frequency which sensitively depended on their structures.Nanoclusters such as Anderson,Keggin,Dawson,and a few lacunary Dawson polyoxometalates with very similar structures,even with only a two-atom difference,could be discriminated.This nanopore device could simultaneously measure multiple nanoclusters in a mixture qualitatively and quantitatively.Furthermore,molecular dynamics(MD)simulations provided microscopic understandings of the nanocluster translocation dynamics and yielded 2D dwell time–current blockage spectra in close agreement with experiments.The nanopore platform provides a novel powerful tool for nanocluster characterization.

基于纳米孔读取的DNA存储发展与展望

现代社会正处于数据爆炸的时代,全球对数据存储的需求已经远远超过了已有的存储能力。DNA是一种天然的遗传信息载体,可实现稳定、高效、低功耗的数据存储。目前的DNA存储过程主要分为6个环节:编码、写入、保存、检索、读取、解码。纳米孔测序技术被广泛应用于读取DNA中所存储的信息。该文系统性地介绍了纳米孔分子检测技术的原理和发展历史,及其在DNA存储中的应用。此外,该文总结了机器学习在纳米孔检测技术中的应用,着重介绍了结合机器学习的新型纳米孔检测技术。该文为纳米孔检测技术的发展提供了新方向,也为新型实用化DNA存储系统的发展奠定了基础。

基于固态纳米孔道的单颗粒散射光谱分析

固态纳米孔道单分子分析技术因其尺寸可控、无标记、高通量等优点,在近几年得到了快速的发展,已经被广泛应用于金属离子、DNA、RNA、蛋白质等物质的研究中。本文主要聚焦基于氮化硅固态纳米孔道的单颗粒散射光谱分析技术,简要介绍了近年来的研究进展,总结了固态纳米孔道的制备方法、单分子分析原理以及光电协同应用,并对拉曼散射光谱技术在固态纳米孔道单分子检测技术进行了展望。

Exponentially modified Gaussian relevance to the distributions of translocation events in nanopore-based single molecule detection

Nanopore technique plays an important role in single molecule detection, which illuminates the properties of an individual molecule by analyzing the blockage durations and currents. However, the traditional exponential function is lack of efficiency to describe the distributions of blockage durations in nanopore experiments. Herein, we introduced an exponentially modified Gaussian （EMG） function to fit the duration histograms of both simulated events and experimental events. In comparison with the traditional exponential function, our results demonstrated that the EMG provides a better fit while covers the entire range of the distributions. In particular, the fitted parameters of EMG could be directly used to discriminate the sequence length of the oligonucleotides at single molecule level.

A solid-state nanopore-based single-molecule approach for label-free characterization of plant polysaccharides

Polysaccharides are important biomacromolecules existing in all plants,most of which are integrated into a fibrillar structure called the cell wall.In the absence of an effective methodology for polysaccharide analysis that arises from compositional heterogeneity and structural flexibility,our knowledge of cell wall architecture and function is greatly constrained.Here,we develop a single-molecule approach for identifying plant polysaccharides with acetylated modification levels.We designed a solid-state nanopore sensor supported by a free-standing SiNx membrane in fluidic cells.This device was able to detect cell wall polysaccharide xylans at concentrations as low as 5 ng/mL and discriminate xylans with hyperacetylated and unacetylated modifications.We further demonstrated the capability of this method in distinguishing arabinoxylan and glucuronoxylan in monocot and dicot plants.Combining the data for categorizing polysaccharide mixtures,our study establishes a single-molecule platformfor polysaccharide analysis,opening a new avenue for understanding cell wall structures,and expanding polysaccharide applications.

Temperature effect on translocation speed and capture rate of nanopore-based DNA detection

We study the effects of electrolyte temperature on DNA molecule translocation experimentally without and with a temperature gradient across nanopore membranes.The same temperatures on both electrolyte chambers are first considered.The DNA molecule translocation time is measured to be 2.44 ms at 2°C in both chambers,which is 1.57 times longer than at 20°C.Then the temperature difference effect is characterized in both chambers.The results show that the DNA translocation speed can be slowed down as long as one side temperature is lowered,irrespective of the temperature gradient direction.This indicates that the thermophoretic driving force generated by a temperature gradient has no obvious effect on the threading speed of DNA molecules,while the main reason for the slowed DNA translocation speed is the increased viscosity.Interestingly,the capture rate of DNA molecules is enhanced under a temperature gradient condition,and the capture rate during DNA translocation from hot side at 21°C to cold one at 2°C is 1.7 times larger than that under the condition of both chambers at 20°C.Finally,an optimized configuration is proposed to acquire higher capture rates and lower DNA translocation speeds.

肽适体修饰纳米孔道对L⁃精氨酸的灵敏检测

氨基酸是生命之源,其中L⁃精氨酸(L⁃Arg)是生物体进行新陈代谢的一种重要氨基酸,同时也是重要的肿瘤标志物之一.因此,开发高选择性的L⁃Arg检测方法在生物分析领域十分重要.本文在Uniprot数据库29185个蛋白质序列中筛选出特异性结合L⁃Arg的多肽序列(序列为CFGHIHEGY),经ITC验证后,将其作为识别元件固定在子弹形纳米孔道尖端表面.在纳米空间限域效应下,利用多肽与L⁃Arg特异性结合前后构象由伸展状态向蜷缩状态的变化,调控纳米孔道离子输运特性变化,从而实现对L⁃Arg的选择性检测.实验结果表明,多肽修饰纳米孔道对L⁃Arg具有高灵敏度和高选择性,线性范围为1~100 nmol/L,检出限低至1 nmol/L.该研究为氨基酸高选择性、高灵敏检测提供了新方法,同时也为多肽修饰仿生离子通道的构建提供了新思路.

共价有机框架作为通道材料的有机电化学晶体管的制备及性能

有机电化学晶体管(OECT)由于具有器件制造简单、可拉伸、相对较低的工作电压和良好的开关电流比等优点,被广泛应用于生物医学、环境监测、保健产品、水处理和食品检测等领域。然而有机电化学晶体管材料往往受限于沟道材料的低化学稳定性以及低电子和离子迁移率等。共价有机框架(COF)由于具有稳定的共价键、良好的面内π-π共轭以及面外有序结构有望成为新一代的OECT沟道材料。文章通过一种表面引发方法在硅片表面原位生长COF薄膜,并用于OECT器件的组装,具有约100倍的开关比、0.4 V的低阈值电压和0.53 cm^(2)(V·s)的场效应迁移率。该研究结果为共价有机框架薄膜应用于电子器件领域拓宽了道路。

大肠杆菌内毒素的固态纳米孔单分子检测

细菌内毒素又称脂多糖,是革兰氏阴性菌细胞外壁的主要成分之一。微量的内毒素就可以引起生物体发热,是炎症反应、脓毒症、败血症等疾病的主要诱发因子。本文提出了一种基于固态氮化硅纳米孔检测单分子脂多糖的技术,利用电介质击穿法制备目标孔径,通过膜片钳监测脂多糖单分子的易位信息,分析得到在不同电压、不同孔径下脂多糖的构象变化,最终得到4.5 nm的最适检测孔径。本研究可用于临床医学检测、环境生物标志物的测定等。

In Situ Monitoring of Transmetallation in Electric Potential-Promoted Oxidative Coupling in a Single-Molecule Junction

To monitor and investigate chemical reactions in real time and in situ is a long-standing,challenging goal in chemistry.Herein,an electric potential-promoted oxidative coupling reaction of organoboron compounds without the addition of base is reported,and the transmetallation process involved is monitored in real time and in situ with the scanning tunneling microscopy break single-molecule junctions(STMBJ)technique.We found that the electric potential applied determined the transmetallation.At low-bias voltage,the first-step transmetallation process occurred and afforded Au─C-bonded aryl gold intermediates.The electronic properties of organoboron compounds have a strong influence on the transmetallation process,and electron-rich compounds facilitate this transformation.At high-bias voltage,the second-step transmetallation process took place,and the corresponding intermediate(highly reactive diaryl metal complex)was detected with the assistance of Pd(OAc)_(2).Our work demonstrates the applications of STMBJ on in situ monitoring and catalyzing of chemical reactions and provides a new methodology to fabricate singlemolecule devices.

纳米多孔银的制备及其表面增强拉曼散射特性研究

表面增强拉曼散射(SERS)因其具有高达单分子检测量级的灵敏度,在医学诊断、食品安全、环境监测等领域有着较大的应用前景。制备具有高密度“热点”的SERS基底是这项技术走向实际应用的关键。双连续结构的纳米多孔金属由于近邻纳米结构之间的耦合效应,所以具有很好的SERS增强特性。采用溅射方法制备了银锌合金前驱体,采用自由脱合金工艺和电化学脱合金工艺制备了具有纳米多孔结构的银基底,通过调制脱合金参数,获得了具有高增强因子的SERS基底。所制备的纳米多孔银基底对结晶紫的检测极限达到了10−12 mol/L,可应用于超灵敏检测。

Microelectrode glucose biosensor based on nanoporous platinum/graphene oxide nanostructure for rapid glucose detection of tomato and cucumber fruits

A microelectrode glucose biosensor based on a three-dimensional hybrid nanoporous platinum/graphene oxide nanostructure was developed for rapid glucose detection of tomato and cucumber fruits.The nanostructure was fabricated by a two-step modification method on a microelectrode for loading a larger amount of glucose oxidase.The nanoporous structure was prepared on the surface of the platinum microelectrode by electrochemical etching,and then graphene oxide was deposited on the prepared nanoporous electrode by electrochemical deposition.The nanoporous platinum/graphene oxide nanostructure had the advantage of improving the effective surface area of the electrode and the loading quantity of glucose oxidase.As a result,the biosensor achieved a wide range of 0.1-20.0 mmol/L in glucose detection,which had the ability to accurately detect the glucose content.It was found that the three-dimensional hybrid nanostructure on the electrode surface realized the rapid direct electrochemistry of glucose oxidase.Therefore,the biosensor achieved high glucose detection sensitivity 11.64μA·L/(mmol.cm^(2)),low detection limit(13μmol/L)and rapid response time(reaching 95%steady-state response within 3 s),when calibrating in glucose standard solution.In agricultural application,the as-prepared biosensor was employed to detect the glucose concentration of tomato and cucumber samples.The results showed that the relative deviation of this method was less than 5%when compared with that of high-performance liquid chromatography,implying high accuracy of the presented biosensor in glucose detection in plants.

蔬菜种子病害及其识别方法的研究进展

蔬菜种子病害对经济收益及民众健康的危害呈现出愈演愈烈之势,必须引起高度重视。在种子病害通用分类的基础上,指出传染性病害中的真菌性病害、细菌性病害和病毒性病害及对应蔬菜种子病害的主要性状及其传播途径。为此,分类论述了其病原机理、主要特征、危害方式等,并在此基础上详细介绍了以分子生物学检测、光谱检测技术、机器视觉检测技术、纳米孔检测技术等为代表的蔬菜种子病害识别研究的发展现状、应用范围及技术特征,尤其着重介绍了近年来基于机器视觉的人工智能识别方案,该方法简单易行、快速灵活且可塑性突出,并同时对各项检测技术存在的问题进行了分析,以期推进蔬菜种子病害识别技术的广泛应用与新技术的传播。