Identification of single nucleotides in MoS2 nanopores

Ultrathin membranes have drawn much attention due to their unprecedented spatial resolution for DNA nanopore sequencing. However, the high translocation velocity (3000-50000 nt/ms) of DNA molecules moving across such membranes limits their usability. To this end, we have introduced a viscosity gradient system based on room-temperature ionic liquids (RTILs) to control the dynamics of DNA translocation through a nanometer-size pore fabricated in an atomically thin MoS2 membrane. This allows us for the first time to statistically identify all four types of nucleotides with solid state nanopores. Nucleotides are identified according to the current signatures recorded during their transient residence in the narrow orifice of the atomically thin MoS2 nanopore. In this novel architecture that exploits high viscosity of RTIL, we demonstrate single-nucleotide translocation velocity that is an optimal speed (1-50 nt/ms) for DNA sequencing, while keeping the signal to noise ratio (SNR) higher than 10. Our findings pave the way for future low-cost and rapid DNA sequencing using solid-state nanopores.Comment: Manuscript 24 pages, 4 Figures Supporting Information 24 pages, 12 Figures, 2 Tables Manuscript in review Nature Nanotechnology since May 27th 201

Plasmonic Biosensors for Single-Molecule Biomedical Analysis

Review[EN] The rapid spread of epidemic diseases (i.e., coronavirus disease 2019 (COVID-19)) has contributed to focus global attention on the diagnosis of medical conditions by ultrasensitive detection methods. To overcome this challenge, increasing efforts have been driven towards the development of single-molecule analytical platforms. In this context, recent progress in plasmonic biosensing has enabled the design of novel detection strategies capable of targeting individual molecules while evaluating their binding affinity and biological interactions. This review compiles the latest advances in plasmonic technologies for monitoring clinically relevant biomarkers at the single-molecule level. Functional applications are discussed according to plasmonic sensing modes based on either nanoapertures or nanoparticle approaches. A special focus was devoted to new analytical developments involving a wide variety of analytes (e.g., proteins, living cells, nucleic acids and viruses). The utility of plasmonic-based single-molecule analysis for personalized medicine, considering technological limitations and future prospects, is also overviewedS

Adaptive nanopores: A bioinspired label-free approach for protein sequencing and identification

AbstractSingle molecule protein sequencing would tremendously impact in proteomics and human biology and it would promote the development of novel diagnostic and therapeutic approaches. However, its technological realization can only be envisioned, and huge challenges need to be overcome. Major difficulties are inherent to the structure of proteins, which are composed by several different amino-acids. Despite long standing efforts, only few complex techniques, such as Edman degradation, liquid chromatography and mass spectroscopy, make protein sequencing possible. Unfortunately, these techniques present significant limitations in terms of amount of sample required and dynamic range of measurement. It is known that proteins can distinguish closely similar molecules. Moreover, several proteins can work as biological nanopores in order to perform single molecule detection and sequencing. Unfortunately, while DNA sequencing by means of nanopores is demonstrated, very few examples of nanopores able to perform reliable protein-sequencing have been reported so far. Here, we investigate, by means of molecular dynamics simulations, how a re-engineered protein, acting as biological nanopore, can be used to recognize the sequence of a translocating peptide by sensing the "shape" of individual amino-acids. In our simulations we demonstrate that it is possible to discriminate with high fidelity, 9 different amino-acids in a short peptide translocating through the engineered construct. The method, here shown for fluorescence-based sequencing, does not require any labelling of the peptidic analyte. These results can pave the way for a new and highly sensitive method of sequencing

Chemiresistive Nanosensors with Convex/Concave structures

航空航天学院陈松月副教授和化学化工学院、物理科学与技术学院双聘教授侯旭共同在国际著名期刊Nano Today (纳米科学领域权威刊物，IF=17.476)上发表了该文章。随着纳米技术和新材料的涌现，纳米传感器自90年代末出现后，因其高比表面积带来的优越性受到了越来越多的研究上和应用上的关注。本论文综述了基于一维结构（特殊凹凸结构）的化学电阻式纳米传感器，包括纳米线、纳米管和纳米孔道结构。根据传感器的不同材料和结构，分别讨论了它们的传感原理、材料和结构设计、界面设计、在不同领域中的应用。在此基础上讨论了各种纳米传感器在应用中表现出的优缺点。并提出了未来的发展将更注重传感器的稳定性、灵敏度、特异性以及器件的可集成性。【Abstract】Nanosensors have attracted tremendous, scientific and application, interests promoted by the advances in nanotechnology and emerging new nanomaterials. There has been rapid progress in developing chemiresistive nanosensors, and these sensor technologies are being transferred among a variety of different fields, from energy, environment to life science. This review presents nanomaterials with special convex/concave structures used for chemiresistive sensors, which mainly composed of one-dimensional conductive structures, e.g. nanowires, nanotubes, nanopores and nanochannels. Furthermore, designing, operation, and applications of current chemiresistive nanosensors are discussed to give an outlook of this field, especially for ionic solution and gas as the working chemical environments. The authors hope this review could inspire the active interest in the scientific field of sensor development and application.This work was supported by the National Natural Science Foundation of China (grant numbers 61601387, 21673197, U1505243), the Natural Science Foundation of Fujian Province of China (grant number 2017J05107), Young Overseas High-level Talents Introduction Plan, the 111 Project (grant number B16029), the Open Funding of State Key Laboratory of Precision Measuring Technology and Instruments (grant number pilab1709), and the Fundamental Research Funds for the Central Universities of China (grant number 20720170050). 该工作得到了国家自然科学基金（项目批准号: 61601387, 21673197, U1505243），福建省自然科学基金（项目批准号: 2017J05107），高等学校学科创新引智计划（项目批准号: B16029），精密测试技术及仪器国家重点实验室开放基金（项目批准号: pilab1709）和厦门大学校长基金（项目批准号: 20720170050）等资助与支持

Label-free single-molecule nanopore analysis of protein post-translational modifications

Despite significant advancements and refinements in single-molecule nanopore technology for DNA and RNA sequencing, its applicability to protein analysis remains limited. Proteins, consisting of 20 diverse building blocks, undergo post-translational modifications, fold into complex three-dimensional structures, and lack uniform charge distribution. These factors complicate signal interpretation and hinder their capture and movement through nanopore sensors. In the endeavor to enhance protein sequencing methodologies, ongoing research predominantly classifies into two primary strategies. Firstly, there are initiatives aimed at emulating the ordered translocation events observed in DNA/RNA nanopore sequencing, a process guided by enzymatic mechanisms. These approaches frequently employ molecular motors, necessitating preliminary sample processing steps to introduce recognition tags and labels. Alternatively, more straightforward strategies center on the examination of short peptides. Nonetheless, a substantial challenge persists in decoding the signal and reconstructing the original protein sequence from these peptide fragments, presenting a notable bottleneck in this methodology. Electro-osmotic flow (EOF), which occurs when an applied potential induces liquid motion in narrow tube-like conduits, is a phenomenon reported in nanopores for decades. EOF generates forces capable of increasing the capture rate and intra-pore residence time of neutral molecules and folded protein substrates. Despite promising results, limited work has explored the utilization of EOF for driving proteins across nanopores in a manner compatible with sequence-level readouts. In this study, we capitalize on the robust and well-characterized heptameric protein nanopore, αHL. We engineer this nanopore by introducing charged residues near its constriction, creating a series of ion-selective pores with varying EOF strengths. We then use a model protein, Trx, to construct concatemeric molecules featuring 2, 4, 6, 8, and 9 repetitions of a Trx-linker unit, resulting in chain lengths ranging from approximately 300 to over 1200 amino acids. These constructs are transported across the engineered pores in a label-free, enzyme-less manner under mild denaturing conditions. This process generates distinct current signatures corresponding to the number of units within each construct, indicating a mechanism of co-translocational unfolding. In an effort to assess the applicability of this approach, we introduce three types of post-translational modifications (PTMs)—phosphorylations, glutathionylation, and a glycosylation mimic—at five different positions within the middle Trx-linker unit of a nonameric concatemer. We engineer a universal modification site at these locations. The detection and discrimination of the three different modifications are successfully demonstrated at two of these positions. While this approach may not provide de novo sequence information, with further optimizations, it holds the potential to become a powerful method for fingerprinting PTMs. It could enable the characterization of underivatized full-length proteoforms from cells and tissues, serving as a valuable tool for proteomic analysis

Lysenin Channels as Sensors for Ions and Molecules

Lysenin is a pore-forming protein extracted from the earthworm Eisenia fetida, which inserts large conductance pores in artificial and natural lipid membranes containing sphingomyelin. Its cytolytic and hemolytic activity is rather indicative of a pore-forming toxin; however, lysenin channels present intricate regulatory features manifested as a reduction in conductance upon exposure to multivalent ions. Lysenin pores also present a large unobstructed channel, which enables the translocation of analytes, such as short DNA and peptide molecules, driven by electrochemical gradients. These important features of lysenin channels provide opportunities for using them as sensors for a large variety of applications. In this respect, this literature review is focused on investigations aimed at the potential use of lysenin channels as analytical tools. The described explorations include interactions with multivalent inorganic and organic cations, analyses on the reversibility of such interactions, insights into the regulation mechanisms of lysenin channels, interactions with purines, stochastic sensing of peptides and DNA molecules, and evidence of molecular translocation. Lysenin channels present themselves as versatile sensing platforms that exploit either intrinsic regulatory features or the changes in ionic currents elicited when molecules thread the conducting pathway, which may be further developed into analytical tools of high specificity and sensitivity or exploited for other scientific biotechnological applications