Detection of intrinsically disordered peptides by biological nanopore.

Intrinsically disordered protein regions (IDPRs) are pivotal in the regulation of transcription and the facilitation of signal transduction. Because of their multiple conformational states of structure, characterizing the highly flexible structures of IDPRs becomes challenging. Herein, we employed the wild-type (WT) aerolysin nanopore as a real-time biosensor for the identification and monitoring of long peptides containing IDPRs. This sensor successfully identified three intrinsically disordered peptides, with the lengths up to 43 amino acids, by leveraging the unique signatures of blockade current and translocation duration time. The analysis of the binding constant revealed that interactions between the nanopore and peptide are critical for peptide translocation, suggesting that mechanisms beyond mere volume exclusion are at play. Furthermore, by examining the detailed current traces of blockade events, we were able to compare the conformational stabilities of various IDPRs. Our approach can detect the conformational changes of IDPR in a confined nanopore space. These insights broaden the understanding of peptide structural changes under the confined space. This nanopore biosensor showed the potential to study the conformations change of IDPRs, IDPRs transmembrane interactions, and protein drug discovery.

Single-Digit Nanobubble Sensing via Nanopore Technology.

Nanobubbles play an important role in diverse fields, including engineering, medicine, and agriculture. Understanding the characteristics of individual nanobubbles is essential for comprehending fluid dynamics behaviors and advancing nanoscale science across various fields. Here, we report a strategy based on nanopore sensors for characterizing single-digit nanobubbles. We investigated the sizes and diffusion coefficients of nanobubbles at different voltages. Additionally, the finite element simulation and molecular dynamics simulation were introduced to account for counterion concentration variation around nanobubbles in the nanopore. In particular, the differences in stability and surface charge density of nanobubbles under various solution environments have been studied by the ion-stabilized model and the DLVO theory. Additionally, a straightforward method to mitigate nanobubble generation in the bulk for reducing current noise in nanopore sensing was suggested. The results hold significant implications for enhancing the understanding of individual nanobubble characterizations, especially in the nanofluid field.

Selective Capture and Manipulation of DNA through Double Charged Nanopores.

In the past few decades, nanometer-scale pores have been employed as powerful tools for sensing biological molecules. Owing to its unique structure and properties, solid-state nanopores provide interesting opportunities for the development of DNA sequencing technology. Controlling DNA translocation in nanopores is an important means of improving the accuracy of sequencing. Here we present a proof of principle study of accelerating DNA captured across targeted graphene nanopores using surface charge density and find the intrinsic mechanism of the combination of electroosmotic flow induced by charges of nanopore and electrostatic attraction/repulsion between the nanopore and ssDNA. The theoretical study performed here provides a new means for controlling DNA transport dynamics and makes better and cheaper application of graphene in molecular sequencing.

Trapping and recapturing single DNA molecules with pore-cavity-pore device.

Single-molecule detection technology is a technique capable of detecting molecules at the single-molecule level, characterized by high sensitivity, high resolution, and high specificity. Nanopore technology, as one of the single-molecule detection tools, is widely used to study the structure and function of biomolecules. In this study, we constructed a small-sized nanopore with a pore-cavity-pore structure, which can achieve a higher reverse capture rate. Through simulation, we investigated the electrical potential distribution of the nanopore with a pore-cavity-pore structure and analyzed the influence of pore size on the potential distribution. Accordingly, different pore sizes can be designed based on the radius of gyration of the target biomolecules, restricting their escape paths inside the chamber. In the future, nanopores with a pore-cavity-pore structure based on two-dimensional thin film materials are expected to be applied in single-molecule detection research, which provides new insights for various detection needs.

Actuation mechanism of a nanoscale drilling rig based on nested carbon nanotubes.

With the increasing emphasis on health and the continuous improvement of medical standards, more and more micro/nano devices are being used in the medical field. However, the existing micro/nano devices cannot effectively solve various problems encountered in medical processes and achieve specific therapeutic effects. Based on this, this article designs a new type of nanoscale drilling rig. The nanoscale drilling rig is composed of double-layer nested carbon nanotubes with multiple electrodes, and is powered by an external power source, making it easy to perform long-term surgery in the human body. Through coding strategies, we can adjust the surface charge density and distribution of the nanoscale drilling rig, thereby controlling its periodical rotation and achieving precise medical treatment. In addition, in order to control the length of the nanoscale drill bit, meet the treatment needs of different parts of the human body, and reduce damage to the human body, we have designed a structure of ion electric double layers so that the drill bit can be fixed in different positions, reducing the risk of treatment to a certain extent. This drilling rig enriches the functions of micro/nano devices, which is beneficial for the development of the medical industry.

Electroosmotic Sensing of Uncharged Peptides and Differentiating Their Phosphorylated States Using Nanopores.

The correct characterization and identification of different kinds of proteins is crucial for the survival and development of living organisms, and proteomics research promotes the analysis and understanding of future genome functions. Nanopore technique has been proved to accurately identify individual nucleotides. However, accurate and rapid protein sequencing is difficult due to the variability of protein structures that contains more than 20 amino acids, and it remains very challenging especially for uncharged peptides as they can not be electrophoretically driven through the nanopore. Graphene nanopores have the advantages of high accuracy, sensitivity and low cost in identifying protein phosphorylation modifications. Here, by using all-atom molecular dynamics simulations, charged graphene nanopores are employed to electroosmotically capture and sense uncharged peptides. By further mimicking AFM manipulation of single molecules, it is also found that the uncharged peptides and their phosphorylated states could also be differentiated by both the ionic current and pulling force signals during their pulling processes through the nanopore with a slow and constant velocity. The results shows ability of using nanopores to detect and discriminate single amino acid and its phosphorylation, which is essential for the future low-cost and high-throughput sequencing of protein residues and their post-translational modifications.

Conformation Influence of DNA on the Detection Signal through Solid-State Nanopores.

The detection and identification of nanoscale molecules are crucial, but traditional technology comes with a high cost and requires skilled operators. Solid-state nanopores are new powerful tools for discerning the three-dimensional shape and size of molecules, enabling the translation of molecular structural information into electric signals. Here, DNA molecules with different shapes were designed to explore the effects of electroosmotic forces (EOF), electrophoretic forces (EPF), and volume exclusion on electric signals within solid-state nanopores. Our results revealed that the electroosmotic force was the main driving force for single-stranded DNA (ssDNA), whereas double-stranded DNA (dsDNA) was primarily dominated by electrophoretic forces in nanopores. Moreover, dsDNA caused greater amplitude signals and moved faster through the nanopore due to its larger diameter and carrying more charges. Furthermore, at the same charge level and amount of bases, circular dsDNA exhibited a tighter structure compared to brush DNA, resulting in a shorter length. Consequently, circular dsDNA caused higher current-blocking amplitudes and faster passage speeds. The characterization approach based on nanopores allows researchers to get molecular information about size and shape in real time. These findings suggest that nanopore detection has the potential to streamline nanoscale characterization and analysis, potentially reducing both the cost and complexity.

Confined Transport Behavior of Biomolecules within Tilted Nanopores.

The transport behavior of biomolecules at the confined nanoscale is very different from that of the bulk state. Numerous disease diagnostics and targeted drug treatments are performed based on nanochannels in cells. The specific structure and shape of nanochannels play an important role in the behavior and efficiency of substance transport. In this paper, we fabricated nanopores with different tilt angles and the same diameters using focused ion beam. The capture frequency and the blocking current amplitude of λ-DNA within large-angle nanopores decrease obviously, suggesting an increase in the energy barrier of large-angle nanopores and the fact that they stretch biomolecules to thinness. Most importantly, large-angle nanopores slow down λ-DNA transport by 2-4 times. MD simulations find that the sloped electroosmotic flow inside the tilted nanopores is the main factor contributing to the transport phenomena. The increase in the capture time of biomolecules by nanopores assists in obtaining more biological information from the current trajectories. Our study provides a new understanding of substance transport in specially shaped nanopores, which can be instrumental in providing fresh inspiration and approaches to the biomedical field.

Protein Deceleration and Sequencing Using Si3N4-CNT Hybrid Nanopores.

Protein sequencing is crucial for understanding the complex mechanisms driving biological functions and is of utmost importance in molecular diagnostics and medication development. Nanopores have become an effective tool for single molecule sensing, however, the weak charge and non-uniform charge distribution of protein make capturing and sensing very challenging, which poses a significant obstacle to the development of nanopore-based protein sequencing. In this study, to facilitate capturing of the unfolded protein, highly charged peptide was employed in our simulations, we found that the velocity of unfolded peptide translocating through a hybrid nanopore composed of silicon nitride membrane and carbon nanotube is much slower compared to bare silicon nitride nanopore, it is due to the significant interaction between amino acids and the surface of carbon nanotube. Moreover, by introducing variations in the charge states at the boundaries of carbon nanotube nanopores, the competition and combination of the electrophoretic and electroosmotic flows through the nanopores could be controlled, we then successfully regulated the translocation velocity of unfolded proteins through the hybrid nanopores. The proposed hybrid nanopore effectively retards the translocation velocity of protein through it, facilitates the acquisition of ample information for accurate amino acid identification.

Unfolding of protein using MoS2/SnS2heterostructure for nanopore-based sequencing.

Protein sequencing is crucial for understanding the complex mechanisms driving biological functions. However, proteins are usually folded in their native state and the mechanism of fast protein conformation transitions still remains unclear, which make protein sequencing challenging. Molecular dynamics simulations with accurate force field are now able to observe the entire folding/unfolding process, providing valuable insights into protein folding mechanisms. Given that proteins can be unfolded, nanopore technology shows great potential for protein sequencing. In this study, we proposed to use MoS2/SnS2heterostructures to firstly unfold proteins and then detect them by a nanopore in the heterostructural membrane. All-atom molecular dynamics simulations performed in this work provided rich atomic-level information for a comprehensive understanding of protein unfolding process and mechanism on the MoS2/SnS2heterostructure, it was found that the strong binding of protein to SnS2nanostripe and hydrogen bond breaking were the main reasons for unfolding the protein on the heterostructure. After the protein was fully unfolded, it was restrained on the nanostripe because of the affinity of protein to the SnS2nanostripe. Thus by integrating the proposed unfolding technique with nanopore technology, detection of linear unfolded peptide was realized in this work, allowing for the identification of protein components, which is essential for sequencing proteins in the near future.

Ion Concentration-Dependent Surface Charge Density Inside a Nanopore.

Surface charges shape the electrical double layer (EDL) structure at solid-liquid interfaces, critically influencing the performance of energy storage and micro/nanofluidic devices. However, accurately measuring surface charge density in nanoconfined spaces continues to be a challenge. Here, we introduce a methodology via solid-state nanopores that can investigate the dependence of surface charge density on salt concentrations and nanopore diameters. Measurements, complemented by a theoretical model, reveal that the surface charge density decreases as both the salt concentration in bulk solutions and the nanopore sizes are reduced. Notably, when the salt concentration in the bulk solution drops below 10-3 M, protons dominate ion conductance in a nanopore, resulting in a constant surface charge density. This study introduces an effective approach to surface charge characterization and may serve in the design of electrokinetically driven nanofluidic systems.

Surface Roughness Effects on Confined Nanoscale Transport of Ions and Biomolecules.

Biological channels, especially membrane proteins, play a crucial role in metabolism, facilitating the transport of nutrients and other materials across cell membranes in a bio-electrolyte environment. Artificial nanopores are employed to study ion and biomolecule transport behavior inside. While the non-specific interaction between the nanopore surface and transport targets has garnered significant attention, the impact of surface roughness is overlooked. In this study, Nanopores with different levels of inner surface roughness is created by adjusting the FIB (Focus Ion Beam) fabrication parameters. Experiments and molecular dynamics (MD) simulations are employed to demonstrate that greater roughness results from larger FIB beam currents and shorter processing times. Lower roughness increases the capture rate of biomolecules, while greater roughness enhances the normalized blockade current (ΔI/I0 ). The phenomenon of rougher nanopores are attributed to a barrier-dominated capture mechanism and more likely to induce DNA folding. This transport barrier exists in rough nanopores by utilizing steer molecular dynamics (SMD) simulations to investigate the force profile of a dA10 DNA molecule during translocation is demonstrated. This work illustrates how surface roughness influences the ionic current features and the translocation of biomolecules, paving a new way for tunning the molecule transport in nanopores.

DNA Carrier-Assisted Molecular Ping-Pong in an Asymmetric Nanopore.

Nanopore analysis relies on ensemble averaging of translocation signals obtained from numerous molecules, requiring a relatively high sample concentration and a long turnaround time from the sample to results. The recapture and subsequent re-reading of the same molecule is a promising alternative that enriches the signal information from a single molecule. Here, we describe how an asymmetric nanopore improves molecular ping-pong by promoting the recapture of the molecule in the trans reservoir. We also demonstrate that the molecular recapture could be improved by linking the target molecule to a long DNA carrier to reduce the diffusion, thereby achieving over 100 recapture events. Using this ping-pong methodology, we demonstrate its use in accurately resolving nanostructure motifs along a DNA scaffold through repeated detection. Our method offers novel insights into the control of DNA polymer dynamics within nanopore confinement and opens avenues for the development of a high-fidelity DNA detection platform.

Nanopore actuation of a DNA-tracked nanovehicle.

As a kind of nanomachine that has great potential for applications in nanoscale sensing and manipulation, nanovehicles with unique shapes and functions have received extensive attention in recent years. Different from the existing common method of using synthetic chemistry to design and manufacture a nanovehicle, here we theoretically report a molecularly assembled DNA-tracked nanovehicle that can move on a solid-state surface using molecular dynamics simulations. A graphene membrane with four nanopores acts as the chassis of the nanoscale vehicle, and two circular ssDNAs across the nanopores serve as the wheels. The electroosmotic flows induced by independently charged nanopores with different surface charge densities under external electric fields were found to be the main power to actuate the controlled rotary motion of circular ssDNAs across every two nanopores. By tuning the rotary speed of each circular ssDNA, the linear and turning movements of the designed nanovehicle were realized. The designed nanovehicle makes it possible to have access to almost everywhere in the human body, which would lead to significant breakthroughs in the fields of nanoscale surgery, drug delivery and so on. The research not only enriches the family of nanorobots, but also opens another way for designing nanovehicles.

Precise control of CNT-DNA assembled nanomotor using oppositely charged dual nanopores.

Inspired by nature, nanomotors have been developed that have great potential in microfluidics and biomedical applications. The development of the rotary nanomotor, which is an important type of nanomotor, is an essential step towards intelligent nanomachines and nanorobots. Carbon nanotubes (CNTs) are a crucial component of rotary nanomotors because of their excellent mechanical properties and adaptability to the human body. Herein, we introduce a convenient manipulation method for controlling the rotation of a nanomotor assembled from CNT-DNA, which uses the electroosmosis effect within oppositely charged dual nanopores. The central components of this nanomotor consist of a double-walled carbon nanotube (DWCNT) and a circular single-stranded DNA (ssDNA), which acts as the driving element for the nanomotor. Selective ion transport through charged nanopores can generate a robust electroosmotic flow (EOF), which serves as the primary power for the movement of circular ssDNA. The tangential force on the ssDNA is transmitted via electrostatic adsorption to the outer surface of the CNT, known as the rotor, resulting in the rotation of the nanomotor. By simply adjusting the electric field and surface charge density of each nanopore, rotational variables including speed, output power and torque can be readily regulated in this work. This proof-of-concept research provides a promising foundation for the future development of the precise control of nanomotors.

Detection of DNA translocations in a nanopore series circuit using a current clamp.

The advancement of nanopore sensing technology over the past 20 years has been impressive, particularly in the field of nucleic acid sequencing, which has already been used in commercial diagnostic tests. A traditional configuration of nanopore sensing records the current through a single nanopore using a voltage clamp, which hits a bottleneck in expanding its functions, while integrating several nanopores to build a nanopore circuit may be an effective solution. Here, we report a new strategy combining a nanopore series circuit and a current clamp to record the current signal and the voltage signal of DNA translocation through a nanopore simultaneously, which could increase the fidelity of event analysis. We observed a capacitor-like charging and discharging behavior in the voltage signals and proposed a detailed microscopic mechanism to elucidate it. Our strategy could benefit the development of nanopore technology and contribute to understanding the working principles of the units in a nanopore circuit system.

A controllable nanoscale telescopic arm designed by encoding the nested multi-walled carbon nanotubes.

Micro/nano manipulation technologies have shown enormous potential in the field of accurate surgery, which is expected to promote the development of precision medicine. Therefore, scientists have been devoted to designing and manipulating nanoscale devices and tools which can conduct surgical functions, such as penetration, drilling and cleaving targeting either single cells or biological tissues. To enrich the functionality of the family of nanomachines, a theoretical nanoscale telescopic arm manipulated by charge-tunable multi-walled carbon nanotubes is designed in this work. By using predesigned encoding strategies that could periodically alternate the external electric fields and surface charge densities of the nanorings embedded in the carbon nanotubes, well controlled manipulations of the telescopic arm are realized in MD simulations to mimic nanoscale surgeries. The telescopic arm can stretch out by the external electric force and draw back by vdW attraction between the nested nanotubes. Meanwhile, the electric double layer formed around the nanoring area in the nanotube is used as a brake during the retraction process to make the nanotube halt stably at the target position. The working distance could also be tuned by changing the number of the nested nanotubes, which presents a promising avenue for varieties of biomedical applications.

Large-area 2D bismuth antimonide with enhanced thermoelectric properties via multiscale electron-phonon decoupling.

It is a challenge to obtain high thermoelectric efficiency owing to the conflicting parameters of the materials that are required. In this work, the composition-adjustable 2D bismuth antimonide (Bi100-xSbx) is synthesized using an e-beam evaporation system with homemade targets. Engineering multiscale defects is done to optimize the thermoelectric performance in the compound. Sb alloying introduces atomic defects, lattice distortion and increased grain boundary. They drastically decrease the thermal conductivity, with an ultralow value of ∼0.23 W m-1 K-1 obtained for the composition with x = 18. It is noticed that the atomic and nanoscale defects do not deteriorate the electrical conductivity (105 S m-1), and the value is even comparable to the bulk counterpart over a wide composition range (0 ≤ x ≤ 35). Annealing induces pore structure with microscale defects, which increase the Seebeck coefficient by 84% due to the energy barrier. The resultant ZT of 0.13 is enhanced by 420% in the annealed Bi82Sb18 when compared with the as-grown Bi. This work demonstrates a cost-effective and controllable way to decouple electrons and phonons in the thermoelectric field.

Portable nanopore-sequencing technology: Trends in development and applications.

Sequencing technology is the most commonly used technology in molecular biology research and an essential pillar for the development and applications of molecular biology. Since 1977, when the first generation of sequencing technology opened the door to interpreting the genetic code, sequencing technology has been developing for three generations. It has applications in all aspects of life and scientific research, such as disease diagnosis, drug target discovery, pathological research, species protection, and SARS-CoV-2 detection. However, the first- and second-generation sequencing technology relied on fluorescence detection systems and DNA polymerization enzyme systems, which increased the cost of sequencing technology and limited its scope of applications. The third-generation sequencing technology performs PCR-free and single-molecule sequencing, but it still depends on the fluorescence detection device. To break through these limitations, researchers have made arduous efforts to develop a new advanced portable sequencing technology represented by nanopore sequencing. Nanopore technology has the advantages of small size and convenient portability, independent of biochemical reagents, and direct reading using physical methods. This paper reviews the research and development process of nanopore sequencing technology (NST) from the laboratory to commercially viable tools; discusses the main types of nanopore sequencing technologies and their various applications in solving a wide range of real-world problems. In addition, the paper collates the analysis tools necessary for performing different processing tasks in nanopore sequencing. Finally, we highlight the challenges of NST and its future research and application directions.

AutoNanopore: An Automated Adaptive and Robust Method to Locate Translocation Events in Solid-State Nanopore Current Traces.

Solid-state nanopore sequencing has shown impressive performances in several research scenarios but is still challenging, mainly due to the ultrafast speed of DNA translocation and significant noises embedded in raw signals. Hence, event detection, aiming to locate precisely these translocation events, is the fundamental step of data analysis. However, existing event detection methods use either a user-defined global threshold or an adaptive threshold determined by the data, assuming the baseline current to be stable over time. These disadvantages limit their applications in real-world application scenarios, especially considering that the results of different methods are often inconsistent. In this study, we develop an automated adaptive method called AutoNanopore, for fast and accurate event detection in current traces. The method consists of three consecutive steps: current trace segmentation, current amplitude outlier identification by straightforward statistical analyses, and event characterization. Then we propose ideas/metrics on how to quantitatively evaluate the performance of an event detection method, followed by comparing the performance of AutoNanopore against two state-of-the-art methods, OpenNanopore and EventPro. Finally, we examine if one method can detect the overlapping events detected by the other two, demonstrating that AutoNanopore has the highest coverage ratio. Moreover, AutoNanopore also performs well in detecting challenging events: e.g., those with significantly varying baselines.