Solid-state Nanopore Sensors Research Articles

Brownian Motion Paving the Way for Molecular Translocation in Nanopores.

Tracing fast nanopore-translocating analytes requires a high-frequency measurement system that warrants a temporal resolution better than 1µs. This constraint may practically shift the challenge from increasing the sampling bandwidth to dealing with the rapidly growing noise with frequencies typically above 10kHz, potentially making it still uncertain if all translocation events are unambiguously captured. Here, a numerical simulation model is presented as an alternative to discern translocation events with different experimental settings including pore dimension, bias voltage, the charge state of the analyte, salt concentration, and electrolyte viscosity. The model allows for simultaneous analysis of forces exerting on a large analyte cohort along their individual trajectories; these forces are responsible for the analyte movement leading eventually to the nanopore translocation. Through tracing the analyte trajectories, the Brownian force is found to dominate the analyte movement in electrolytes until the last moment at which the electroosmotic force determines the final translocation act. The mean dwell time of analytes mimicking streptavidin decreases from ≈6 to ≈1µs with increasing the bias voltage from ±100 to ±500mV. The simulated translocation events qualitatively agree with the experimental data with streptavidin. The simulation model is also helpful for the design of new solid-state nanopore sensors.

CRISPR-assisted solid-state nanopore sensor for rapid and sensitive point-of-care amendable of monkeypox virus detection via RPA amplification

CRISPR-assisted solid-state nanopore sensor for rapid and sensitive point-of-care amendable of monkeypox virus detection via RPA amplification

Solid-State Nanopore/Nanochannel Sensors with Enhanced Selectivity through Pore-in Modification.

Nanopore sensing technology, as an emerging analytical method, has the advantages of simple operation, fast output, and label-free and has been widely used in fields such as protein analysis, gene sequencing, and biomarker detection. Inspired by biological ion channels, scientists have prepared various artificial solid-state nanopores/nanochannels. Biological ion channels have extremely high ion transport selectivity, while solid-state nanopores/nanochannels have poor selectivity. The selectivity of solid-state nanopores and nanochannels can be enhanced by modifying channel charge, varying pore size, incorporating specific chemical functionality, and adjusting operating (or solution) conditions. This Perspective highlights pore-in modification strategies for enhancing the selectivity of solid-state nanopore/nanochannel sensors by summarizing the articles published in the last 10 years. The future development prospects and challenges of pore-in modification in solid-state nanopore and nanochannel sensors are discussed. This Perspective helps readers better understand nanopore sensing technology, especially the importance of detection selectivity. We believe that solid-state nanopore/nanochannel sensors will soon enter our homes after various challenges.

Targeted Analysis of the Size Distribution of Heavy Chain-Modified Hyaluronan with Solid-State Nanopores.

The glycosaminoglycan hyaluronan (HA) plays important roles in diverse physiological functions where the distribution of its molecular weight (MW) can influence its behavior and is known to change in response to disease conditions. During inflammation, HA undergoes a covalent modification in which heavy chain subunits of the inter-alpha-inhibitor family of proteins are transferred to its structure, forming heavy chain-HA (HC•HA) complexes. While limited assessments of HC•HA have been performed previously, determining the size distribution of its HA component remains a challenge. Here, we describe a selective method for extracting HC•HA from mixtures that yields material amenable to MW analysis with a solid-state nanopore sensor. After demonstrating the approach in vitro, we validate extraction of HC•HA from osteoarthritic human synovial fluid as a model complex biological matrix. Finally, we apply our technique to pathophysiology by measuring the size distributions of HC•HA and total HA in an equine model of synovitis.

Aggregation and Oligomerization Characterization of ß-Lactoglobulin Protein Using a Solid-State Nanopore Sensor.

Protein aggregation is linked to many chronic and devastating neurodegenerative human diseases and is strongly associated with aging. This work demonstrates that protein aggregation and oligomerization can be evaluated by a solid-state nanopore method at the single molecule level. A silicon nitride nanopore sensor was used to characterize both the amyloidogenic and native-state oligomerization of a model protein ß-lactoglobulin variant A (βLGa). The findings from the nanopore measurements are validated against atomic force microscopy (AFM) and dynamic light scattering (DLS) data, comparing βLGa aggregation from the same samples at various stages. By calibrating with linear and circular dsDNA, this study estimates the amyloid fibrils' length and diameter, the quantity of the βLGa aggregates, and their distribution. The nanopore results align with the DLS and AFM data and offer additional insight at the level of individual protein molecular assemblies. As a further demonstration of the nanopore technique, βLGa self-association and aggregation at pH 4.6 as a function of temperature were measured at high (2 M KCl) and low (0.1 M KCl) ionic strength. This research highlights the advantages and limitations of using solid-state nanopore methods for analyzing protein aggregation.

Solid-State Nanopore Sensors with Enhanced Sensitivity through Nucleic Acid Amplification.

Solid-state nanopores have wide applications in DNA sequencing, energy conversion and storage, seawater desalination, sensors, and reactors due to their high stability, controllable geometry, and a variety of pore-forming materials. Solid-state nanopore sensors can be used for qualitative and quantitative analyses of ions, small molecules, proteins, and nucleic acids. The combination of nucleic acid amplification and solid-state nanopores to achieve trace detection of analytes is gradually attracting attention. This review outlines nucleic acid amplification strategies for enhancing the sensitivity of solid-state nanopore sensors by summarizing the articles published in the past 10 years. The future development prospects and challenges of nucleic acid amplification in solid-state nanopore sensors are discussed. This review helps readers better understand the field of solid-state nanopore sensors. We believe that solid-state nanopore sensors will break through the bottleneck of traditional detection and become a powerful single-molecule detection platform.

Ensuring fair assessment of solid-state nanopore sensors with reporting baseline current

In developing solid-state nanopore sensors for single molecule detection, comprehensive evaluation of the nanopore quality is important. Existing studies typically rely on comparing the noise root mean square or power spectrum density values. Nanopores exhibiting lower noise values are generally considered superior. This evaluation is valid when the single molecule signal remains consistent. However, the signal can vary, as it is strongly related to the solid-state nanopore size, which is hard to control during fabrication consistently. This work emphasized the need to report the baseline current for evaluating solid-state nanopore sensors. The baseline current offers insight into several experimental conditions, particularly the nanopore size. Our experiments show that a nanopore sensor with more noise is not necessarily worse when considering the signal-to-noise ratio (SNR), particularly when the pore size is smaller. Our findings suggest that relying only on noise comparisons can lead to inaccurate evaluations of solid-state nanopore sensors, considering the inherent variability in fabrication and testing setups among labs and measurements. We propose that future studies should include reporting baseline current and sensing conditions. Additionally, using SNR as a primary evaluation tool for nanopore sensors could provide a more comprehensive understanding of their performance.

Enhanced Pulley Effect for Translocation: The Interplay of Electrostatic and Hydrodynamic Forces.

Solid-state nanopore sensors remain a promising solution to the rising global demand for genome sequencing. These single-molecule sensing technologies require single-file translocation for high resolution and accurate detection. In a previous publication, we discovered a hairpin unraveling mechanism, namely, the pulley effect, in a pressure-driven translocation system. In this paper, we further investigate the pulley effect in the presence of pressure-driven fluid flow and an opposing force provided by an electrostatic field as an approach to increase single-file capture probability. A hydrodynamic flow is used to move the polymer forward, and two oppositely charged electrostatic square loops are used to create an opposing force. By optimizing the balance between forces, we show that the single-file capture can be amplified from about 50% to almost 95%. The force location, force strength, and flow rate are used as the optimizing variables.

Our health in our hands: Surface decorated metasurfaces for sensing exhaled breath markers & solid-state nanopores towards complex sample sensing.

The development of highly sensitive, portable and selective sensing technologies capable of detecting disease biomarkers is vital for early detection and monitoring of adverse health conditions like diabetes and multiple sclerosis. ‘Our Health in Our Hands’ (OHIOH) is a research initiative by the Australian National University to develop innovative technologies to this end, and in turn we explore several platforms i.e.: optical metasurfaces, chemiresistive devices, electrochemical sensors and solid-state nanopore sensors. Here we discuss our work on dielectric metasurfaces for label-free sensing. Metasurfaces are nanostructured 2D surfaces that possess intriguing optical properties. With low losses, high quality-factors and electric field extended beyond the surface following light interaction, these metasurfaces are highly sensitive to the refractive index (RI) change induced by placing target molecules on the metasurface. However, metasurfaces lack the selectivity towards a target molecule, that mitigates its real-world applications in complex sample testing. Additionally, they are not sensitive enough for gas sensing due to the ultra-small RI change caused by gas molecule adsorption on bare metasurfaces. To overcome these challenges, we use surface decorated metasurfaces—by organic functional layers and metal textures—for highly sensitive and selective bio and gas sensing to monitor biological samples and volatile organic compounds (VOCs) in breath. In gas sensing, our decorated metasurfaces operate at ambient conditions vs most gas sensors and can be regenerated when VOC is removed making the sensor reusable and pragmatic. We demonstrated the textured metasurfaces elevating the sensor response significantly and sensing acetone down to 0.1% concentration. Moreover, we explore solid-state nanopores to characterize proteins at single-molecular level with bandwidths up to 10 MHz to uncover unattenuated and ultra-fast translocation events towards developing technologies to monitor adverse health conditions.

Recent progress of biosensors for the detection of lung cancer markers.

Lung cancer is one of the most common cancers worldwide and the leading cause of death. Early screening of lung cancer is exceptionally essential for later treatment. Abnormal lung cancer tumor markers are validated to assess their diagnostic utility in non-small cell lung cancer (NSCLC) patients. Therefore, tumor markers can be identified in the early stage of lung cancer through biosensor technology and timely diagnosis. This review discusses cutting-edge methods for detecting various types of lung cancer tumor markers using multiple biosensors. The biosensors working at the molecular level are mainly introduced, which can be divided into three categories according to the types of markers: DNA biosensors, RNA biosensors, and protein biosensors. This review focuses on critical electrochemical methods such as electrochemical impedance spectroscopy (EIS), field-effect transistors (FET), cyclic voltammetry (CV), necessary optical sensors such as surface enhancement Raman spectroscopy (SERS), surface-plasmon resonance (SPR), fluorescence methods, and some novel sensing platforms such as biological nanopore and solid-state nanopore sensors and these sensors detect lung cancer tumor markers, such as microRNA (miRNA), DNA mutations (EGFR, KRAS and p53), DNA methylation, circulating tumor DNA (ctDNA), cytokeratin fragment 21-1 (CYFRA21-1), carcinoembryonic antigen (CEA), matrix metallopeptidase 9 (MMP-9), and vascular endothelial growth factor (VEGF). The advantages and disadvantages of different methods are summarized and prospected on this basis, which provides important insights for developing pioneering optoelectronic biosensors for the early diagnosis of lung cancer.

Metal-Organic Cage as Single-Molecule Carrier for Solid-State Nanopore Analysis.

The ability to detect biomolecules at the single-molecule level is at the forefront of biological research, precision medicine, and early diagnosis. Recently, solid-state nanopore sensors have emerged as a promising technique for label-free and precise diagnosis assay. However, insufficient sensitivity and selectivity for small analytes are a great challenge for clinical diagnosis applications via solid-state nanopores. Here, for the first time, a metal-organic cage, PCC-57, is employed as a carrier to increase the sensitivity and selectivity of solid-state nanopores based on the intrinsic interaction of the nanocage with biomolecules. Firstly, it is found that the carrier itself is undetectable unless bound with the target analytes and used oligonucleotides as linkers to attach PCC-57 and target analytes. Secondly, two small analytes, oligonucleotide conjugated angiopep-2 and polyphosphoric acid, are successfully distinguished using the molecular carrier. Finally, selectivity of nanopore detection is achieved by attaching PCC-57 to oligonucleotide-tailed aptamers, and the human alpha-thrombin sample is successfully detected. It is believed that the highly designable metal-organic cage could serve as a rich carrier repository for a variety of biomolecules, facilitating single-molecule screening of clinically relevant biomolecules based on solid-state nanopores in the future.

Application of Nanopore Sensors for Biomolecular Interactions and Drug Discovery.

Biomolecular interactions, such as protein-protein, protein-nucleic acid, and protein/nucleic acid-ligand interactions, play crucial roles in various cellular signaling and biological processes, and offer attractive therapeutic targets in numerous human diseases. Currently, drug discovery is limited by the low efficiency and high cost of conventional ensemble-averaging-based techniques for biomolecular interaction analysis and high-throughput drug screening. Nanopores are an emerging technology for single-molecule sensing of biomolecules. Owing to the notable merits of single-molecule sensing, nanopore sensors have contributed tremendously to nucleic acid sequencing and disease diagnostics. In this minireview, we summarize the recent developments and outlooks in single-molecule sensing of various biomolecular interactions for drug discovery applications using biological and solid-state nanopore sensors.

Sensing with ion current rectifying solid-state nanopores

This review gives a current opinion on the state of the art of ion rectifying solid-state nanopore sensors, as well as on the recent directions and challenges of the field, focusing in particular on the progress made in the last two to three years. The review explains the phenomenon of ion current rectification in geometrically asymmetric nanopores and the principle of sensing with these systems. Aside from the conventional approach of analyte immobilization onto the pore surface, some intriguing sensing schemes that decouple the pore selectivity from the surface and promise a more flexible sensing approach are also presented. Lastly, an overview of the recent effort towards amplifying the ion currents and the rectification of these sensors is given, followed by a brief discussion of future perspectives for the field. • Decoupling the selectivity of detection from the pore surface can lead to universal sensors. • Maximizing ion current magnitude and rectification ratios is needed to enhance sensitivity. • Various mesopores and micropores have shown enhanced current magnitudes or rectification. • Examples of practical ion current rectifying nanopore sensors highlight real-world potential.

Molecular Dynamics Simulation of a Single Carbon Chain through an Asymmetric Double-Layer Graphene Nanopore for Prolonging the Translocation Time.

In recent years, sensing technology based on nanopores has become one of the trustworthy options for characterization and even identification of a single biomolecule. In nanopore based DNA sequencing technology, the DNA strand in the electrolyte solution passes through the nanopore under an applied bias electric field. Commonly, the ionic current signals carrying the sequence information are difficult to detect effectively due to the fast translocation speed of the DNA strand, so that slowing down the translocation speed is expected to make the signals easier to distinguish and improve the sequencing accuracy. Modifying the nanopore structure is one of the effective methods. Through all-atom molecular dynamics simulations, we designed an asymmetric double-layer graphene nanopore structure to regulate the translocation speed of a single carbon chain. The structure consists of two nanopores with different sizes located on two layers. The simulation results indicate that the asymmetric nanopore structure will affect the chain’s translocation speed and the ionic current value. When the single carbon chain passes from the smaller pore to the larger pore, the translocation time is significantly prolonged, which is about three times as long as the chain passing from the larger pore to the smaller pore. These results provide a new idea for designing more accurate and effective single-molecule solid-state nanopore sensors.

Multi-layered heterostructure nanopore sensor for single biomolecule detection

Nanopores have shown to be a promising tool for fast and real-time sequencing of DNA. Due to the chemical and mechanical robustness and design flexibility, two-dimensional (2D) solid-state nanopore (SSNP) sensors are a contesting candidate for next-generation sequencing devices. However, the major hindrance in detecting DNA nucleotides via 2D SSNPs lies in the low signal-to-noise ratio (SNR) due to thermal noise and the lack of statistically significant current signals. In this regard, we propose a stacked 2D SSNP heterostructure to naturally quench the conformational fluctuations of the translocating biomolecule while obtaining multiple independent electronic in-plane current signals.

Controlled laser-induced fabrication of multichannel solid-state nanopore sensors for studying single-molecule protein dynamics

Nanopores have emerged in recent years as a sensitive and informative sensor for probing biomolecules at a single-molecule level. Nanopore sensing is achieved by recording the ion flux through a small pore submerged in an aqueous electrolyte by applying a voltage bias across it. The essential parameters of an analyte are then deduced through resistive pulses in the ionic current as it is electrokinetically captured and driven through the pore. Solid-state nanopores have recently gained traction as high-resolution sensors for protein characterization because unlike their lipid-embedded biological pore analogs, the solid-state membrane is stable at higher voltages and the pore size can be tailored to be compatible with the studied molecule of choice.

Investigation on the competition of duplex/ G-quadruplex/ i-motif in telomere sequences and c-MYC gene with a solid-state nanopore sensor

Apart from duplex helix, the coexisted noncanonical DNA may be involved in inhibiting DNA replication, and controlling gene expression. The competition of duplex/ G4/ i-motif depends on many factors, including sequence information, external conditions and binding effects. This work looked into the human telomere sequences of different length (hTelo-1, 2) and intramolecular G-rich and C-rich strands (hTelo-3, 4, 5). Nanopore translocation properties suggested that in the presence of their complementary sequences in the mixed K+/Cs+ buffer, G4 was the major species, and G4 from hTelo-2 that holds multiple G-quartets formed slower than shorter hTelo-1. The intramolecular G/C-rich strands hTelo-3, 4, 5 predominantly formed duplex rather than quadruplex under K+ or acidic conditions, indicating that intramolecular quadruplex is less stable than duplex due to the strand strain. Translocation behaviors of mixed proto-oncogene cMYC/c-cMYC illustrated that G4 was formed in both K+ and Cs+, and Cs+ inhibited the duplex formation in K+/Cs+ buffer. The increase of the ratio of Cs+ will decrease the duplexes species. Our work demonstrated the relative stability of duplex/quadruplex under various conditions with distinct strands, which is useful for the investigation of DNA secondary structural dynamics and transition mechanism in biological function regions, and paved the way for the potential of disease-targeted diagnosis.

On the Behavior of Nanoparticles beyond the Nanopore Interface.

Recent advances in solid-state and biological nanopore sensors have produced a deluge of analytical techniques for in situ characterization of bio-nano colloidal dispersions; however, the transport forces governing particle movement into and out of the nanopore are not yet fully understood. Herein, we study the motion of particles outside the smaller opening of an elastomeric size-tunable nanopore and relate this motion to existing transport forces known to act on particles within the pore. Subsequently, we develop a combined optoelectronic approach which allows the comparison of both resistive pulse sensing and single particle tracking-based techniques for particle size characterization and, intriguingly, measurements of the ensemble particle motion induced by a combination of particle electrophoresis as well as pressure-driven and electroosmotic flows through the sensor nanopore. We find evidence suggesting that although bulk fluid flow from the pore tends to drive particle motion, in certain circumstances, electrophoretically driven motion can dominate bulk fluid flow-driven motion even at large distances from the pore opening. By permitting direct observation of the behavior of fluids at the nanopore interface, this approach enables a greater understanding of the transport forces acting on particles as they migrate toward and move through nanopore sensors-with implications for future particle characterization systems and for nanopore methods in general.

Recent advances in integrated solid-state nanopore sensors.

The advent of single-molecule probing techniques has revolutionized the biomedical and life science fields and has spurred the development of a new class of labs-on-chip based on powerful biosensors. Nanopores represent one of the most recent and most promising single molecule sensing paradigms that is seeing increased chip-scale integration for improved convenience and performance. Due to their physical structure, nanopores are highly sensitive, require low sample volume, and offer label-free, amplification-free, high-throughput real-time detection and identification of biomolecules. Over the last 25 years, nanopores have been extensively employed to detect a variety of biomolecules with a growing range of applicatons ranging from nucleic acid sequencing to ultrasensitive diagnostics to single-molecule biophysics. Nanopores, in particular those in solid-state membranes, also have the potential for integration with other technologies such as optics, plasmonics, microfluidics, and optofluidics to perform more complex tasks for an ever-expanding demand. A number of breakthrough results using integrated nanopore platforms have already been reported, and more can be expected as nanopores remain the focus of innovative research and are finding their way into commercial instruments. This review provides an overview of different aspects and challenges of nanopore technology with a focus on chip-scale integration of solid-state nanopores for biosensing and bioanalytical applications.

Author Correction: Solid-state nanopore sensors

Author Correction: Solid-state nanopore sensors