Nanopore Engineering Research Articles

Blobs form during the single-file transport of proteins across nanopores

The transport of biopolymers across nanopores is an important biological process currently under investigation for the rapid analysis of DNA and proteins. While the transport of DNA is generally understood, methods to induce unfolded protein translocation have only recently been discovered (Yu et al., 2023, Sauciuc et al., 2023). Here, we found that during electroosmotically driven translocation of polypeptides, blob-like structures typically form inside nanopores, often obstructing their transport and preventing addressing individual amino acids. This is in contrast with the electrophoretic transport of DNA, where the formation of such structures has not been reported. Comparisons between different nanopore sizes and shapes and modifications by different surface chemistries allowed formulating a mechanism for blob formation. We also show that single-file transport can be achieved by using 1) nanopores that have an entry and an internal diameter smaller than the persistence length of the polymer, 2) nanopores with a nonsticky (i.e., nonaromatic) inner surface, and 3) moderate translocation velocities. These experiments provide a basis for understanding polypeptide transport under confinement and for improving the design and engineering of nanopores for protein analysis.

Beta-Barrel Nanopores as Diagnostic Sensors: An Engineering Perspective.

Biological nanopores are ultrasensitive and highly attractive platforms for disease diagnostics, including the sequencing of viral and microbial genes and the detection of biomarkers and pathogens. To utilize biological nanopores as diagnostic sensors, they have been engineered through various methods resulting in the accurate and highly sensitive detection of biomarkers and disease-related biomolecules. Among diverse biological nanopores, the β-barrel-containing nanopores have advantages in nanopore engineering because of their robust structure, making them well-suited for modifications. In this review, we highlight the engineering approaches for β-barrel-containing nanopores used in single-molecule sensing for applications in early diagnosis and prognosis. In the highlighted studies, β-barrel nanopores can be modified by genetic mutation to change the structure; alter charge distributions; or add enzymes, aptamers, and protein probes to enhance sensitivity and accuracy. Furthermore, this review discusses challenges and future perspectives for advancing nanopore-based diagnostic sensors.

Electrical Resistivity Modification of Electrodeposited Mo and Mo–Co Nanowires for Interconnect Applications

Achieving historically anticipated improvement in the performance of integrated circuits is challenging, due to the increasing cost and complexity of the required technologies with each new generation. To overcome this limitation, the exploration and development of novel interconnect materials and processes are highly desirable in the microelectronics field. Molybdenum (Mo) is attracting attention as an advanced interconnect material due to its small resistivity size effect and high cohesive energy; however, effective processing methods for such materials have not been widely investigated. Here, we investigate the electrochemical behavior of ions in the confined nanopores that affect the electrical properties and microstructures of nanoscale Mo and Mo–Co alloys prepared via template-assisted electrodeposition. Additives in an electrolyte allow the deposition of extremely pure metal materials, due to their interaction with metal ions and nanopores. In this study, boric acid and tetrabutylammonium bisulfate (TBA) were added to an acetate bath to inhibit the hydrogen evolution reaction. TBA accelerated the reduction of Mo at the surface by inducing surface conduction on the nanopores. Metallic Mo nanowires with a 130 nm diameter synthesized through high-aspect-ratio nanopore engineering exhibited a resistivity of (63.0 ± 17.9) μΩ·cm. We also evaluated the resistivities of Mo–Co alloy nanowires at various compositions toward replacing irreducible conventional barrier/liner layers. An intermetallic compound formed at a Mo composition of 28.6 at%, the resistivity of the Mo–Co nanowire was (58.0 ± 10.6) μΩ·cm, indicating its superior electrical and adhesive properties in comparison with those of conventional barriers such as TaN and TiN. Furthermore, density functional theory and non-equilibrium Green’s function calculations confirmed that the vertical resistance of the via structure constructed from Mo-based materials was 21% lower than that of a conventional Cu/Ta/TaN structure.

Functional Nanopore Screen: A Versatile High-Throughput Assay to Study and Engineer Protein Nanopores in Escherichia coli.

Nanopores comprise a versatile class of membrane proteins that carry out a range of key physiological functions and are increasingly developed for different biotechnological applications. Yet, a capacity to study and engineer protein nanopores by combinatorial means has so far been hampered by a lack of suitable assays that combine sufficient experimental resolution with throughput. Addressing this technological gap, the functional nanopore (FuN) screen now provides a quantitative and dynamic readout of nanopore assembly and function in the context of the inner membrane of Escherichia coli. The assay is based on genetically encoded fluorescent protein sensors that resolve the nanopore-dependent influx of Ca2+ across the inner membrane of E. coli. Illustrating its versatile capacity, the FuN screen is first applied to dissect the molecular features that underlie the assembly and stability of nanopores formed by the S2168 holin. In a subsequent step, nanopores are engineered by recombining the transmembrane module of S2168 with different ring-shaped oligomeric protein structures that feature defined hexa-, hepta-, and octameric geometries. Library screening highlights substantial plasticity in the ability of the S2168 transmembrane module to oligomerize in alternative geometries, while the functional properties of the resultant nanopores can be fine-tuned through the identity of the connecting linkers. Overall, the FuN screen is anticipated to facilitate both fundamental studies and complex nanopore engineering endeavors with many potential applications in biomedicine, biotechnology, and synthetic biology.

The sorting and characterizing of engineered nanopore sensors

In the past couple of decades, the nanopore field has used transmembrane proteins as scaffolds for functional biosensors. These sensors have proven highly sensitive and accurate. Yet, the engineering of nanopores requires a strenuous procedure to characterize their function. This sorting process has become more relevant, as the field expands to sensing without steric hindrance of the nanopore confinement. We address this problem by introducing a detailed procedure with key indicators to determine a nanopore’s functionality as a sensor before thorough testing.

Influence of Nitrogen and Boron on the magnetization of nanoporous graphene : A first-principle investigation

Spin-polarized density functional theory (DFT) calculations with the generalized gradient approximation (GGA) of the Perdew–Burke–Ernzerhof (PBE) flavour, i.e., GGA-PBE has been performed to study the interactions of nitrogen (N) and boron (B) dopants with model graphene nanopores. We study the formation energy, magnetic and electronic properties of nanoporous graphene containing varying concentration of N and B as dopants. Magnetism in nanoporous graphene is induced by σ and π dangling bonds due to missing carbon atoms following the creation of the nanopores. Here, we show that progressive substitution of carbon atoms carrying dangling bonds, with either B or N atoms, results to the progressive decrease in the magnetic moment of the doped nanopore defect structures. In the N doped nanopore, for every single N substituting the C atom carrying dangling bonds at the perimeter of the nanopore, the magnetic moment reduces by about 1 μB. The magnetization of the nanopore is completely annihilated after full substitution of the carbon atoms carrying dangling bonds. In the case of B doped nanopore however, the magnetic moment decreases rapidly and vanishes before all the C atoms carrying dangling bonds are substituted. Löwdin charge analysis shows that N doping leads to charge transfer from the C to the N atom while B doping leads to charge transfer from B to the neighbouring C atoms. Thus, the electron withdrawing characteristics of N atom contributes to reduce the magnetization. On the other hand, B atom donates its charges which contribute to saturate the C dangling bonds and thus reduces the magnetization. Our work shows the possibility of achieving finite magnetic moment in graphene via a combination of nanopore engineering, B or N doping. Such nanoscale control of magnetization in a low-dimensional structure such as graphene, has important consequence for spintronics and related applications.

In situ nanopores enrichment of Mesh-like palladium nanoplates for bifunctional fuel cell reactions: A joint etching strategy

Two-dimensional (2D) nanomaterials with nanopore display an enhancement effect on electrocatalysis behavior, whereas the nanopore engineering for 2D nanocatalysts remains an insurmountable challenge. We advance the synthesis of multilayer Pd nanoplates (Pd NPs) and two types of meshy nanoplates (Pd LMNPs/MNPs) with escalating nanopores from none and sparse to porous. Specially, an in situ nanopore enrichment on these Pd nanoplates hinges on a joint etching strategy with integrated manipulation of reaction kinetics. The optimized Pd MNPs exhibit exceptional oxygen reduction reaction performance, owing to the enhanced intermediates protonation on Pd site neighboring nanopore, which has been elucidated by density functional theory calculations. In addition, Pd MNPs also deliver excellent performances in fuel cell anodic reactions, including ethanol oxidation reaction and formic acid oxidation reaction. This study highlights a new strategy for in situ nanopores engineering, providing a prospect for designing superior nanocatalysts.

Quantitative insights into the growth mechanisms of nanopores in hexagonal boron nitride

The formation of nanopores under electron irradiation is an ideal process to quantify chemical bonds in two-dimensional materials. Nowadays, high-resolution transmission electron microscopy (HRTEM) allows investigating such nucleation and growth phenomena with incomparable spatial and temporal resolution. Moreover, theoretical calculations are usually exploited to confirm characteristic features of these atomic-scale observations. Nevertheless, the full understanding of the ejection mechanisms of atoms requires a detailed investigation of the interplay between the very dynamic edge structure of expanding nanopores and the displacement energy of edge atoms $({E}_{D})$. Here, the dynamics of triangular nanopores in hexagonal boron nitride (h-BN) under various electron dose rates was followed by aberration-corrected HRTEM with high temporal resolution to provide new in situ insights into their growth processes. We reveal that the ejection of atomic pairs is an elemental mechanism that considerably speeds up the expansion of nanopores. Atomic-scale calculations were exploited to quantify the structure-dependent ${E}_{D}$ of all the ejected edge atoms. They revealed strong variations of this threshold energy during the growth processes. This quantitative study reconciles theoretical and experimental measurements of the ejection rate of atoms in h-BN under electron irradiation, which is essential for nanopore engineering in this atomically thin membrane.

Electrical recognition of the twenty proteinogenic amino acids using an aerolysin nanopore.

Efforts to sequence single protein molecules in nanopores1-5 have been hampered by the lack of techniques with sufficient sensitivity to discern the subtle molecular differences among all twenty amino acids. Here we report ionic current detection of all twenty proteinogenic amino acids in an aerolysin nanopore with the help of a short polycationic carrier. Application of molecular dynamics simulations revealed that the aerolysin nanopore has a built-in single-molecule trap that fully confines a polycationic carrier-bound amino acid inside the sensing region of the aerolysin. This structural feature means that each amino acid spends sufficient time in the pore for sensitive measurement of the excluded volume of the amino acid. We show that distinct current blockades in wild-type aerolysin can be used to identify 13 of the 20 natural amino acids. Furthermore, we show that chemical modifications, instrumentation advances and nanopore engineering offer a route toward identification of the remaining seven amino acids. These findings may pave the way to nanopore protein sequencing.

Intensification of Pseudocapacitance by Nanopore Engineering on Waste-Bamboo-Derived Carbon as a Positive Electrode for Lithium-Ion Batteries.

Nanoporous carbon, including redox-active functional groups, can be a promising active electrode material (AEM) as a positive electrode for lithium-ion batteries owing to its high electrochemical performance originating from the host-free surface-driven charge storage process. This study examined the effects of the nanopore size on the pseudocapacitance of the nanoporous carbon materials using nanopore-engineered carbon-based AEMs (NE-C-AEMs). The pseudocapacitance of NE-C-AEMs was intensified, when the pore diameter was ≥2 nm in a voltage range of 1.0~4.8 V vs Li+/Li under the conventional carbonate-based electrolyte system, showing a high specific capacity of ~485 mA·h·g−1. In addition, the NE-C-AEMs exhibited high rate capabilities at current ranges from 0.2 to 4.0 A·g−1 as well as stable cycling behavior for more than 300 cycles. The high electrochemical performance of NE-C-AEMs was demonstrated by full-cell tests with a graphite nanosheet anode, where a high specific energy and power of ~345 Wh·kg−1 and ~6100 W·Kg−1, respectively, were achieved.

Theranostic nanomedicine by surface nanopore engineering

Theranostic nanomedicine that integrates diagnostic and therapeutic agents into one nanosystem has gained considerable momentum in the field of cancer treatment. Among diverse strategies for achieving theranostic capabilities, surface-nanopore engineering based on mesoporous silica coating has attracted great interest because of their negligible cytotoxicity and chemically active surface that can be easily modified to introduce various functional groups (e.g., −COOH, −NH2, −SH, etc.) via silanization, which can satisfy various requirements of conjugating biological molecules or functional nanoparticles. In addition, the nanopore-engineered biomaterials possess large surface area and high pore volume, ensuring desirable loading of therapeutic guest molecules. In this review, we comprehensively summarize the synthetic procedure/paradigm of nanopore engineering and further broad theranostic applications. Such nanopore-engineering strategy endows the biocompatible nanocomposites (e.g., Au, Ag, graphene, upconversion nanoparticles, Fe3O4, MXene, etc.) with versatile functional moieties, which enables the development of multifunctional nanoplatforms for multimodal diagnostic bio-imaging, photothermal therapy, photodynamic therapy, targeted drug delivery, synergetic therapy and imaging-guided therapies. Therefore, mesoporous silica-based surface-nanopore engineering integrates intriguing unique features for broadening the biomedical applications of the single mono-functional nanosystem, facilitating the development and further clinical translation of theranostic nanomedicine.

Cancer Therapy: Surface Nanopore Engineering of 2D MXenes for Targeted and Synergistic Multitherapies of Hepatocellular Carcinoma (Adv. Mater. 25/2018)

Cancer Therapy: Surface Nanopore Engineering of 2D MXenes for Targeted and Synergistic Multitherapies of Hepatocellular Carcinoma (Adv. Mater. 25/2018)

Surface Nanopore Engineering of 2D MXenes for Targeted and Synergistic Multitherapies of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common and deadly gastrointestinal malignancies. Given its insensitivity to traditional systematic chemotherapy, new therapeutic strategies for efficient HCCs treatment are urgently needed. Here, the development of a novel 2D MXene-based composite nanoplatform for highly efficient and synergistic chemotherapy and photothermal hyperthermia against HCC is reported. A surface-nanopore engineering strategy is developed for the MXenes' surface functionalization, which achieves the uniform coating of a thin mesoporous-silica layer onto the surface of 2D Ti3 C2 MXene (Ti3 C2 @mMSNs). This strategy endows MXenes with well-defined mesopores for on-demand drug release/delivery, enhanced hydrophilicity/dispersity, and abundant surface chemistry for targeting engineering. Systematic in vitro and in vivo evaluations have demonstrated the high active-targeting capability of arginine-glycine-aspartic acid (RGD)-targeting Ti3 C2 @mMSNs into tumor, and the synergistic chemotherapy (contributed by the mesoporous shell) and photothermal hyperthermia (contributed by the Ti3 C2 MXene core) completely eradicate the tumor without obvious reoccurrence. This work not only provides a novel strategy for efficiently combating HCC by developing MXene-based composite nanoplatforms, but also paves a new way for extending the biomedical applications of MXenes by surface-nanopore engineering.

Porphyrin-Assisted Docking of a Thermophage Portal Protein into Lipid Bilayers: Nanopore Engineering and Characterization

Porphyrin-Assisted Docking of a Thermophage Portal Protein into Lipid Bilayers: Nanopore Engineering and Characterization

Remote Activation of a Nanopore for High-Performance Genetic Detection Using a pH Taxis-Mimicking Mechanism.

Aerolysin protein pore has been widely used for sensing peptides and proteins. However, only a few groups explored this nanopore for nucleic acids detection. The challenge is the extremely low capture efficiency for nucleic acids (>10 bases), which severely lowers the sensitivity of an aerolysin-based genetic biosensor. Here we reported a simple and easy-to-operate approach to noncovalently transform aerolysin into a highly nucleic acids-sensitive nanopore. Through a remote pH-modulation mechanism, we simply lower the pH on one side of the pore, then aerolysin is immediately "activated" and enabled to capture target DNA/RNA efficiently from the opposite side of the pore. This mechanism also decelerates DNA translocation, a desired property for sequencing and gene detection, allowing temporal separation of DNAs in different lengths. This method provides insight into the nanopore engineering for biosensing, making aerolysin applicable in genetic and epigenetic detections of long nucleic acids.

Porphyrin-Assisted Docking of a Thermophage Portal Protein into Lipid Bilayers: Nanopore Engineering and Characterization.

Nanopore-based sensors for nucleic acid sequencing and single-molecule detection typically employ pore-forming membrane proteins with hydrophobic external surfaces, suitable for insertion into a lipid bilayer. In contrast, hydrophilic pore-containing molecules, such as DNA origami, have been shown to require chemical modification to favor insertion into a lipid environment. In this work, we describe a strategy for inserting polar proteins with an inner pore into lipid membranes, focusing here on a circular 12-subunit assembly of the thermophage G20c portal protein. X-ray crystallography, electron microscopy, molecular dynamics, and thermal/chaotrope denaturation experiments all find the G20c portal protein to have a highly stable structure, favorable for nanopore sensing applications. Porphyrin conjugation to a cysteine mutant in the protein facilitates the protein's insertion into lipid bilayers, allowing us to probe ion transport through the pore. Finally, we probed the portal interior size and shape using a series of cyclodextrins of varying sizes, revealing asymmetric transport that possibly originates from the portal's DNA-ratchet function.

Design and Engineering of Nanopores with Emergent Functions

Engineering new functions into existing assemblies is a first step to build artificial biological systems. Further, the emergence of an enzymatic function can reveal functional insights and allows engineering biological systems with enhanced properties. Biological nanopores with known structure are ideal building elements for this task because they have a robust assembly that allows precise engineering. For example, we recently incorporated DNA molecules atop a ClyA nanopore to build artificial transporters that are able to selectively shuttle DNA or proteins across a lipid membrane. Nanopores have additional advantages, as the ionic flux through the nanopore can be exploited to recognize molecules or study biological and chemical processes at the single-molecule level.Here we engineered an alpha hemolysin nanopore to operate as GroES, a co-chaperonin that in complex with GroEL forms a two-stroke protein-folding nanomachine. The new function was introduced bottom-up by adding modular elements into an unrelated globular structure, suggesting a possible path during GroEL-GroES evolution in which the co-chaperonin function appeared sequentially by incorporating hydrophobic polypeptide loops into pre-existing protein assemblies. The binding of GroEL to individual GroES-nanopores prompted large changes to the unitary nanopore current, most likely reflecting the allosteric transitions of the chaperonin nanomachine. In the presence of unfolded proteins the pattern of current transitions changed, suggesting a possible mechanism in which the free energy of ATP binding and hydrolysis is expended only when substrate proteins are occupied.

Nanostructural Engineering of Nanoporous Anodic Alumina for Biosensing Applications.

Modifying the diameter of the pores in nanoporous anodic alumina opens new possibilities in the application of this material. In this work, we review the different nanoengineering methods by classifying them into two kinds: in situ and ex situ. Ex situ methods imply the interruption of the anodization process and the addition of intermediate steps, while in situ methods aim at realizing the in-depth pore modulation by continuous changes in the anodization conditions. Ex situ methods permit a greater versatility in the pore geometry, while in situ methods are simpler and adequate for repeated cycles. As an example of ex situ methods, we analyze the effect of changing drastically one of the anodization parameters (anodization voltage, electrolyte composition or concentration). We also introduce in situ methods to obtain distributed Bragg reflectors or rugate filters in nanoporous anodic alumina with cyclic anodization voltage or current. This nanopore engineering permits us to propose new applications in the field of biosensing: using the unique reflectance or photoluminescence properties of the material to obtain photonic barcodes, applying a gold-coated double-layer nanoporous alumina to design a self-referencing protein sensor or giving a proof-of-concept of the refractive index sensing capabilities of nanoporous rugate filters.

Three-dimensional structural engineering of nanoporous alumina by controlled sprinkling of an electrolyte on a porous anodic alumina (PAA) template

The significant effect of anodization current on the morphology of nanopores encourages us to consider a novel method for engineering these nanostructures. In this paper we study a controlled anodization process using controlled electrolyte stirring. This is done using an electrical pump with variable speed for sprinkling an electrolyte on a membrane. The variable voltage applied to the pump can control the temperature gradient and diffusion of the ions which produce the anodization current. Here we show the significant role of pumping and how this effect can be used in the engineering of nanopores as a three-dimensional nanostructure. These experiments are carried out for a variety of anodization voltages and various electrolyte concentrations, and thus we investigate how these parameters in the vicinity of pumping can control the porosity of the template in three dimensions.