Macromolecular Crowding Facilitates ssDNA Capture within Biological Nanopores: Role of Size Variation and Solution Heterogeneity.

Genetic sequencing is a vital process that requires the transport of charged nucleic acids through transmembrane nanopores. Single-molecule studies show that macromolecular bulk crowding facilitates the capture of these polymers, leading to a high throughput of nanopore sensors. Motivated by these observations, a minimal discrete-state stochastic framework was developed to describe the role of poly(ethylene glycol) (PEG) crowders in varying concentrations in the transport of ssDNA through α-hemolysin nanopores. This theory suggested that the cooperative partitioning of polycationic PEGs controls the capture of ssDNA due to underlying electrostatic interactions. Herein, we investigate the impact of the size variation of PEGs on the capture event. Even though larger crowders attract ssDNA strongly to enhance its capture, our results show that considerable cooperative partitioning of PEGs is also required to achieve high interevent frequency. The exact analytical results are supported by existing single-molecule studies. Since real cellular conditions are heterogeneous, its influence on the ssDNA capture rate is studied by introducing a binary mixture of crowders. Our results indicate that the "polymer-pushing-polymer" concept possibly affects the capture rate depending on the mixture composition. These new findings provide valuable insights into the microscopic mechanism of the capture process, which eventually allows for accurate genome sequencing in crowded solutions.

Capacidad de procesamiento de los nanofiltros empleados en la purificación de la proteína recombinante del dominio de unión al receptor del coronavirus tipo 2 del síndrome respiratorio agudo severo / Processability of nanofilters used in the purification of the recombinant receptor-binding domain protein of severe acute respiratory syndrome type 2 coronavirus

En el Centro de Inmunología Molecular se producen el ingrediente farmacéutico activo y la materia prima biológica empleados para la formulación de las vacunas SOBERANAS®. El antígeno de estas vacunas es la proteína del dominio de unión al receptor del coronavirus tipo 2 del síndrome respiratorio agudo severo. La producción de esta proteína recombinante se basa en el cultivo de células de ovario de hámster chino en biorreactores tipo tanque agitado. El proceso tecnológico a escala industrial consta de varias etapas: preparación de medios de cultivo y soluciones, fermentación, clarificación de sobrenadante y purificación. En los procesos biotecnológicos derivados de líneas celulares de origen animal, la contaminación viral endógena o adventicia constituye un riesgo potencial. Por tal motivo, en el proceso de purificación se emplea un paso específico para la remoción viral mediante la nanofiltración. Los nanofiltros empleados son materiales desechables que influyen significativamente en el costo del proceso. Actualmente se desconoce la capacidad de procesamiento de los nanofiltros en el proceso de purificación en cuestión, siendo el objetivo de la presente investigación con vistas a reducir los costos de producción. Se determinó la capacidad de procesamiento de los filtros Virosart CPV, siendo de 239,74 g/m2 (71,67 por ciento de saturación) y 1.259 g/m2 (67,82 por ciento de saturación) para la especie dímero y la mezcla, respectivamente. Se determinó la disminución del costo de producción de la etapa de nanofiltración, que representa una disminución del 54,85 por ciento del costo de filtración de la especie dímero y un 25 por ciento de la mezcla(AU)

The active pharmaceutical ingredient and the biological raw material, used for the formulation of the SOBERANA® vaccines, are produced at the Molecular Immunology Center. The antigen of these vaccines is the receptor-binding domain protein of the severe acute respiratory syndrome type 2 coronavirus. The production of this recombinant protein is based on the culture of Chinese hamster ovary cells in stirred tank bioreactors. The technological process on an industrial scale consists of several stages: preparation of culture media and solutions, fermentation, clarification of supernatant and purification. In biotechnological processes derived from cell lines of animal origin, endogenous or adventitious viral contamination is a potential risk. For this reason, a specific step for viral removal by nanofiltration is used in the purification process. The nanofilters used are disposable materials that significantly influence the cost of the process. The processing capacity of the nanofilters in the purification process in question is currently unknown, being the objective of the present investigation with a view to reducing production costs. The processing capacity of the Virosart CPV filters was determined to be 239.74 g/m2 (71.67percent saturation) and 1,259 g/m2 (67.82percent saturation) for the dimer species and the mixture, respectively. The decrease in the production cost of the nanofiltration stage was determined, representing a 54.85percent decrease in the filtration cost for the dimer species and a 25percent decrease for the mixture(AU)

Glass Capillary-Based Nanopores for Single Molecule/Single Cell Detection.

A glass capillary-based nanopore (G-nanopore), due to its tapered tip, easy tunability in orifice size, and especially its flexible surface modifications that can be tailored to effectively capture and enhance the ionic current signal of single entities (single molecules, single cells, and single particles), offers a powerful and nanoconfined sensing platform for diverse biological measurements of single cells and single molecules. Compared with other artificial two-dimensional solid-state nanopores, its conical tip and high spatial and temporal resolution characteristics facilitate noninvasive single molecule and selected area (subcellular) single cell detections (e.g., DNA mutations, highly expressed proteins, and small molecule markers that reflect the change characteristics of the tumor), as a small G-nanopore (≤100 nm) does negligible damage to cell functions and cell membrane integrity when inserted through the cell membrane. In this brief review, we summarize the preparation of G-nanopores and discuss the advantages of them as solid-state sensing platforms for single molecule and single cell detection applications as well as for cancer diagnosis and treatment applications. We also describe the current bottlenecks that limit the widespread use of G-nanopores in clinical applications and provide an outlook on future developments. The brief review will provide the reader with a quick survey of this field and facilitate the rapid development of a G-nanopore sensing platform for future tumor diagnosis and personalized medicine based on single-molecule/single-cell bioassay.

Equilibrium and Kinetic Study of l- and d-Valine Adsorption in Supramolecular-Templated Chiral Mesoporous Materials.

Adsorption kinetic studies are conducted to investigate the potential to use chiral mesoporous materials nanoporous guanosine monophosphate material-1 (NGM-1) and nanoporous folic acid material-1 (NFM-1) for the enantiomeric separation of l- and d-valine. A pseudo-second-order (PSO) kinetic model is applied to test the experimental adsorption equilibrium isotherms, according to both the Langmuir and Freundlich models and the characteristic parameters for each model are determined. The calcined versions of both NGM-1 and NFM-1 fit the Langmuir model with maximum sorption capacities of 0.36 and 0.26 g/g for the preferred adsorption enantiomers, d-valine and l-valine, respectively. Experimental results and the analysis of adsorption models suggest a strong adsorbate-adsorbent interaction, and the formation of a monolayer of tightly packed amino acid on the internal mesopore surface for the preferred enantiomers.

Design, assembly, and characterization of membrane-spanning DNA nanopores.

DNA nanopores are bio-inspired nanostructures that control molecular transport across lipid bilayer membranes. Researchers can readily engineer the structure and function of DNA nanopores to synergistically combine the strengths of DNA nanotechnology and nanopores. The pores can be harnessed in a wide range of areas, including biosensing, single-molecule chemistry, and single-molecule biophysics, as well as in cell biology and synthetic biology. Here, we provide a protocol for the rational design of nanobarrel-like DNA pores and larger DNA origami nanopores for targeted applications. We discuss strategies for the pores' chemical modification with lipid anchors to enable them to be inserted into membranes such as small unilamellar vesicles (SUVs) and planar lipid bilayers. The procedure covers the self-assembly of DNA nanopores via thermal annealing, their characterization using gel electrophoresis, purification, and direct visualization with transmission electron microscopy and atomic force microscopy. We also describe a gel assay to determine pore-membrane binding and discuss how to use single-channel current recordings and dye flux assays to confirm transport through the pores. We expect this protocol to take approximately 1 week to complete for DNA nanobarrel pores and 2-3 weeks for DNA origami pores.

Polymer escape through a three dimensional double-nanopore system.

We study the escape dynamics of a double-stranded DNA (dsDNA) through an idealized double nanopore geometry subject to two equal and opposite forces (tug-of-war) using Brownian dynamics (BD) simulation. In addition to the geometrical restrictions imposed on the cocaptured dsDNA segment in between the pores, the presence of tug-of-war forces at each pore results in a variation of the local chain stiffness for the segment of the chain in between the pores, which increases the overall stiffness of the chain. We use the BD simulation results to understand how the intrinsic chain stiffness and the tug-of-war forces affect the escape dynamics by monitoring the local chain persistence length ℓp, the residence time of the individual monomers W(m) in the nanopores, and the chain length dependence of the escape time ⟨τ⟩ and its distribution. Finally, we generalize the scaling theory for the unbiased single nanopore translocation for a fully flexible chain for the escape of a semi-flexible chain through a double nanopore in the presence of tug-of-war forces. We establish that the stiffness dependent part of the escape time is approximately independent of the translocation mechanism so that ⟨τ⟩∼ℓp 2/D+2, and therefore, the generalized escape time for a semi-flexible chain can be written as ⟨τ⟩=ANαℓp 2/D+2. We use the BD simulation results to compare the predictions of the scaling theory. Our numerical studies supplemented by scaling analysis provide fundamental insights to design new experiments where a dsDNA moves slowly through a series of graphene nanopores.

Native Structure of the Plant Cell Wall Utilized for Top-Down Assembly of Aligned Cellulose Nanocrystals into Micrometer-Sized Nanoporous Particles.

Despite their sustainable appeal, biomass components are currently undervalued in nanotechnology because means to control the assembly of bio-based nanoparticles are lagging behind the synthetic counterparts. Here, micrometer-sized particles consisting of aligned cellulose nanocrystals (CNCs) are prepared by crosslinking cellulose in cotton linter fibers that are prehydrolyzed with gaseous HCl, resulting in chemical cleavage necessary for CNC formation but retaining the morphology of the native fibers. That way, the intrinsic alignment of cellulose microfibrils within the fiber cell wall can be retained and utilized for top-down CNC alignment. Subsequent crosslinking with citric acid cements the alignment and preserves it, following the dispersion of CNCs trapped end-to-end, connected, and crosslinked within the colloidally stable micrometer-sized particles. Furthermore, thermoporosimetry and cryogenic transmission electron microscopy (Cryo TEM) shows that the particles possess mainly nanoporous (<2 nm) character in water. The approach challenges the current paradigm of predominantly bottom-up methods for nanoparticle assembly.

Flexible Nanoporous Template for the Design and Development of Reusable Anti-COVID-19 Hydrophobic Face Masks.

Since the outbreak of the severe respiratory disease caused by the novel coronavirus (COVID-19), the use of face masks has become ubiquitous worldwide to control the rapid spread of this pandemic. As a result, the world is currently facing a face mask shortage, and some countries have placed limits on the number of masks that can be bought by each person. Although the surgical grade N95 mask provides the highest level of protection currently available, its filtration efficiency for sub-300 nm particles is around 85% due to its wider pore size (∼300 nm). Because the COVID-19 virus shows a diameter of around 65-125 nm, there is a need for developing more efficient masks. To overcome these issues, we demonstrate the development of a flexible, nanoporous membrane to achieve a reusable N95 mask with a replaceable membrane and enhanced filtration efficiency. We first developed a flexible nanoporous Si-based template on a silicon-on-insulator wafer using KOH etching and then used the template as a hard mask during a reactive ion etching process to transfer the patterns onto a flexible and lightweight (<0.12 g) polymeric membrane. Pores with sizes down to 5 nm were achieved with a narrow distribution. Theoretical calculations show that airflow rates above 85 L/min are possible through the mask, which confirms its breathability over a wide range of pore sizes, densities, membrane thicknesses, and pressure drops. Finally, the membrane is intrinsically hydrophobic, which contributes to antifouling and self-cleaning as a result of droplets rolling and sliding on the inclined mask area.

Durable, Stable, and Functional Nanopores Decorated by Self-Assembled Dipeptides.

Nanopores have become an important tool for the detection and analysis of molecules at the single-molecule level. Surface modification of solid-state nanopores can improve their durability and efficiency. Peptides are ideal for surface modifications as they allow tailoring of multiple properties by a rational design of their sequence. Here, silicon nitride nanopores were coated by a dipeptide layer where a l-3,4-dihydroxyphenylalanine (DOPA) residue is the anchoring element and the other amino acid moiety is the functional element. DOPA binds tightly to many types of surfaces and allows a one-step functionalization of surfaces by simple immersion. As a result, the lifetime of coated nanopores increased from hours to months and the current-stability has significantly improved with respect to uncoated pores. This improvement is achieved by controlling the surface wettability and charge. Peptide-coated nanopores can be utilized as sensitive sensors that can be adjusted based on the choice of the functional moiety of the coated peptide. In addition, the coating slows down dsDNA translocation because of the DNA interaction with the pore coating.

DNA nanotechnology assisted nanopore-based analysis.

Nanopore technology is a promising label-free detection method. However, challenges exist for its further application in sequencing, clinical diagnostics and ultra-sensitive single molecule detection. The development of DNA nanotechnology nonetheless provides possible solutions to current obstacles hindering nanopore sensing technologies. In this review, we summarize recent relevant research contributing to efforts for developing nanopore methods associated with DNA nanotechnology. For example, DNA carriers can capture specific targets at pre-designed sites and escort them from nanopores at suitable speeds, thereby greatly enhancing capability and resolution for the detection of specific target molecules. In addition, DNA origami structures can be constructed to fulfill various design specifications and one-pot assembly reactions, thus serving as functional nanopores. Moreover, based on DNA strand displacement, nanopores can also be utilized to characterize the outputs of DNA computing and to develop programmable smart diagnostic nanodevices. In summary, DNA assembly-based nanopore research can pave the way for the realization of impactful biological detection and diagnostic platforms via single-biomolecule analysis.

Ultrasensitive and regenerable nanopore sensing based on target induced aptamer dissociation.

For ionic current rectification (ICR) based sensing, nanopore functionalizations are mostly designed for directly binding target molecules to generate detectable signals from surface charge variation. However, this strategy is highly dependent on the charge difference between the captured molecules and surface functionalization layers, which will increase the nanopore design difficulty and subsequently limit the nanopore applicability. Another key challenge for ICR based sensing is the nanopore regenerability that is critical if online monitoring or repeated determination needs to be performed with one sensor. Though some types of nanopore regeneration have been realized on some specific targets or with harsh conditions, it is still highly favored to develop a regenerability using mild conditions for various targets. To address these two challenges, we developed a novel and universal sensing strategy for aptamer-functionalized nanopore that can be easily regenerated after each usage without any harsh conditions and independent of target molecule charge or size for ICR based nanopore sensing. Ochratoxin A (OTA) was used as a model analyte and its corresponding aptamer partially hybridized with the pre-immobilized complementary DNA (cDNA) onto the nanopore inner surface. We demonstrated that the recognition and conjugation of OTA with its aptamer resulted in rectified ionic current variations due to the dissociation between the OTA aptamer and its partially paired cDNA. The performance of this nanopore sensor including sensitivity, selectivity, regenerability, and applicability was characterized using rectified ionic current. This nanopore sensing strategy will provide a promising platform for extensive targets and online sensing applications.

Polynucleotide differentiation using hybrid solid-state nanopore functionalizing with α-hemolysin.

We report results from full atomistic molecular dynamics simulations on the properties of biomimetic nanopores. This latter result was obtained through the direct insertion of an α-hemolysin protein inside a hydrophobic solid-state nanopore. Upon translocation of different DNA strands, we demonstrate here that the theoretical system presents the same discrimination properties as the experimental one obtained previously. This opens an interesting way to promote the stability of a specific protein inside a solid nanopore to develop further biomimetic applications for DNA or protein sequencing.

Hybridization chain reaction (HCR) for amplifying nanopore signals.

Circulating tumor DNA (ctDNA) in the blood is an important biomarker for noninvasive diagnosis, assessment, prediction and treatment of cancer. However, sensing performance of solid nanopore is limited by the fast kinetics of small DNA targets and unmatched dimensions. Here, we combines hybridization chain reaction (HCR) with nanopore detection to translate the presence of a small DNA target to characteristic nanopore signals of a long nicked DNA polymer. The amplification of nanopore signals obtained by HCR not only overcomes the functional limitation of solid nanopore, but also significantly elevates both selectivity and signal-to-noise ratio, which allows to detect ctDNA at a detection limit of 2.8 fM (S/N = 3) and the single-base resolution. Furthermore, the proposed method can apply in detection of ctDNA of KRAS G12DM in serum sample.

A novel electrochemical sensor for determination of hydroxyl radicals in living cells by coupling nanoporous gold layer with self-assembled 6-(Ferrocenyl) hexanethiol.

The detection of hydroxyl radicals (•OH) in live cells is significant to study its physiological and pathological roles, while it is full of challenge due to the extremely low concentration and short lifetime of •OH. Herein, we have developed a novel electrochemical sensor based on 6-(Ferrocenyl) hexanethiol (6-FcHT) self-assembled nanoporous gold layer (NPGL) modified GE (6-FcHT/NPGL/GE), which can detect the release of •OH from living cells with high sensitivity and selectivity. The superior sensitivity can stem from the unique porous architecture of NPGL, which enlarged electrode surface area and expedited electron transportation during electrochemical reactions. Additionally, NPGL provides more active binding sites for the assembly of capture agent (6-FcHT) of •OH, thus ensuring high selectivity. For comparison, 6-FcHT/GE was applied to detect •OH, and the obtained sensitivity was 0.0305 mA nM-1 and detection limit was 0.133 nM in the linear range of 0.4 nM-70 nM. After modification of NPGL, the sensitivity of 6-FcHT/NPGL/GE to the •OH response was increased to 0.1364 mA nM-1, detection limit was reduced to 0.316 pM and the linear range was extended from 1 pM to 100 nM. It is worth mentioning that a plenty of extra merits has also been validated like reproducibility, repeatability and stability, enabling to direct electrochemical detection of •OH in HepG2 cells.

Synthetic protein-conductive membrane nanopores built with DNA.

Nanopores are key in portable sequencing and research given their ability to transport elongated DNA or small bioactive molecules through narrow transmembrane channels. Transport of folded proteins could lead to similar scientific and technological benefits. Yet this has not been realised due to the shortage of wide and structurally defined natural pores. Here we report that a synthetic nanopore designed via DNA nanotechnology can accommodate folded proteins. Transport of fluorescent proteins through single pores is kinetically analysed using massively parallel optical readout with transparent silicon-on-insulator cavity chips vs. electrical recordings to reveal an at least 20-fold higher speed for the electrically driven movement. Pores nevertheless allow a high diffusive flux of more than 66 molecules per second that can also be directed beyond equillibria. The pores may be exploited to sense diagnostically relevant proteins with portable analysis technology, to create molecular gates for drug delivery, or to build synthetic cells.

Active DNA unwinding and transport by a membrane-adapted helicase nanopore.

Nanoscale transport through nanopores and live-cell membranes plays a vital role in both key biological processes as well as biosensing and DNA sequencing. Active translocation of DNA through these nanopores usually needs enzyme assistance. Here we present a nanopore derived from truncated helicase E1 of bovine papillomavirus (BPV) with a lumen diameter of c.a. 1.3 nm. Cryogenic electron microscopy (cryo-EM) imaging and single channel recording confirm its insertion into planar lipid bilayer (BLM). The helicase nanopore in BLM allows the passive single-stranded DNA (ssDNA) transport and retains the helicase activity in vitro. Furthermore, we incorporate this helicase nanopore into the live cell membrane of HEK293T cells, and monitor the ssDNA delivery into the cell real-time at single molecule level. This type of nanopore is expected to provide an interesting tool to study the biophysics of biomotors in vitro, with potential applications in biosensing, drug delivery and real-time single cell analysis.

Loop-Mediated Isothermal Amplification-Coupled Glass Nanopore Counting Toward Sensitive and Specific Nucleic Acid Testing.

Solid-state nanopores have shown great promise and achieved tremendous success in label-free single-molecule analysis. However, there are three common challenges in solid-state nanopore sensors, including the nanopore size variations from batch to batch that makes the interpretation of the sensing results difficult, the incorporation of sensor specificity, and the impractical analysis time at low analyte concentration due to diffusion-limited mass transport. Here, we demonstrate a novel loop-mediated isothermal amplification (LAMP)-coupled glass nanopore counting strategy that could effectively address these challenges. By using the glass nanopore in the counting mode (versus the sizing mode), the device fabrication challenge is considerably eased since it allows a certain degree of pore size variations and no surface functionalization is needed. The specific molecule replication effectively breaks the diffusion-limited mass transport thanks to the exponential growth of the target molecules. We show the LAMP-coupled glass nanopore counting has the potential to be used in a qualitative test as well as in a quantitative nucleic acid test. This approach lends itself to most amplification strategies as long as the target template is specifically replicated in numbers. The highly sensitive and specific sensing strategy would open a new avenue for solid-state nanopore sensors toward a new form of compact, rapid, low-cost nucleic acid testing at the point of care.

Machine Learning Prediction on Properties of Nanoporous Materials Utilizing Pore Geometry Barcodes.

In this work, we propose a computational framework for machine learning prediction on structural and performance properties of nanoporous materials for methane storage application. For our machine learning prediction, two descriptors based on pore geometry barcodes were developed; one descriptor is a set of distances from a structure to the most diverse set in barcode space, and the second descriptor extracts and uses the most important features from the barcodes. First, to identify the optimal condition for machine learning prediction, the effects of training set preparation method, training set size, and machine learning models were investigated. Our analysis showed that kernel ridge regression provides the highest prediction accuracy, and randomly selected 5% structures of the entire set would work well as a training set. Our results showed that both descriptors accurately predicted performance and even structural properties of zeolites. Furthermore, we demonstrated that our approach predicts accurately properties of metal-organic frameworks, which might indicate the possibility of this approach to be easily applied to predict the properties of other types of nanoporous materials.

Plasmonic Nanopores for Single-Molecule Detection and Manipulation: Toward Sequencing Applications.

Solid-state nanopore-based sensors are promising platforms for next-generation sequencing technologies, featuring label-free single-molecule sensitivity, rapid detection, and low-cost manufacturing. In recent years, solid-state nanopores have been explored due to their miscellaneous fabrication methods and their use in a wide range of sensing applications. Here, we highlight a novel family of solid-state nanopores which have recently appeared, namely plasmonic nanopores. The use of plasmonic nanopores to engineer electromagnetic fields around a nanopore sensor allows for enhanced optical spectroscopies, local control over temperature, thermophoresis of molecules and ions to/from the sensor, and trapping of entities. This Mini Review offers a comprehensive understanding of the current state-of-the-art plasmonic nanopores for single-molecule detection and biomolecular sequencing applications and discusses the latest advances and future perspectives on plasmonic nanopore-based technologies.

Enzyme-Decorated Covalent Organic Frameworks as Nanoporous Platforms for Heterogeneous Biocatalysis.

Sustainability in chemistry heavily relies on heterogeneous catalysis. Enzymes, the main catalyst for biochemical reactions in nature, are an elegant choice to catalyze reactions due to their high activity and selectivity, although they usually suffer from lack of robustness. To overcome this drawback, enzyme-decorated nanoporous heterogeneous catalysts were developed. Three different approaches for Candida antarctica lipase B (CAL-B) immobilization on a covalent organic framework (PPF-2) were employed: physical adsorption on the surface, covalent attachment of the enzyme in functional groups on the surface and covalent attachment into a linker added post-synthesis. The influence of the immobilization strategy on the enzyme uptake, specific activity, thermal stability, and the possibility of its use through multiple cycles was explored. High specific activities were observed for PPF-2-supported CAL-B in the esterification of oleic acid with ethanol, ranging from 58 to 283 U mg-1 , which was 2.6 to 12.7 times greater than the observed for the commercial Novozyme 435.