Translocation Of Biomolecules Research Articles

Cell Penetrating Peptide Enhances the Aphidicidal Activity of Spider Venom-Derived Neurotoxin.

HxTx-Hv1h, a neurotoxic peptide derived from spider venom, has been developed for use in commercial biopesticide formulations. Cell Penetrating Peptides (CPPs) are short peptides that facilitate the translocation of various biomolecules across cellular membranes. Here, we evaluated the aphidicidal efficacy of a conjugated peptide, HxTx-Hv1h/CPP-1838, created by fusing HxTx-Hv1h with CPP-1838. Additionally, we aimed to establish a robust recombinant expression system for HxTx-Hv1h/CPP-1838. We successfully achieved the secretory production of HxTx-Hv1h, its fusion with Galanthus nivalis agglutinin (GNA) forming HxTx-Hv1h/GNA and HxTx-Hv1h/CPP-1838 in yeast. Purified HxTx-Hv1h exhibited contact toxicity against Megoura crassicauda, with a 48 h median lethal concentration (LC50) of 860.5 μg/mL. Fusion with GNA or CPP-1838 significantly enhanced its aphidicidal potency, reducing the LC50 to 683.5 μg/mL and 465.2 μg/mL, respectively. The aphidicidal efficacy was further improved with the addition of surfactant, decreasing the LC50 of HxTx-Hv1h/CPP-1838 to 66.7 μg/mL-over four times lower compared to HxTx-Hv1h alone. Furthermore, we engineered HxTx-Hv1h/CPP-1838 multi-copy expression vectors utilizing the BglBrick assembly method and achieved high-level recombinant production in laboratory-scale fermentation. This study is the first to document a CPP fusion strategy that enhances the transdermal aphidicidal activity of a natural toxin like HxTx-Hv1h and opens up the possibility of exploring the recombinant production of HxTx-Hv1h/CPP-1838 for potential applications.

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.

Translocation of Proteins through Solid-State Nanopores Using DNA Polyhedral Carriers.

The detection of biomolecules at the single molecule level has important applications in the fields of biosensing and biomedical diagnosis. The solid-state nanopore (SS nanopore) is a sensitive tool for detecting single molecules because of its unique label-free and low sample consumption properties. SS nanopore translocation of small biomolecules is typically driven by an electronic field force and is thus influenced by the charge, shape, and size of the target molecules. Therefore, it remains challenging to control the translocation of biomolecules through SS nanopores, particularly for different proteins with complex conformations and unique charges. Toward this problem, a DNA polyhedral carrier coating strategy to assist protein translocation through SS nanopores is developed, which facilitates target protein detection. The current signal-to-noise ratios are improved significantly using this DNA carrier loading strategy. The proposed method should aid the detection of proteins, which are difficult to translocate through nanopores. This coating-assisted method offers a wide range of applications for SS nanopore detection and promotes the development of single-molecule detection.

Thermally Switchable Nanogate Based on Polymer Phase Transition.

Mimicking and extending the gating properties of biological pores is of paramount interest for the fabrication of membranes that could be used in filtration or drug processing. Here, we build a selective and switchable nanopore for macromolecular cargo transport. Our approach exploits polymer graftings within artificial nanopores to control the translocation of biomolecules. To measure transport at the scale of individual biomolecules, we use fluorescence microscopy with a zero-mode waveguide set up. We show that grafting polymers that exhibit a lower critical solution temperature creates a toggle switch between an open and closed state of the nanopore depending on the temperature. We demonstrate tight control over the transport of DNA and viral capsids with a sharp transition (∼1 °C) and present a simple physical model that predicts key features of this transition. Our approach provides the potential for controllable and responsive nanopores in a range of applications.

The impact of Rhodiola rosea on biomarkers of diabetes, inflammation, and microbiota in a leptin receptor-knockout mouse model

Type 2 diabetes is the most prevalent endocrine disease in the world, and recently the gut microbiota have become a potential target for its management. Recent studies have illustrated that this disease may predispose individuals to certain microbiome compositions, and treatments like metformin have been shown to change gut microbiota and their associated metabolic pathways. However, given the limitations and side effects associated with pharmaceuticals currently being used for therapy of diabetes, there is a significant need for alternative treatments. In this study, we investigated the effects of a root extract from Rhodiola rosea in a Leptin receptor knockout (db/db) mouse model of type 2 diabetes. Our previous work showed that Rhodiola rosea had anti-inflammatory and gut microbiome-modulating properties, while extending lifespan in several animal models. In this study, treatment with Rhodiola rosea improved fasting blood glucose levels, altered the response to exogenous insulin, and decreased circulating lipopolysaccharide and hepatic C-reactive protein transcript levels. We hypothesize that these changes may in part reflect the modulation of the microbiota, resulting in improved gut barrier integrity and decreasing the translocation of inflammatory biomolecules into the bloodstream. These findings indicate that Rhodiola rosea is an attractive candidate for further research in the management of type 2 diabetes.

Lifetime-based analysis of binary fluorophores mixtures in the low photon count limit

SummarySingle biomolecule sensing often requires the quantification of multiple fluorescent species. Here, we theoretically and experimentally use time-resolved fluorescence via Time Correlated Single Photon Counting (TCSPC) to accurately quantify fluorescent species with similar chromatic signatures. A modified maximum likelihood estimator is introduced to include two fluorophore species, with compensation of the instrument response function. We apply this algorithm to simulated data of a simplified two-fluorescent species model, as well as to experimental data of fluorophores' mixtures and to a model protein, doubly labeled with different fluorophores' ratio. We show that 100 to 200 photons per fluorophore, in a 10-ms timescale, are sufficient to provide an accurate estimation of the dyes' ratio on the model protein. Our results provide estimation for the desired photon integration time toward implementation of TCSPC in systems with fast occurring events, such as translocation of biomolecules through nanopores or single-molecule burst analyses experiments.

Single-Molecule, Non-Chromatic Discrimination in Binary Fluorophores Mixtures

SummarySingle biomolecule sensing often involves quantification of multiple fluorescent species, coupled for example to different DNAs or proteins resides. While chromatic separation of the fluorescent species is well-established and broadly used it involves several limitations, particularly when the total photon budget is limited. Here we theoretically and experimentally explore the use of time-resolved fluorescence via Time Correlated Single Photon Counting (TCSPC) to accurately quantify multiple fluorescent species. A modified maximum likelihood estimator (MLE) is introduced to include two fluorophore species, with the compensation of the instrument response. We apply this algorithm to simulated data of a simplified two fluorescent species model, as well as to experimental data of free fluorophores mixtures and doubly labelled proteins in order to estimate the errors in the two species ratios as a function of photon budget available. Moreover, focusing on the case of labeled proteins in which the ratios of the two species per molecule is a discrete variable subject to the chemical labeling numbers of the two dyes, we obtain that between 1,000 to 2,000 photons can already provide an accurate estimation of the dyes’ ratio in labelled Ovalbumin proteins. These values correspond to roughly 100 to 200 photons per fluorophore suggesting a realistic path for dyes ratio determination from a single passage of a protein in the detection volume in roughly 10 ms timescale. Our results highlight the way in which temporal limitations of the TCSPC technique could hinder its implementation in systems with fast occurring events such as translocation of biomolecules through nanopores, or fast diffusion in single-molecule burst analyses experiments and provide estimation for the desired photon integration time.

Optimized electrochemical breakdown etching using temporal voltage variation for formation of nanopores in a silicon membrane

Abstract Dielectric breakdown etching is a well-known method of making nanopores on thin (∼50 nm) dielectric membranes. However, voltage driven translocation of biomolecules through such nanopores becomes extremely fast. For improved detection, for instance by the current blockage, a high-aspect-ratio nanopore could be beneficial for slowing down the translocation. High-aspect-ratio nanopore on silicon fabrication requires a well-controlled process and is dependent on specific crystal orientation, dopant type and resistivity of substrate. Therefore, an optimized method of processing high-aspect-ratio nanopores is necessary considering the advantage of a silicon membrane being able to be integrated with standard CMOS processing. Here, we present an optimized fabrication method for mass-producing a single and an array of nanopores on a thick (2 μm) silicon device layer based on a silicon-on-insulator (SOI) wafer. A method of temporal voltage variation is exploited to optimize the etching parameters for the nanopore formation during electrochemical breakdown etching, diameters of nanopores around 12 nm have been achieved. Besides, the correlation between the parameters of etching and nanopore diameter is deduced. The processed high-aspect-ratio nanopore enables applications in single-molecule sensing such as DNA, exosomes, viruses, and protein markers. The developed process is inexpensive, fast and can be batch fabricated.

How Nanopore Translocation Experiments Can Measure RNA Unfolding

Electrokinetic translocation of biomolecules through solid-state nanopores represents a label-free single-molecule technique that may be used to measure biomolecular structure and dynamics. Recent investigations have attempted to distinguish individual transfer RNA (tRNA) species based on the associated pore translocation times, ion-current noise, and blockage currents. By manufacturing sufficiently smaller pores, each tRNA is required to undergo a deformation to translocate. Accordingly, differences in nanopore translocation times and distributions may be used to infer the mechanical properties of individual tRNA molecules. To bridge our understanding of tRNA structural dynamics and nanopore measurements, we apply molecular dynamics simulations using a simplified “structure-based” energetic model. Calculating the free-energy landscape for distinct tRNA species implicates transient unfolding of the terminal RNA helix during nanopore translocation. This provides a structural and energetic framework for interpreting current experiments, which can aid the design of methods for identifying macromolecules using nanopores.

Unbalanced Ion Flushing Effect in MoS2 Nanopore Biosensors

In MoS2 nanopores, in addition to the widely measured ionic current signal to detect DNA nucleotides or epigenetic malformations, recent efforts have focused on the use of the transverse electronic current along the membrane for biomolecules sensing. This approach provides larger current magnitude and allows detection at the observed DNA translocation speed in the solid-state nanopore, which are crucial for accurate biomolecule sensing and faster DNA sequencing. As of today, no existing model is able to relate the variations of the transverse current to the interaction between ions, biomolecules and the MoS2 membrane. In this presentation, we report a comprehensive transport model based on the Poisson-Boltzmann transport formalism that considers the effect of biomolecules, electrolyte, and ion screening on conducting carriers along a MoS2 membrane. By accounting for membrane geometry and material properties used in recent experiment, we are able to extract the different electrical contributions to the transverse current variation during a DNA translocation event. In particular, we show that the major contribution to the electrical signal is caused by an unbalanced-ion-flushing effect that arises when the ion double-layer around the nanopore edge is swept out of the pore by the biomolecule translocation. By optimizing the MoS2 membrane geometry and nanopore size, we predict higher sensitivity to detect the translocation event in the corresponding transverse current signal. Thus,the identification of the unbalanced ion effect on the transverse electronic current foresees a critical new direction to improving the signal-to-noise ratio in solid-state nanopore sensing device.

BPEI-Induced Delocalization of PBP4 Potentiates β-Lactams against MRSA.

With its high morbidity rate and increasing resistance to treatment, methicillin-resistant Staphylococcus aureus (MRSA) is a grave concern in the medical field. In methicillin-susceptible strains, β-lactam antibiotics disable the penicillin binding proteins (PBPs) that cross-link the bacterial cell wall. However, methicillin-resistant strains have PBP2a and PBP4, which continue enzymatic activity in the presence of β-lactam antibiotics. The activity of PBP2a and PBP4 is linked to the presence of wall teichoic acid (WTA); thus, WTA has emerged as a target for antibiotic drug discovery. In this work, we disable WTA in situ using its anionic phosphodiester backbone to attract cationic branched polyethylenimine (BPEI). Data show that BPEI removes β-lactam resistance in common MRSA strains and clinical isolates. Fluorescence microscopy was used to investigate this mechanism of action. The results indicate that BPEI prevents the localization of PBP4 to the cell division septum, thereby changing the cellular morphology and inhibiting cell division. Although PBP4 is not required for septum formation, proper cell division and morphology require WTA; BPEI prevents this essential function. The combination of BPEI and β-lactams is bactericidal and synergistic. Because BPEI allows us to study the role of WTA in the cell wall without genetic mutation or altered translocation of biomolecules and/or their precursors, this approach can help revise existing paradigms regarding the role of WTA in prokaryotic biochemistry at every growth stage.

Localized detection of ions and biomolecules with a force-controlled scanning nanopore microscope.

Proteins, nucleic acids and ions secreted from single cells are the key signalling factors that determine the interaction of cells with their environment and the neighbouring cells. It is possible to study individual ion channels by pipette clamping, but it is difficult to dynamically monitor the activity of ion channels and transporters across the cellular membrane. Here we show that a solid-state nanopore integrated in an atomic force microscope can be used for the stochastic sensing of secreted molecules and the activity of ion channels in arbitrary locations both inside and outside a cell. The translocation of biomolecules and ions through the nanopore is observed in real time in live cells. The versatile nature of this approach allows us to detect specific biomolecules under controlled mechanical confinement and to monitor the ion-channel activities of single cells. Moreover, the nanopore microscope was used to image the surface of the nuclear membrane via high-resolution scanning ion conductance measurements.

Fabrication and practical applications of molybdenum disulfide nanopores.

Among the different developed solid-state nanopores, nanopores constructed in a monolayer of molybdenum disulfide (MoS2) stand out as powerful devices for single-molecule analysis or osmotic power generation. Because the ionic current through a nanopore is inversely proportional to the thickness of the pore, ultrathin membranes have the advantage of providing relatively high ionic currents at very small pore sizes. This increases the signal generated during translocation of biomolecules and improves the nanopores' efficiency when used for desalination or reverse electrodialysis applications. The atomic thickness of MoS2 nanopores approaches the inter-base distance of DNA, creating a potential candidate for DNA sequencing. In terms of geometry, MoS2 nanopores have a well-defined vertical profile due to their atomic thickness, which eliminates any unwanted effects associated with uneven pore profiles observed in other materials. This protocol details all the necessary procedures for the fabrication of solid-state devices. We discuss different methods for transfer of monolayer MoS2, different approaches for the creation of nanopores, their applicability in detecting DNA translocations and the analysis of translocation data through open-source programming packages. We present anticipated results through the application of our nanopores in DNA translocations and osmotic power generation. The procedure comprises four parts: fabrication of devices (2-3 d), transfer of MoS2 and cleaning procedure (24 h), the creation of nanopores within MoS2 (30 min) and performing DNA translocations (2-3 h). We anticipate that our protocol will enable large-scale manufacturing of single-molecule-analysis devices as well as next-generation DNA sequencing.

Detection of metal corrosion characteristics in chlorine solution using solid state nanopore

Nanopore structures were originally proposed for detection of biomolecule translocation through nanometer-scale pores that perforate membranes by transient changes in ionic current. In this study, these changes are utilized to detect corrosion of different metals in aqueous chlorine media. The corrosion behaviors of Cu, Al, Ti, and Cr were analyzed by monitoring the changes in ion current resulting from ion concentration variations in solutions due to corrosion of the metals. We observed that the Cu layer passivated by CuOx was severely corroded when a drastic change of ion current was induced, from 10 to 30 nS to the level of 104 nS, as soon as the bias voltage was applied. In the case of Al passivated by thin AlOx, the conductance increased from 10–30 to 166 ± 52 nS and became saturated. Highly localized pitting corrosion was observed on the periphery of the nanopore, where the electrical field was most concentrated. Finally, we observed that Ti and Cr passivated by oxide showed long-term stability without corrosion in 1 M KCl in the pH range of 4–11.

Improved model of ionic transport in 2-D MoS2 membranes with sub-5 nm pores

Solid-state nanopores made of two-dimensional materials such as molybdenum disulfide are of great interest thanks in part to promising applications such as ion filtration and biomolecule translocation. Controlled fabrication and tunability of nanoporous membranes require a better understanding of their ionic conductivity capabilities at the nanoscale. Here, we developed a model of ionic conductivity for a KCl electrolyte through sub 5-nm single-layer MoS2 nanopores using equilibrium all-atom molecular dynamics simulations. We investigate the dynamics of K+ and Cl− ions inside the pores in terms of concentration and mobility. We report that, for pore dimensions below 2.0 nm, which are of particular interest for biomolecule translocation applications, the behaviors of the concentration and mobility of ions strongly deviate from bulk properties. Specifically, we show that the free-energy difference for insertion of an ion within the pore is proportional to the inverse surface area of the pore and that the inverse mobility scales linearly as the inverse diameter. Finally, we provide an improved analytical model taking into account the deviation of ion dynamics from bulk properties, suitable for direct comparison with experiments.

Translocation of DNA and protein through a sequentially polymerized polyurea nanopore.

Here, we investigated the translocation of biomolecules, such as DNA and protein, through a sequentially polymerized polyurea nanopore, with a thin (<10 nm) polymer membrane of uniform thickness. The polyurea membrane was synthesized by molecular layer deposition using p-phenylenediisocyanate (PDI) and p-phenylenediamine (PDA) as sequential precursors. The membrane exhibited a hydrophobic surface with a highly negative surface charge density (-51 mC m-2 at pH 8). It was particularly noted that the high surface charge density of the membrane resulted in a highly developed electro-osmotic flow which, in turn, strongly influenced the capture probability of biomolecules, depending on the balance between the electro-osmotic and electrophoretic forces. For instance, the capture frequency of negatively charged DNA was demonstrated to be quite low, since these two forces more or less cancelled each other, whereas that of positively charged MDM2 was much higher, since these two forces were additive. We also identified that the mean translocation time of MDM2 through the polyurea nanopore was 26.1 ± 3.7 μs while that of the SiN nanopore was 14.2 ± 2.0 μs, hence suggesting that the enhanced electrostatic interaction between positively charged MDM2 and the negatively charged pore surface affects the translocation speed.

DNA capture into the ClyA nanopore: diffusion-limited versus reaction-limited processes

The capture and translocation of biomolecules through nanometer-scale pores are processes with a potentially large number of applications, and hence they have been intensively studied in recent years. The aim of this paper is to review existing models of the capture process by a nanopore, together with some recent experimental data of short single- and double-stranded DNA captured by the Cytolysin A (ClyA) nanopore. ClyA is a transmembrane protein of bacterial origin which has been recently engineered through site-specific mutations, to allow the translocation of double- and single-stranded DNA. A comparison between theoretical estimations and experiments suggests that for both cases the capture is a reaction-limited process. This is corroborated by the observed salt dependence of the capture rate, which we find to be in quantitative agreement with the theoretical predictions.

Thermophoresis-Controlled Size-Dependent DNA Translocation through an Array of Nanopores.

Large arrays of nanopores can be used for high-throughput biomolecule translocation with applications toward size discrimination and sorting at the single-molecule level. In this paper, we propose to discriminate DNA length by the capture rate of the molecules to an array of relatively large nanopores (50-130 nm) by introducing a thermal gradient by laser illumination in front of the pores balancing the force from an external electric field. Nanopore arrays defined by photolithography were batch processed using standard silicon technology in combination with electrochemical etching. Parallel translocation of single, fluorophore-labeled dsDNA strands is recorded by imaging the array with a fast CMOS camera. The experimental data show that the capture rates of DNA molecules decrease with increasing DNA length due to the thermophoretic effect of the molecules. It is shown that the translocation can be completely turned off for the longer molecule using an appropriate bias, thus allowing a size discrimination of the DNA translocation through the nanopores. A derived analytical model correctly predicts the observed capture rate. Our results demonstrate that by combining a thermal and a potential gradient at the nanopores, such large nanopore arrays can potentially be used as a low-cost, high-throughput platform for molecule sensing and sorting.

Zero-Depth Interfacial Nanopore Capillaries.

High-fidelity analysis of translocating biomolecules through nanopores demands shortening the nanocapillary length to a minimal value. Existing nanopores and capillaries, however, inherit a finite length from the parent membranes. Here, nanocapillaries of zero depth are formed by dissolving two superimposed and crossing metallic nanorods, molded in polymeric slabs. In an electrolyte, the interface shared by the crossing fluidic channels is mathematically of zero thickness and defines the narrowest constriction in the stream of ions through the nanopore device. This novel architecture provides the possibility to design nanopore fluidic channels, particularly with a robust 3D architecture maintaining the ultimate zero thickness geometry independently of the thickness of the fluidic channels. With orders of magnitude reduced biomolecule translocation speed, and lowered electronic and ionic noise compared to nanopores in 2D materials, the findings establish interfacial nanopores as a scalable platform for realizing nanofluidic systems, capable of single-molecule detection.

Lipid Flip-Flop and Pore Nucleation on Zwitterionic Bilayers are Asymmetric under Ionic Imbalance.

Lipid flip-flop and its associated transient pore formation are key thermodynamic properties of living cell membranes. However, there is a lack of understanding of whether ionic imbalance that exists ubiquitously across cell membranes affects lipid flip-flop and its associated functions. Potential of mean force calculations show that the free-energy barrier of lipid flip-flop on the extracellular leaflet reduces with the presence of ionic imbalance, whereas the barrier on the intracellular leaflet is generally not affected. The linear decrease of the activation energy of lipid flip-flop on the extracellular leaflet is consistent with the experimentally measured conductance-voltage relationship of zwitterionic lipid bilayers. This suggests: 1) lipid flip-flop has a directionality under physiological conditions and phospholipids accumulate at a rate on the order of 105 µm-2 h-1 on the cytoplasmic side of cell membranes; 2) ion permeation across a lipid membrane is moderated by lipid flip-flop; 3) the energy barrier of pore formation is aligned with the weaker leaflet that has a lower energy of lipid flip-flop. The asymmetry of lipid flip-flop and pore nucleation may have substantial implications for protein translocation, signaling, enzymatic activities, vesicle fusion, and transportation of biomolecules on cell membranes.