Glass Nanopore Research Articles

Hydrogel interfaced glass nanopore for high-resolution sizing of short DNA fragments

Solid-state nanopores, known for their label-free detection and operational simplicity, face challenges in accurately sizing the short nucleic acids due to fast translocation and a lack of enzyme-based control mechanisms as compared to their biological counterparts with sizing resolutions still limited to ≥100 bp. Here, we present a facile polyethylene glycol-dimethacrylate (PEG-DMA) hydrogel interfaced glass nanopore (HIGN) system by inserting glass nanopore into the hydrogel to achieve sub-100 base pair (bp) resolution in short DNA sizing analysis. We systematically investigated the effects of hydrogel mesh size, spatial configurations of glass nanopores about the hydrogel, applied bias voltage, and analyte concentration on the transport dynamics of 200 bp double-stranded DNA (dsDNA). A 7.5 w/v% PEG-DMA hydrogel induced ∼11x increase in the mean dwell times compared with bare solution nanopore system. The insertion locations and depths of the glass nanopore into the hydrogel resulted in 7.16% and 5.28% coefficients of variation (CV) for mean normalized event frequencies. This enhancement of dwell times and invariability in translocation characteristics enables precise sizing of dsDNA fragments under 400 bp using HIGN, with an achieved size resolution of 50 bp with observable mean normalized peak amplitude (ΔI/Io) of ∼0.005. Furthermore, we have demonstrated the capability of HIGN to perform multiplex detection of influenza A virus (IAV) and severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) through reverse transcriptase-polymerase chain reaction (RT-PCR). These results demonstrated the potential of HIGN as a versatile tool in nucleic acid analysis and multiplexed label-free molecular diagnostics.

Numerical Modelling of Nanopores

Nanopores are used for resistive pulse sensing of single analytes and as nanopipette probes for electrochemical scanned probe microscopy. The current-voltage responses of nanopores display a rich range of behaviors, which arise due to interactions between the electric fields (applied potential and surface charges), ion concentrations, and fluid flows.1 To understand these behaviors and interpret experimental results, numerical simulation of the coupled physics are typically performed.2 Yet despite simulations of conical nanopores being commonplace, they remain challenging. As the electric double layer requires resolution of concentrations and electric fields at sub-nm length scales, while concentration enhancements and depletions occur 10s of μm inside the pore simulations must resolve differences over length scales that vary by ~5 orders of magnitude. While various strategies have been employed to make these simulations more manageable including simulating a small portion of the pore, ignoring surface charge beyond a certain distance [others]. However, no consensus exists as to best practices or the impact of these simplifications.In this work we assess the impact of commonly made simplifications and describe best practices for efficient modelling of conical nanopores to ensure accurate results are readily obtained. Suggestions include effective choices of mesh, initial conditions, and the handling of semi-infinite boundaries.(1) Lan, W.-J.; Holden, D. A.; White, H. S. Pressure-Dependent Ion Current Rectification in Conical-Shaped Glass Nanopores. J. Am. Chem. Soc. 2011, 133 (34), 13300–13303. https://doi.org/10.1021/ja205773a.(2) White, H. S.; Bund, A. Ion Current Rectification at Nanopores in Glass Membranes. Langmuir 2008, 24 (5), 2212–2218. https://doi.org/10.1021/la702955k.

Detection of Glutamate Decarboxylase Antibodies and Simultaneous Multi-Molecular Translocation Exploration by Glass Nanopores.

Glutamic acid decarboxylase antibody (GADAb) has emerged as a significant biomarker for clinical diagnosis and prognosis in type 1 diabetes (T1D). In this study, we investigated the potential utilization of glass capillary solid-state nanopores as a cost-effective and easily preparable platform for the detection of individual antigens, antibodies, and antigen-antibody complexes without necessitating any modifications to the nanopores. Our findings revealed notable characteristic variations in the translocation events of glutamic acid decarboxylase (GAD65) through nanopores under different voltage conditions, discovered that anomalous phenomenon of protein translocation events increasing with voltage may potentially be caused by the crowding of multiple proteins in the nanopores, and demonstrated that there are multiple components in the polyclonal antibodies (GADAb-poly). Furthermore, we achieved successful differentiation between GAD65, GADAb, and GADAb-GAD65 complexes. These results offer promising prospects for the development of a rapid and reliable GADAb detection method, which holds the potential to be applied in patient serum samples, thereby facilitating a label-free, cost-effective, and early diagnosis of type I diabetes.

Size counting analysis of short nucleic acid molecules using high-resolution hydrogel-interfaced glass nanopore

Size counting analysis of short nucleic acid molecules using high-resolution hydrogel-interfaced glass nanopore

(Invited) Single-Molecule Electrical Currents Associated with Valinomycin Transport of K+

A quantitative description of ionophore-mediated ion transport is important in understanding ionophore activity in biological systems and developing new ionophore applications. We present the direct measurement of the electrical current resulting from K+ transport mediated by individual valinomycin (val) ionophores. Step fluctuations in current measured across a 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) bilayer suspended over a ~400 nm radius glass nanopore result from dynamic partitioning of val between the bilayer and torus region, effectively increasing or decreasing the total number of val present in the membrane. Approximately 30 val are present in the membrane on average with a val entering or leaving the bilayer approximately every 50 seconds, allowing measurement of changes in electrical current associated with individual val. The single-molecule val(K+) transport current at 0.1 V applied potential is (1.3 ±0.6)×10-15 A, consistent with estimates of the transport kinetics based on large val ensembles. This methodology for analyzing single ionophore transport is general and can be applied to other carrier-type ionophores.

DNA Volume, Topology, and Flexibility Dictate Nanopore Current Signals.

Nanopores have developed into powerful single-molecule sensors capable of identifying and characterizing small polymers, such as DNA, by electrophoretically driving them through a nanoscale pore and monitoring temporary blockades in the ionic pore current. However, the relationship between nanopore signals and the physical properties of DNA remains only partly understood. Herein, we introduce a programmable DNA carrier platform to capture carefully designed DNA nanostructures. Controlled translocation experiments through our glass nanopores allowed us to disentangle this relationship. We vary DNA topology by changing the length, strand duplications, sequence, unpaired nucleotides, and rigidity of the analyte DNA and find that the ionic current drop is mainly determined by the volume and flexibility of the DNA nanostructure in the nanopore. Finally, we use our understanding of the role of DNA topology to discriminate circular single-stranded DNA molecules from linear ones with the same number of nucleotides using the nanopore signal.

Metal-Organic Framework Sub-Nanochannels Formed inside Solid-State Nanopore with Proton Ultra-High Selectivity.

Metal-Organic frameworks (MOFs) have the advantages of high porosity, angstrom-scale pore size, and unique structure. In this work, a kind of MOFs, UiO-66 and its derivatives (including aminated UiO-66-(NH2 )2 and sulfonated UiO-66-(NH-SAG)2 ), were constructed on the inner surface of solid-state nanopores for ultra-selective proton transport. UiO-66 and UiO-66-(NH2 )2 nanocrystal particles were in-situ grown at the orifice of glass nanopores firstly, which were used to investigate the ionic current responses in LiCl and HCl solutions when the monovalent anions (Cl- ) were unchanged. Compared with UiO-66-modifed nanopores, the aminated MOFs modification (UiO-66-(NH2 )2 ) can improve the proton selectivity obviously. However, when the UiO-66-(NH-SAG)2 nanopore is prepared by further post-modification with sulfo-acetic acid, lithium ions can hardly pass through the channel, and the interaction between protons and sulfonic acid groups can promote the transport of protons, thus achieving ultra-high selectivity to protons. This work provides a new way to achieve sub-nanochannels with high selectivity, which can widely be used in ion separation, sensing and energy conversion.

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Herein, a single molecule detection technology based on glass nanopore is established to analyze xylan dissolved in ionic liquid. The nanopore detection technology shows the structural difference of heterogenous branched chain and homogeneous straight chain based on the single characteristic current blocking signal and statistical information, providing a research basis for the structural analysis of water insoluble polysaccharides. More details are discussed in the article by He et al. on page 1720—1726.image

Application of Nanopore Single Molecule Detection Technology in Analysis of Xylan Dissolved in Ionic Liquid†

Comprehensive SummaryXylan is the most abundant hemicellulose in nature. As a new type of green organic solvent, ionic liquid shows good preservation ability for the functional groups of hemicellulose. In this paper, a single molecule detection technology based on glass nanopore was established to analyze xylan dissolved in ionic liquid. Arabino‐xylan (AX) and beech xylan (BX) are respectively taken as the representatives of heterogeneous xylan and homogeneous xylan. Firstly, unmodified glass nanopore was used to detect the dissolved xylan in ionic liquid, and then poly(ethylene imine) (PEI) was used to modify the nanopore to change the surface charge in the nanopore and further enhance the interaction between the nanopore and the xylan molecule. It was found that before and after nanopore modification, at negative voltage and low positive voltage, AX didn't generate current blocking signal. On the contrary, BX didn't generate current blocking signal at positive voltage. This phenomenon may be due to the current disturbance driven by electrophoresis and electroosmosis of xylan molecules with weak negative charge. After statistics analysis, the current blocking signal of AX showed that the modified nanopore showed multiple peaks. It indicates that heterogeneous xylan and PEI modified nanopore had stronger interaction. The results show that the nanopore detection technology can show the structural difference of heterogenous branched chain and homogeneous straight chain based on the single characteristic current blocking signal and statistical information, providing a research basis for the structural analysis of water insoluble polysaccharides.

Single-Molecule Electrical Currents Associated with Valinomycin Transport of K+

A quantitative description of ionophore-mediated ion transport is important in understanding ionophore activity in biological systems and developing ionophore applications. Herein, we describe the direct measurement of the electrical current resulting from K+ transport mediated by individual valinomycin (val) ionophores. Step fluctuations in current measured across a 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) bilayer suspended over a ∼400 nm radius glass nanopore result from dynamic partitioning of val between the bilayer and torus region, effectively increasing or decreasing the total number of val present in the membrane. In our studies, approximately 30 val are present in the membrane on average with a val entering or leaving the bilayer approximately every 50 s, allowing measurement of changes in electrical current associated with individual val. The single-molecule val(K+) transport current at 0.1 V applied potential is (1.3 ± 0.6) × 10-15 A, consistent with estimates of the transport kinetics based on large val ensembles. This methodology for analyzing single ionophore transport is general and can be applied to other carrier-type ionophores.

Analysis of starch dissolved in ionic liquid by glass nanopore at single molecular level

In this paper, the glass nanopore technology was proposed to detect a single molecule of starch dissolved in ionic liquid [1-butyl-3-methylimidazolium chloride (BmimCl)]. Firstly, the influence of BmimCl on nanopore detection is discussed. It is found that a certain amount of strong polar ionic liquids will disturb the charge distribution in nanopores and increase the detection noise. Then, by analysis of the characteristic current signal of the conical nanopore, the motion behaviour of starch near the entrance of the nanopore was studied and analysis the dominant ion of starch in the BmimCl dissolution process. Finally, based on nuclear magnetic resonance (NMR) and Fourier transform infrared (FTIR) spectroscopy simply discussed the mechanism of amylose and amylopectin dissolved in BmimCl. These results confirm that branched chain structure would affect the dissolution of polysaccharides in ionic liquids and the contribution of anions to the dissolution of polysaccharides are dominant. It is further proved that the current signal can be used to judge the charge and structure information of the analyte, and the dissolution mechanism can be assist analyzed at the single molecule level.

Probing voltage-dependent stability of nucleosomes using glass nanopores.

The packaging of DNA in the form of chromatin helps the cell to compact and regulate its genetic material. It is known that the nucleosomes exist in various structural and positional combinations that leads to chromatin assuming locally folded structures. This allows for its efficient packaging and also helps in the gene regulation. In this work, using resistive pulse technique with our glass nanopores, we have investigated the voltage dependent stability of mono-nucleosomes as well as the 12-mer nucleosome arrays. In experiments with our in vitro assembled mononucleosomes, we show that the octasomal nucleosomes, comprising of octameric histone complexes on DNA, systematically breakdown into its partial structures, which we reason to be hexasomes and tetrasomes, in a voltage dependent manner. We attribute these structural changes to the opposing electrical forces experienced by the positively charged histone protein complex and the negatively charged DNA. We next investigate the stability of 12-mer nucleosome arrays and find discrete multi-level events corresponding to discrete structures on the array molecules. A detailed analysis of these discrete levels revealed that these structures correspond to single, multiple as well as partial nucleosomes on the array molecule. We further show that these structures disassemble in a voltage dependent manner. Our work demonstrates, for the first time, application of the glass nanopore platform to perform stability studies on nucleosome-DNA complexes, both individually as well as on nucleosomal arrays. This opens up future studies to evaluate force dependent structural transitions in the context of epigenetic control of chromatin structure.

Electrical detection and analysis of exosomes by a glass capillary nanopore.

An exosome is a vesicle less than 200 nm in diameter released from a cell and works as a medium for cell-to-cell communication. It is suggested that compositions of encapsulated molecules in exosomes have diversity even from a same cell. Single-particle analysis will be insight into the understanding the diversity, but it is still challenging. Nanopore technology will have a potential to detect and to capture single exosomes because it can discriminate nanoparticle properties including size, shape, and surface properties such as the charge by observation of ionic current change through a nano-sized pore. Here, we proposed to use a glass capillary nanopore (φ∼200 nm) and attempt to capture milk exosomes. Exosomes used in this study were extracted from cow milk using a procedure proposed previously. The particle sizes were confirmed by dynamic light scattering (DLS). A glass capillary nanopore was prepared by a puller. We observed the pore diameter of the capillaries by a scanning electron microscope (SEM). In the nanopore experiments, a glass capillary was filled with and immersed in PBS solution to measure I-V curve for checking the pore conductance, and then it was replaced in the exosome solution to detect the exosomes. The size of extracted exosomes was confirmed by DLS, and it was consistent with that in a previous study (∼130 nm). The outer diameter of the glass nanopores ranged from about 100 to 500 nm determined by SEM observation. For channel current measurement, the current blockages were observed in the presence of exosomes that was depended on the polarity of the applying voltages. We are conducting identification of microRNA encapsulated in exosomes collected from the nanopore by RT-qPCR, and it will be discussed in San Diego.

Bare glassy nanopore for length-resolution reading of PCR amplicons from various pathogenic bacteria and viruses

In this study, it is confirmed that without addition of organic solvent and embedding polymer hydrogel into glass nanopore, bare glass nanopore can faithfully measure various lengths of DNA duplexes from 200 to 3000 base pairs with 200 base pairs resolution, showing well-separated peak amplitudes of blockage currents. Furthermore, motivated by this readout capability of duplex DNA, amplicons from Polymerase Chain Reaction (PCR) amplification are straightforwardly discriminated by bare glassy nanopore without fluorescent labeling. Except simultaneous discrimination of up to 7 different segments of the same lambda genome, various pathogenic bacteria and viruses including SARS-CoV-2 and its mutants in clinical samples can be discriminated at high resolution. Moreover, quantitative measurement of PCR amplicons is obtained with detection range spanning from 0.75 aM to 7.5 pM and detection limit of 7.5 aM, which reveals that bare glass nanopore can faithfully disclose PCR results without any extra labeling.

Monitoring Bipolar Electrochemistry and Hydrogen Evolution Reaction of a Single Gold Microparticle under Sub-Micropipette Confinement.

Herein, an approach to track the process of autorepeating bipolar reactions and hydrogen evolution reaction (HER) on a micro gold bipolar electrode (BPE) is established. Once blocking the channel of the sub-micropipette tip, the formed gold microparticle is polarized into the wireless BPE, which induces the dissolution of the gold at the anode and the HER at the cathode. The current response shows a periodic behavior with three regions: the bubble generation region (I), the bubble rupture/generation region (II), and the channel opening region (III). After a stable low baseline current of region I, a series of positive spike signals caused by single H2 nanobubbles rupture/generation are recorded standing for the beginning of region II. Meanwhile, the dissolution of the gold blocking at the orifice will create a new channel, increasing the baseline current for region III, where the synthesis of gold occurs again, resulting in another periodic response. Finite element simulations are applied to unveil the mechanism thermodynamically. In addition, the integral charge of the H2 nanobubbles in region II corresponds to the consumption of the anode gold. It simultaneously monitors autorepeating bipolar reactions of a single gold microparticle and HER of a single H2 nanobubble electrochemically, which reveals an insightful physicochemical mechanism in nanoscale confinement and makes the glass nanopore an ideal candidate to further reveal the heterogeneity of catalytic capability at the single particle level.

A novel design of DNA duplex containing programmable sensing sites for nanopore-based length-resolution reading and applications for Pb2+ and cfDNA analysis.

Glass nanopore is an ideal candidate for biosensors due to its unique advantages such as label-free analysis, single-molecule sensitivity, and easy operation. Previous studies have shown that glass nanopores can distinguish different lengths of double-stranded DNA (dsDNA) at the same time with the length-resolution ability. Based on this, we proposed a novel design of a dsDNA block containing a programmable sensing site inside, which can be programmed to respond to different target molecules and cleaved into two smaller DNA blocks. When programming the sensing site with different sequences, for example, programming it as the substrate of GR-5 DNAzyme and CRISPR-Cas12a system, the DNA block could realize Pb2+ and cfDNA detection with the length-resolution ability of the glass nanopore. This strategy achieved a Pb2+ detection range from 0.5 nM to 100 nM, with a detection limit of 0.4 nM, and a BRCA-1 detection range from 1 pM to 10 pM, with a detection limit of 1 pM. The programable sensing site is easy to design and has strong expandability, which gives full play to the advantages of glass nanopore in length-resolution ability for dsDNA, and is expected to become an optional design for biosensing strategy for the glass nanopore as a biosensing platform.

An amplification-free, 16S rRNA test for Neisseria gonorrhoeae in urine.

An amplification-free, nanopore-based nucleic acid detection platform has been demonstrated for rapid, 16S rRNA sequence-specific detection of Neisseria gonorrhoeae at 10-100 CFU mL-1 in human urine against background bacterial flora at 1000 CFU mL-1. Gonorrhea is a very common notifiable communicable disease, antibiotic resistant strains have emerged, and the rate of reported gonococcal infections continues to increase. Since rapid clinical identification of bacterial pathogens in clinical samples is needed to guide proper antibiotic treatment and to control disease spread, it is important to engineer rapid, sensitive, selective, and inexpensive point-of-care (POC) diagnostic devices for pathogens such as N. gonorrhoeae. Our detector technology is based on straightforward conductometric detection of sustained blockage of a glass nanopore. Charge neutral, complementary peptide nucleic acid probes are conjugated to polystyrene beads to capture N. gonorrhoeae 16S rRNA selectively. In the presence of an electric field applied externally through a glass nanopore, the PNA-microbead conjugates that acquire substantial negative charge upon target hybridization are driven to the smaller diameter nanopore. At least partial blockage of the nanopore results in a sustained drop in ionic current that can be measured easily with simple electronics. The ability to detect N. gonorrhoeae over the range of 10 to 100 CFU mL-1 spiked in human urine was demonstrated successfully with estimated sensitivity and specificity of ∼98% and ∼100%, respectively. No false positives were observed for the control group of representative background flora (E. coli, K. pneumoniae, and E. faecalis) at 1000 CFU mL-1. Also, N. gonorrhoeae at 50 CFU mL-1 was successfully detected against 1000 CFU mL-1 of background flora in urine. These results suggest that this amplification-free technology may serve as the basis for rapid, inexpensive, low-power detection of pathogens in clinical samples at the POC.

A stimuli-responsive polymer modified nanopore for measuring β-amyloid peptide and zinc ions in brains of live mice with Alzheimer's disease

A copolymer-modified glass nanopore combined with microdialysis was used for assaying cerebral Aβ monomer and Zn2+. In the presence of Aβ monomer, the copolymer stretched. Then, the Zn2+ altered surface charges and fluorescence.

Single-Molecule Investigation of the Protein-Aptamer Interactions and Sensing Application Inside the Single Glass Nanopore.

Solid-state nanopores offer a nanoconfined space for a single-molecule sensing strategy. Evaluating the behavior of proteins and protein-related interactions at the single-molecule level is becoming more and more important for a better understanding of biological processes and diseases. In this work, the aptamer-functionalized nanopore was prepared as the sensing platform for kinetic analysis of the carcinoembryonic antigen (CEA) with its aptamers, which is an important cancer biomarker. CEA molecules were captured by the aptamers immobilized on the inner surface of the nanopore, and there was a complicated interaction between the CEA molecules and the aptamer, which is the process of association and dissociation. This could be used to measure the dynamics of aptamer-protein interactions without labeling. The kinetic analysis could be evaluated at the single-molecule level to interpret the dissociation constants of the binding and dissociation processes. Results showed that the translocation of CEA molecules in a functionalized nanopore had a deep blockades degree and long duration compared with nanopore modified with bare gold, which could be used for CEA sensing. This protein and protein-related interaction we designed provides new insights for evaluating the binding affinity, which will be beneficial for protein sensing and immunoassays.

A highly sensitive and selective artificial nanochannel for in situ detection of hydroxyl radicals in single living cell

As the most aggressive reactive oxygen species (ROS), hydroxyl radical (•OH) can directly modulate the biological ion channel and interfere with the progression of diseases. Inspired by biological •OH-activated ion channel, we reported a novel •OH-regulated glass nanopore functionalized with protoporphyrin Ⅸ (PP) film. This system showed outstanding •OH selective response owing to the ultra-fast reaction between •OH and thiol derivatives. In this case, the PP film is responsible for the changing not only of wettability but also of the inner surface charge. The synergetic effect of the dual transitions can regulate the ion transportation within the nanochannels and enabled tremendous enhancement of responsive efficiency. The detection limit could be achieved down to 1.58 nM. Taking advantage of the excellent analytical performance and mechanical qualities of this glass nanopore, the changes of •OH in single living cells were in situ monitored. Together, this study is beneficial for exploring the role of •OH in pathological events and shows promising potential for biomedical research.