Search

A fundamental question relating to protein folding/unfolding is the time evolution of the folding of a protein into its precisely defined native structure. The proper identification of transition conformations is essential for accurately describing the dynamic protein folding/unfolding pathways. Owing to the rapid transitions and sub-nm conformation differences involved, the acquisition of the transient conformations and dynamics of proteins is difficult due to limited instrumental resolution. Using the electrochemical confinement effect of a solid-state nanopore, we were able to snapshot the transient conformations and trace the multiple transition pathways of a single peptide inside a nanopore. By combining the results with a Markov chain model, this new single-molecule technique is applied to clarify the transition pathways of the β-hairpin peptide, which shows nonequilibrium fluctuations among several blockage current stages. This method enables the high-throughput investigation of transition pathways experimentally to access previously obscure peptide dynamics, which is significant for understanding the folding/unfolding mechanisms and misfolding of peptides or proteins., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

The simultaneous occurrence of multiple heterogeneous DNA phosphorylation statuses, which include 5' end phosphorylation, 5' end dephosphorylation, 3' end phosphorylation, and 3' end dephosphorylation, is crucial for regulating numerous cellular processes. Although there are many methods for detecting a single type of DNA phosphorylation, the direct and simultaneous identification of DNA phosphorylation/dephosphorylation on the 5' and/or 3' ends remains a challenge, let alone the unveiling of the heterogeneous catalysis processes of related phosphatases and kinases. Taking advantage of the charge-sensitive aerolysin nanopore interface, herein, an orientation-dependent sensing strategy is developed to enhance phosphorylation-site-dependent interaction with the nanopore sensing interface, enabling the direct and simultaneous electric identification of four heterogeneous phosphorylation statuses of a single DNA. By using this strategy, we can directly evaluate the heterogeneous dephosphorylation process of alkaline phosphatase (ALP) at the single-molecule level. Our results demonstrate that the ALP in fetal bovine serum preferentially catalyzes the 3' phosphate rather than both ends. The quantification of endogenous ALP activity in fetal bovine serum could reach the submilli-IU/L level. Our aerolysin measurements provide a direct look at the heterogeneous phosphorylation status of DNA, allowing the unveiling of the dynamic single-molecule functions of kinase and phosphatase.

This study aims to understand nanopore technology from the standpoint of capillary electrophoresis separation. The nanopore electrochemical measurements could be regarded as "single molecule electrophoresis". Similar to the case of capillary electrophoresis, the single target molecules migrate inside a nanopore under an external electric field. The recognition ability of the nanopore mainly depends on the charge, shape, and size of the target molecules under the electric force. The confined space of an Aerolysin nanopore matches the size of single biomolecule, while the amino acid residues along the inner wall of the nanopore facilitate electrokinetic regulation inside the nanopore. Under the applied voltage, each molecule enters the nanopore, generating the characteristic migration velocity and trajectory. Therefore, statistical analysis of the current amplitude, duration, frequency, and shape of the electrochemical signals would help differentiate and identify a single analyte from the mixture. Herein, we used an Aerolysin nanopore for identifying the oligonucleotides of 5'-CAA-3' (CA 2 ), 5'-CAAA-3' (CA 3 ), and 5'-CAAAA-3' (CA 4 ), which differ in length only by one nucleotide, as the model system to demonstrate single-molecule electrophoresis. The diameter of the Aerolysin nanopore is around 1 nm, and the pore length is approximately 10 nm. Under an applied voltage of 80 mV, the nanopore experiences a high electric field strength of about 80 kV/cm. The phosphate groups of the nucleotides carry negative charges in an electrolyte buffer solution of 1.0 mol/L KCl, at pH 8. Therefore, CA 2 , CA 3 , and CA 4 carry 2, 3, and 4 negative charges, respectively. During nanopore sensing, CA 2 , CA 3 , and CA 4 are subjected to electrophoretic forces and thus move inside the nanopore. Because the Aerolysin nanopore is anion selective, the direction of electroosmotic flow through the nanopore is consistent with the anion flow direction. Under the combined effects of the electrophoretic force and electroosmotic flow, CA 2 , CA 3 , CA 4 will transverse through the Aerolysin nanopore at different migration velocities. Note that the oligonucleotide shows strong electrostatic interaction with the two sensitive regions of Aerolysin, which comprises polar amino acids around R220 and K238. The strong interaction between the sensitive region of Aerolysin and the analyte would further modulate the translocation of oligonucleotides. Therefore, each oligonucleotide follows a different migration trajectory as it individually transverses through the nanopore. The migration speed and migration trajectory are recorded as ionic blockages in nanopore electrochemistry. The scatter plots of the blockage current and blockage duration of the mixed sample of CA 2 , CA 3 , and CA 4 show three characteristic distributions assigned to each type of oligonucleotide. Since the net charge increases with increasing length of the oligonucleotide, CA 3 and CA 4 experience a stronger electrophoretic force than does CA 2 inside the nanopore, leading to higher migration velocity. Therefore, the blockage duration of CA 3 and CA 4 is 5 times longer than that of CA 2 . By Gaussian fitting, the fitted blockage currents of CA 2 , CA 3 , and CA 4 are 20.7, 15.7, and 12.7 pA, respectively. Similar to our previous results, the blockage current increases with the chain length when the oligonucleotides comprise not more than 14 nucleotides. Therefore, nanopore-based single-molecule electrophoresis allows for the electrochemical identification of CA 2 , CA 3 , and CA 4 that differ in a length by only one nucleotide. Understanding the "single-molecule electrophoresis" concept would promote the application of electrochemically confined effects in single-molecule electrophoresis separation. The combination of single-molecule electrophoresis with a microfluidic system and a nanopore array is expected to aid the separation and identification of single molecules.

Nanobubble nucleation study is important for understanding the dynamic behavior of nanobubble growth, which is instructive for the nanobubble applications. Benefiting from nanopore fabrication, herein, we fabricated a sub-9 nm SiN X nanopore with the comparable size to nanobubbles at early-stage. The confined nanopore interface serves as a generator for producing nanobubbles by the chemical reaction between NaBH 4 and H 2 O and as an ultra-sensitive sensor for monitoring the H 2 nanobubble nucleation process. By carrying out the NaBH 4 concentration-dependent experiments, we found the life-time of nanobubbles decreased 250 times and the frequency of nanobubble generation increased 38 times with the NaBH 4 concentration increasing from 6 to 100 mM. The long-time equilibrium between gas molecules inward flux and outward flux could prolong the life-time of nanobubbles to hundreds of milliseconds at low NaBH 4 concentration. The raw current trace depicted that the transient accumulation and dissolution of cavity occurred during all the life-time of nanobubbles. Therefore, the sub-9 nm SiN X nanopore shows a strong ability for real-time monitoring the nanobubble nucleation at early-stage with high temporal and spatial resolution. This work provides a guide to study the dynamic and stochastic characteristics of nanobubbles., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In this study, we designed SiN X solid-state nanopores to detect the temperature effect on the hydrogen nanobubble formation. Here, we integrated a temperature controller with the highly sensitive nanopore. As the temperature decreases from 25 °C to 5 °C, the occurrence of the nanobubble nucleation inside a 12.3 nm SiN X nanopore confined space decreased from 102 s -1 to 23 s -1 , and the life-time of nanobubbles increased from 1.16 ms to 4.78 ms. The results further gave the activation energy for nanobubble nucleation which was 8.1 × 10 -20 J with a 12.3 nm SiN X nanopore. Our method provides an efficient analytical tool for revealing the temperature-dependent nanobubble nucleation, which further benefits the fundamental understanding of nanobubble nucleation.

Introduction: The characteristic of ABO blood subgroup is crucial for elucidating the mechanisms of such variant phenotypes and offering useful information in blood transfusion., Methods: In total, 211 ABO variants including part of available family members were investigated in this study. The phenotypes of these individuals were typed with serologic methods. The full coding regions of ABO gene and the erythroid cell-specific regulatory elements in intron 1 of them were amplified with polymerase chain reaction and then directly sequenced. The novel alleles were confirmed by cloning and sequencing. Phylogenetic tree was made using CLUSTAL W software. 3D structural analyses of the glycosyltransferases (GTs) with some typical mutations were performed by PyMOL software., Results: Forty-eight distinctly rare ABO alleles were identified in 211 Chinese variant individuals, including 16 novel ABO alleles. All of the alleles were categorized as 5 groups: 16 ABO * A alleles, 23 ABO * B alleles, 4 ABO * BA alleles, 4 ABO * cisAB alleles, and 1 ABO * O alleles. ABO * A2.08 and ABO * BA.02 were the relatively predominant A and B subgroup alleles, respectively. According to the phylogenetic tree, 28 alleles (5 common alleles and 23 alleles identified in our laboratory) were classified into 3 major allelic lineages. The structural analysis of 3D homology modeling predicted reduced protein stability of the mutant GTs and may explain the reduced ABO antigen expression., Conclusions: The molecular basis of ABO variants was analyzed, and 16 novel ABO alleles were identified. The results extended the information of ABO variants and provided a basis for better transfusion strategies and helped to improve blood transfusion safety., Competing Interests: The authors have no conflicts of interest to declare., (Copyright © 2019 by S. Karger AG, Basel.)

Biological nanopore technology has the advantages of high selectivity and high reproducibility for characterizing single biomolecules. However, it is challenging to achieve protein sequencing owing to the heterogeneous charge distributions of the protein and the small structural difference from each amino acid. Here, we took the inherent electrochemically confined sensing interface of the aerolysin nanopore to enhance its interaction with single amino acids. The results showed that single cysteine molecules, a highly reactive amino acid in aging and neurodegenerative diseases, could be captured and monitored by an aerolysin nanopore as it produced distinctive current blockages with a prolonged statistical duration of 0.11 ± 0.02 ms at +120 mV. This is the first report of the detection of a single amino acid molecule by a biological nanopore directly without any modification and labelling. This study facilitates the direct detection of single amino acids by regulating the characteristic interaction between the single amino acids and the designed sensing interface of aerolysin nanopores.

DNA lesions such as metholcytosine( m C), 8-OXO-guanine ( O G), inosine (I), etc. could cause genetic diseases. Identification of the varieties of lesion bases are usually beyond the capability of conventional DNA sequencing which is mainly designed to discriminate four bases only. Therefore, lesion detection remains a challenge due to massive varieties and less distinguishable readouts for structural variations at the molecular level. Moreover, standard amplification and labeling hardly work in DNA lesion detection. Herein, we designed a single molecule interface from the mutant aerolysin (K238Q), whose sensing region shows high compatibility to capture and then directly convert a minor lesion into distinguishable electrochemical readouts. Compared with previous single molecule sensing interfaces, the temporal resolution of the K238Q aerolysin nanopore is enhanced by two orders, which has the best sensing performance in all reported aerolysin nanopores. In this work, the novel K238Q could discriminate directly at least three types of lesions ( m C, O G, I) without labeling and quantify modification sites under the mixed heterocomposition conditions of the oligonucleotide. Such a nanopore electrochemistry approach could be further applied to diagnose genetic diseases at high sensitivity., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

The phosphorylation of oligonucleotides and peptides plays a critical role in regulating virtually all cellular processes. To fully understand these complex and fundamental regulatory pathways, the cellular phosphorylate changes of both oligonucleotides and peptides should be simultaneously identified and characterized. Here, we demonstrated a single-molecule, high-throughput, label-free, general, and one-step aerolysin nanopore method to comprehensively evaluate the phosphorylation of both oligonucleotide and peptide substrates. By virtue of electrochemically confined effects in aerolysin, our results show that the phosphorylation accelerates the traversing speed of a negatively charged substrate for about hundreds of time while significantly enhances the translocation frequency of a positively charged substrate. Thereby, the kinase/phosphatase activity could be directly measured with the aerolysin nanopore from the characteristically dose-dependent event frequency of the substrates. By using this straightforward approach, a model T 4 oligonucleotide kinase (PNK) further achieved the nanopore evaluation of its phosphatase activity and real-time monitoring of its phosphatase-catalyzed dephosphorylation at a single-molecule level. Our study provides a step forward to nanopore enzymology for analyzing the phosphorylation of both oligonucleotides and peptides with significant feasibility in fundamental biochemical researches, clinical diagnosis, and kinase/phosphatase-targeted drug discovery., Competing Interests: There are no conflicts to declare., (Copyright © 2019 Yi-Lun Ying et al.)

Single-molecule measurements have greatly enhanced our understanding of living systems. Biological systems offer nanopores, a sub-class of membrane proteins, the well-defined confined space for accommodating a single molecule. The biological nanopore acts as a single-biomolecule interface for capturing and identifying a single molecule of interest, and thus it can be used as a single-molecule sensor. In this Perspective, we focus on biological nanopore-based single-biomolecule interfaces for single-biomolecule detection. First, we outline the design of the nanopore-based single-biomolecule interface, which provides rich stochastic information regarding each biomolecule. Next, we highlight future research directions beyond DNA sequencing, including detection of rare species, identification of hidden intermediates, spectral analysis of covalent/noncovalent interactions, and tracing of the dynamic pathways of single-biopolymer behaviors. The concept of a "single-molecule ionic spectrum" is discussed, which may allow mapping of noncovalent interactions at an atomic level in the future. We also discuss the challenges and goals for the future to make this measurement possible for addressing entirely new types of biological questions, which would be an exciting area of future research.

We have developed a glass nanopore based single molecule tool to investigate the dynamic oligomerization and aggregation process of Aβ1-42 peptides. The intrinsic differences in the molecular size and surface charge of amyloid aggregated states could be distinguished through single molecule induced characteristic current fluctuation. More importantly, our results reveal that the neurotoxic Aβ1-42 oligomer tends to adsorb onto the solid surface of nanopores, which may explain its instability and highly neurotoxic features., (This journal is © The Royal Society of Chemistry 2019.)

Flavin adenine dinucleotide (FAD) as a cofactor is involved in numerous important metabolic pathways where the biological function is intrinsically related to its transient conformations. The confined space of enzymes requires FAD set in its specific intermediate conformation. However, conventional methods only detect stable conformations of FAD molecules, while transient intermediates are hidden in ensemble measurements. There still exists a challenge to uncover the transient conformation of each FAD molecule, which hinders the understanding of the structure-activity relationship of the FAD mechanism. Here, we employ the electrochemically confined space of an aerolysin nanopore to directly characterize a series of transient conformations of every individual FAD. Based on distinguishable current blockages, the "stack", "open", and four quasi-stacked FADs are clearly determined in solution, which is further confirmed by temperature-dependent experiments and mutant aerolysin assay. Combined with molecular dynamics simulations, we achieved a direct correlation between the residual current ratio ( I / I 0 ) and FAD backbone angle. These results would facilitate further understanding of the structure-activity relationship in the flavoprotein., (This journal is © The Royal Society of Chemistry 2019.)

The hydrogen bond (HB), one of the essential properties of water, tends to link water molecules to form dynamic water clusters. Extrinsic ions could change the size distribution of water clusters by influencing HBs. But the mechanism, especially the influence range of ions on HBs, is still in dispute due to limitation of analytical methods. Herein, we use in situ liquid ToF-SIMS analysis combined with density functional theory calculation to study the influence of different halide anions on HBs at a submicropore confined liquid-vacuum interface. Our experimental results demonstrated that anions show synchronous local and long-range effects on HBs. Specifically, the larger the anion is, the greater degree the long-range HB network and the local hydration number of anions are influenced. More importantly, we found that the long-range effect on the HB network is influenced by nuclear quantum effects, whereas the local effect on water molecules in the first hydration shell is not.

Hydrogen evolution reaction (HER) catalyzed by molybdenum sulfide quantum dots (MoS 2 QDs) has attracted extensive attention in the energy field. Monitoring HER catalyzed by MoS 2 QDs based on a glass nanopore with an electrochemically confined effect was proposed for the first time. MoS 2 QDs inside the glass nanopore is driven toward the orifice of the nanopore and bonded with the Ag nanoparticles (Ag NPs) to form a single nanocomposite. When enough voltage is applied across the orifice, the single Ag NP acts as a single nanoparticle electrode to conduct the electrochemically bipolar reaction on its two extremities. In the process, HER is catalyzed by MoS 2 QDs, and Ag NPs are oxidized at the same time. The appearance of blockages on the elevated ionic current is attributed to the generation of a H 2 bubble. Furthermore, by analyzing the modulations in the ionic current oscillation, the frequency of hydrogen bubble generation that is related to the catalytic efficiency of MoS 2 QDs could be estimated. The results reveal the capability of the glass nanopore for the real-time monitoring electrocatalytic behavior, which makes the glass nanopore an ideal candidate to further reveal the heterogeneity of catalytic capability at the single particle level.

Discrimination between cysteine and homocysteine at the single-molecule level is achieved within a K238Q mutant aerolysin nanopore, which provides a confined space for high spatial resolution to identify the amino acid difference with a 5'-benzaldehyde poly(dA) 4 probe. Our strategy allows potential detection and characterization of various amino acids and their modifications, and provides a crucial step towards developing nanopore protein sequencing devices.

The nanopore technique employs a nanoscale cavity to electrochemically confine individual molecules, achieving ultrasensitive single-molecule analysis based on evaluating the amplitude and duration of the ionic current. However, each nanopore sensing interface has its own intrinsic sensing ability, which does not always efficiently generate distinctive blockade currents for multiple analytes. Therefore, analytes that differ at only a single site often exhibit similar blockade currents or durations in nanopore experiments, which often produces serious overlap in the resulting statistical graphs. To improve the sensing ability of nanopores, herein we propose a novel shapelet-based machine learning approach to discriminate mixed analytes that exhibit nearly identical blockade current amplitudes and durations. DNA oligomers with a single-nucleotide difference, 5'-AAAA-3' and 5'-GAAA-3', are employed as model analytes that are difficult to identify in aerolysin nanopores at 100 mV. First, a set of the most informative and discriminative segments are learned from the time-series data set of blockade current signals using the learning time-series shapelets (LTS) algorithm. Then, the shapelet-transformed representation of the signals is obtained by calculating the minimum distance between the shapelets and the original signals. A simple logistic classifier is used to identify the two types of DNA oligomers in accordance with the corresponding shapelet-transformed representation. Finally, an evaluation is performed on the validation data set to show that our approach can achieve a high F 1 score of 0.933. In comparison with the conventional statistical methods for the analysis of duration and residual current, the shapelet-transformed representation provides clearly discriminated distributions for multiple analytes. Taking advantage of the robust LTS algorithm, one could anticipate the real-time analysis of nanopore events for the direct identification and quantification of multiple biomolecules in a complex real sample (e.g., serum) without labels and time-consuming mutagenesis.

Protein kinases play a critical role in regulating virtually all cellular processes. Here, we developed a novel one-step method based on a wild-type aerolysin nanopore, which enables kinase activity detection without labeling/modification, immobilization, cooperative enzymes and complicated procedures. By virtual of the positively charged confinement of the aerolysin nanopore, the kinase-induced phosphopeptides are specially captured while the positively charged substrate peptides might move away from the pore by the electric field. Combining with internal standard method, the event frequency of the phosphopeptides exhibited a dose-dependent response with kinases. The detection limit of 0.005 U/μL has been achieved with protein kinase A as a model target. This method also allowed kinase inhibitor screening, kinase activity sensing in cell lysates and the real-time monitoring of kinase-catalyzed phosphorylation at singe molecule level, which could further benefit fundamental biochemical research, clinical diagnosis and kinase-targeted drug discovery. Moreover, this nanopore sensor shows strong capacity for the other enzymes that altered substrate charge (e.g., sulfonation, carboxylation, or amidation).

Nanopore is a single-molecule analysis method which also employed electrophoresis has achieved promising single-molecule detections. In this study, we designed two kinds of confined spaces by fabricating solid-state nanopores with desirable diameters to study the structured single-strand DNA of C-rich quadruplex. For the nanopore whose diameter is larger than the quadruplex size, the DNA molecule could directly translocate through the nanopore with extremely high speed. For the nanopore whose diameter is smaller than the quadruplex size, DNA molecule which is captured by nanopore could return to the solution without translocation or unzip the quadruplex structure into single-strand and then pass the nanopore. This study certifies that choosing a suitable sensing interface is the vital importance of observing detailed single-molecule information. The solid-state nanopores hold the great potential to study the structural dynamics of quadruplex DNA molecule., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The aerolysin nanopore displays a charming sensing capability for single oligonucleotide discrimination. When reading from the electrochemical signal, stronger interaction between the aerolysin nanopore and oligonucleotide represent prolonged duration time, thereby amplifying the hidden but intrinsic signal thus improving the sensitivity. In order to further understand and optimize the performance of the aerolysin nanopore, we focus on the investigation of the hydrogen bond interaction between nanopore, and analytes. Taking advantage of site-direct mutagenesis, single residue is replaced. According to whole protein sequence screening, the region near K238 is one of the key sensing regions. Such a positively charged amino acid is then mutagenized into cysteine and tyrosine denoted as K238C, and K238Y. As (dA) 4 traverses the pores, K238C dramatically produces a six times longer duration time than the WT aerolysin nanopore at the voltage of +120 mV. However, K238Y shortens the dwell time which suggests the acceleration of the translocation causing poor sensitivity. Referring to our previous findings in K238G, and K238F, our results suggest that the hydrogen bond does not dominate the dynamic translocation process, but enhances the interaction between pores and analytes confined in such nanopore space. These insights give detailed information for the rational design of the sensing mechanism of the aerolysin nanopore, thereby providing further understanding for the weak interactions between biomolecules and the confined space for nanopore sensing.

Measurements of a single entity underpin knowledge of the heterogeneity and stochastics in the behavior of molecules, nanoparticles, and cells. Electrochemistry provides a direct and fast method to analyze single entities as it probes electron/charge-transfer processes. However, a highly reproducible electrochemical-sensing nanointerface is often hard to fabricate because of a lack of control of the fabrication processes at the nanoscale. In comparison with conventional micro/nanoelectrodes with a metal wire inside, we present a general and easily implemented protocol that describes how to fabricate and use a wireless nanopore electrode (WNE). Nanoscale metal deposition occurs at the tip of the nanopipette, providing an electroactive sensing interface. The WNEs utilize a dynamic ionic flow instead of a metal wire to sense the interfacial redox process. WNEs provide a highly controllable interface with a 30- to 200-nm diameter. This protocol presents the construction and characterization of two types of WNEs-the open-type WNE and closed-type WNE-which can be used to achieve reproducible electrochemical measurements of single entities. Combined with the related signal amplification mechanisms, we also describe how WNEs can be used to detect single redox molecules/ions, analyze the metabolism of single cells, and discriminate single nanoparticles in a mixture. This protocol is broadly applicable to studies of living cells, nanomaterials, and sensors at the single-entity level. The total time required to complete the protocol is ~10-18 h. Each WNE costs ~$1-$3.

Multidrug-resistant bacteria are a great concern and a problem that must be addressed. Extended-spectrum β-lactamases are a common defence mechanism of bacteria to make β-lactam (BL) antibiotics ineffective. β-Lactamase inhibitors (BLIs) are consequently designed and are often clinically prescribed with a BL antibiotic to hinder degradation. Current studies focusing on how BL antibiotics or BLIs interact solely with the bacterial outer membrane nanopores (porins) on reaching the periplasmic side using a nanopore-based sensing technique. In electrochemical studies, the bias voltage allows real-time monitoring of BL antibiotics, BLIs and their mixture through the porin pathway at the single-molecule level. Here we consider the most abundant membrane protein from Escherichia coli (i.e. OmpF), purify and reconstitute the membrane protein in an artificial lipid bilayer and then study its ex vivo electrochemical behaviour. We show the piperacillin/tazobactam mixture interacts with OmpF, whereas the substrate interacts under the maximum bandwidth. The power spectrum analysis of the ionic current trace demonstrates the ampicillin/sulbactram mixture requires more energy than ampicillin alone to pass through the porin pathway. Our results demonstrate that clinically relevant combinations (e.g. piperacillin/tazobactam and ampicillin/sulbactam) interact more strongly with OmpF than either the BL antibiotic or the BLI alone. We suggest a quick and relatively cheap screening method to test the ability of BL antibiotics/BLIs to cross the bacterial cellular membrane.

Experimental techniques to monitor and visualize the behaviors of single nanoparticles have not only revealed the significant spatial and temporal heterogeneity of those individuals, which are hidden in ensemble methods, but more importantly, they have also enabled researchers to elucidate the origin of such heterogeneity. In pursuing the intrinsic structure-function relations of single nanoparticles, the recently developed stochastic collision approach demonstrated some early promise. However, it was later realized that the appropriate sizing of a single nanoparticle by an electrochemical method could be far more challenging than initially expected owing to the dynamic motion of nanoparticles in electrolytes and complex charge-transfer characteristics at electrode surfaces. This clearly indicates a strong necessity to integrate single nanoparticle electrochemistry with high-resolution optical microscopy. Hence, this review aims to give a timely update of the latest progress for both electrochemically sensing and seeing single nanoparticles. A major focus is on collision-based measurements, where nanoparticles or single entities in solution impact on a collector electrode and the electrochemical response is recorded. These measurements are further enhanced with optical measurements in parallel. For completeness, advances in other related methods for single nanoparticle electrochemistry are also included.

Single protein sensing based on solid-state nanopores is promising but challenging, because the fast translocation velocity of a protein is beyond the bandwidth of nanopore instruments. To decelerate the translocation speed, here, we employed a common protein cross-link interaction to achieve a general and robust nanopore sensing platform for single-molecule detection of protein. Benefiting from the EDC/NHS coupling interaction between nanopore and proteins, a 10-fold decrease in speed has been achieved. The clearly distinguishable current signatures further reveal that the anisotropic translocation of a protein, which are horizontal, vertical, and flipping transit inside nanopore confinement. This strategy provides a general platform for rapid detection of proteins as well as exploring fundamental protein dynamics at the single-molecule level.

Measuring the intrinsic features of single nanoparticles by nanoelectrochemistry holds deep fundamental importance and has potential impacts in nanoscience. However, electrochemically analyzing single nanoparticles is challenging, as the sensing nanointerface is uncontrollable. To address this challenge, we describe here the fabrication and characterization of a closed-type wireless nanopore electrode (WNE) that exhibits a highly controllable morphology and outstanding reproducibility. The facile fabrication of WNE enables the preparation of well-defined nanoelectrodes in a general chemistry laboratory without the use of a clean room and expensive equipment. One application of a 30 nm closed-type WNE in analysis of single gold nanoparticles in the mixture is also highlighted, which shows a high current resolution of 0.6 pA and high temporal resolution of 0.01 ms. Accompanied by their excellent morphology and small diameters, more potential applications of closed-type WNEs can be expanded from nanoparticle characterization to single molecule/ion detection and single-cell probing.

Nanopipettes provide a promising confined space that enables advances in electrochemical, optical, and mass spectrometric measurements at the nanoscale. They have been employed to reveal the hidden population properties and dynamics of single molecules and single particles. Moreover, new detection mechanisms based on nanopipettes have led to detailed information on single cells at high spatial and temporal resolution. In this Minireview, we focus on the fabrication and characterization of nanopipettes, summarize their wide applications for the analysis of single entities, and conclude with an outlook for advanced practical sensing., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The field of nanopore sensing at the single-molecular level is in a "boom" period. Such nanopores, which are either composed of biological materials or are fabricated from solid-state substrates, offer a unique confined space that is compatible with the single-molecular scale. Under the influence of an electrical field, such single-biomolecular interfaces can read single-molecular information and, if appropriately fine-tuned, each molecule plays its individual ionic rhythm to compose a "molecular symphony". Over the past few decades, many research groups have worked on nanopore-based single-molecular sensors for a range of thrilling chemical and clinical applications. Furthermore, for the past decade, we have also focused on nanopore-based sensors. In this Minireview, we summarize the recent developments in fundamental research and applications in this area, along with data algorithms and advances in hardware, which act as infrastructure for the electrochemical analysis., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

RNA sensing is of vital significance to advance our comprehension of gene expression and to further benefit medical diagnostics. Taking advantage of the excellent sensing capability of the aerolysin nanopore as a single-biomolecule interface, we for the first time achieved the direct characterization of single native RNA of Poly(A) 4 and Poly(U) 4 . Poly(A) 4 induces ∼10% larger blockade current amplitude than Poly(U) 4 . The statistical duration of Poly(A) 4 is 18.83 ± 1.08 ms, which is 100 times longer than that of Poly(U) 4 . Our results demonstrated that the capture of RNA homopolymers is restricted by the biased diffusion. The translocation of RNA needs to overcome a lower free-energy barrier than that of DNA. Moreover, the strong RNA-aerolysin interaction is attributed to the hydroxyl in pentose, which prolongs the translocation time. This study opens an avenue for aerolysin nanopores to directly achieve RNA sensing, including discrimination of RNA epigenetic modification and selective detection of miRNA.

Wild-type aerolysin (AeL) nanopores allow direct single nucleotide discrimination of very short oligonucleotides (≤10 nt) without labelling, which shows great potential for DNA sensing. To achieve real applications, one major obstacle of AeL is its poor capture ability of long single-stranded DNA (ssDNA, >10 nt). Here, we have proposed a novel and robust strategy for the electrostatic focusing of long ssDNA into a lithium-chloride (LiCl)-active AeL. By using this method, for the first time we have demonstrated AeL detection of ssDNA longer than 100 nt. Due to screening more negative charges, LiCl improves AeL capture ability of long ssDNA ( i.e. 60 nt) by 2.63- to 10.23-fold compared to KCl. Further calculations and molecular dynamics simulations revealed that strong binding between Li + and the negatively charged residue neutralized the AeL, leading to a reduction in the energy barrier for ssDNA capture. These findings facilitate the future high-throughput applications of AeL in genetic and epigenetic diagnostics.

The evolution of the nanobubble from gas molecules in liquid phase is of fundamental interest. However, the lack of sensitive tools hinders the study of growth dynamic of the bubble at nanoscale. Here, we employed a confined glass nanopore to real-time monitor the dynamics behavior of a single nanobubble generated by the reaction between NaBH 4 and H 2 O. By analyzing the characteristic ionic current signal, the formation time and growth time of a single nanobubble could be estimated as 200 and 21 ms, respectively. Further, the nanopore size has been altered to modulate the growth behavior of the nanobubble. The results demonstrate the capability of the nanopore for sensitively tracking the behavior of single nanobubbles in liquid phase, which provides a powerful method for further understanding nanobubble evolution.

Nanopore analysis is a powerful technique for single molecule analysis by virtue of its electrochemically confined effects. As a single molecule translocates through the nanopore, the featured ionic current pattern on the time scale contains single molecule characteristics including volume, charge, and conformational properties. Although the characteristics of a single molecule in a nanopore have been written to the featured ionic current, extracting the dynamic information from a complex current trace is still a big challenge. Here, we present an applicable nanopore analysis method employing the Hilbert-Huang Transform (HHT) to study the vibrational features and interactions of a single molecule during the dynamic translocation process through the confined space of a nanopore. The HHT method is specially developed for analyzing nonlinear and non-stationary data that is highly compatible with nanopore data with a high frequency resolution. To provide proof-of-concept, we applied HHT to measure the frequency response for the wild-type (WT) aerolysin and mutant K238E aerolysin nanopores with and without the presence of poly(dA)4, respectively. The energy-frequency-time distribution spectra demonstrate that the biological nanopore contributes greatly to the characteristics of the high frequency component (>2 kHz) in the current recording. Our results suggest that poly(dA)4 undergoes relatively more consistent and confined interactions with K238E than WT, leading to a prolonging of the duration time. Therefore, the characteristics in frequency analysis could be regarded as an "single-molecule ionic spectrum" inside the nanopore, which encodes the detailed behaviours of single-molecule weak interactions.

Aggregation-induced emission (AIE) as a unique photophysical process has been intensively explored for their features in fields from optical sensing, bioimaging to optoelectronic devices. However, all AIE luminogens (AIEgens) hardly recover into the initial dispersed state after illuminating at the ultimate aggregated state, which limits AIEgens to achieve reversible sensing and reproducible devices. To real-time manipulate the emission of AIEgen, here we take the advantage of confined space in the quartz nanopore to achieve a nanopore-size-dependent restriction of AIEgens for reversible conversions of "on-to-off" and "off-to-on" emission. By electrochemically manipulating 26 fL AIEgen solution inside nanopore confinement, AIE illuminates while moves along nanopore from the constricted tip to inside cavity at a velocity of 1.4-2.2 μm s -1 , and vice versa. We further apply this dynamic manipulation for a target delivery of AIEgen into single cells, which opens up new possibility to design powerful and practical AIE applications.

The nanopore can generate an electrochemical confinement for single-molecule sensing that help understand the fundamental chemical principle in nanoscale dimensions. By observing the generated ionic current, individual bond-making and bond-breaking steps, single biomolecule dynamic conformational changes and electron transfer processes that occur within pore can be monitored with high temporal and current resolution. These single-molecule studies in nanopore confinement are revealing information about the fundamental chemical and biological processes that cannot be extracted from ensemble measurements. In this Concept article, we introduce and discuss the electrochemical confinement effects on single-molecule covalent reactions, conformational dynamics of individual molecules and host-guest interactions in protein nanopores. Then, we extend the concept of nanopore confinement effects to confine electrochemical redox reactions in solid-state nanopores for developing new sensing mechanisms., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The aerolysin nanopore channel is one of the confined spaces for single molecule analysis which displays high spatial and temporal resolution for the discrimination of single nucleotides, identification of DNA base modification, and analyzing the structural transition of DNAs. However, to overcome the challenge of achieving the ultimate goal of the widespread real analytical application, it is urgent to probe the sensing regions of the aerolysin to further improve the sensitivity. In this paper, we explore the sensing regions of the aerolysin nanopore by a series of well-designed mutant nanopore experiments combined with molecular dynamics simulations-based electrostatic analysis. The positively charged lumen-exposed Lys-238, identified as one of the key sensing sites due to the presence of a deep valley in the electrostatic potentials, was replaced by different charged and sized amino acids. The results show that the translocation time of oligonucleotides through the nanopore can be readily modulated by the choice of the target amino acid at the 238 site. In particular, a 7-fold slower translocation at a voltage bias of +120 mV is observed with respect to the wild-type aerolysin, which provides a high resolution for methylated cytosine discrimination. We further determine that both the electrostatic properties and geometrical structure of the aerolysin nanopore are crucial to its sensing ability. These insights open ways for rationally designing the sensing mechanism of the aerolysin nanopore, thus providing a novel paradigm for nanopore sensing.

To enable electrochemistry-based sensors to be applied under size and cost sensitive conditions, we developed a thumb-size electrochemistry instrument system (MiniEC) with integrated components including an electrochemical sensing interface, signal transduction, amplification, filtering, processing and transmission with a cost of around only $15. We showed its potential application for electrochemical studies and on-site environmental monitoring in a wide range of environments such as laboratory, household, countryside, industry or wild settings. By using the MiniEC with screen printed electrodes, the LOD (limit of detection) for Cd2+ and Pb2+ is as low as 1 μg L-1 and 0.5 μg L-1, respectively.

An aerolysin nanopore is employed as a sensitive tool for single-molecule analysis of short oligonucleotides (≤10 nucleotides), poly(ethylene glycol) (PEGs), peptides, and proteins. However, the direct analysis of long oligonucleotides with the secondary structure (e.g., G-quadruplex topology) remains a challenge, which impedes the further practical applications of the aerolysin nanopore. Here, a simple and applicable method of aerolysin nanopore is presented to achieve a direct analysis of structured oligonucleotides that are extended to 30 nucleotides long by a cation-regulation mechanism. By regulating the cation type in electrolyte solution, the structured oligonucleotides are unfolded into linear form which ensures the successive translocation. The results show that each model oligonucleotide of 5'-(TTAGGG) n -3' can produce a well-resolved current blockade in its unfolded solution of MgCl 2 . The length between 6 and 30 nucleotides long of model oligonucleotides is proportional to the duration time, showing a translocation velocity as low as 0.70-0.13 ms nt -1 at +140 mV. This method exhibits an excellent sensitivity and a sufficient temporal resolution, provides insight into the aerolysin nanopore methodology for genetic and epigenetic biosensing, making aerolysin applicable in practical diagnosing with long and structured nucleic acids., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Selectivity and sensitivity are two key parameters utilized to describe the performance of a sensor. In order to investigate selectivity and sensitivity of the aerolysin nanosensor, we manipulated its surface charge at different locations via single site-directed mutagenesis. To study the selectivity, we replaced the positively charged R220 at the entrance of the pore with negatively charged glutamic acid, resulting in barely no current blockages for sensing negatively charged oligonucleotides. For the sensitivity, we substituted the positively charged lumen-exposed amino acid K238 located at trans-ward third of the β-barrel stem with glutamic acid. This leads to a surprisingly longer duration time at +140 mV, which is about 20 times slower in translocation speed for Poly(dA) 4 compared to that of wild-type aerolysin, indicating the stronger pore-analyte interactions and enhanced sensitivity. Therefore, it is both feasible and understandable to rationally design confined biological nanosensors for single molecule detection with high selectivity and sensitivity.

Capturing real-time electron transfer, enzyme activity, molecular dynamics, and biochemical messengers in living cells is essential for understanding the signaling pathways and cellular communications. However, there is no generalizable method for characterizing a broad range of redox-active species in a single living cell at the resolution of cellular compartments. Although nanoelectrodes have been applied in the intracellular detection of redox-active species, the fabrication of nanoelectrodes to maximize the signal-to-noise ratio of the probe remains challenging because of the stringent requirements of 3D fabrication. Here, we report an asymmetric nanopore electrode-based amplification mechanism for the real-time monitoring of NADH in a living cell. We used a two-step 3D fabrication process to develop a modified asymmetric nanopore electrode with a diameter down to 90 nm, which allowed for the detection of redox metabolism in living cells. Taking advantage of the asymmetric geometry, the above 90% potential drop at the two terminals of the nanopore electrode converts the faradaic current response into an easily distinguishable bubble-induced transient ionic current pattern. Therefore, the current signal was amplified by at least 3 orders of magnitude, which was dynamically linked to the presence of trace redox-active species. Compared to traditional wire electrodes, this wireless asymmetric nanopore electrode exhibits a high signal-to-noise ratio by increasing the current resolution from nanoamperes to picoamperes. The asymmetric nanopore electrode achieves the highly sensitive and selective probing of NADH concentrations as low as 1 pM. Moreover, it enables the real-time nanopore monitoring of the respiration chain (i.e., NADH) in a living cell and the evaluation of the effects of anticancer drugs in an MCF-7 cell. We believe that this integrated wireless asymmetric nanopore electrode provides promising building blocks for the future imaging of electron transfer dynamics in live cells.

Identification of the configuration for the photoresponsive oligonucleotide plays an important role in the ingenious design of DNA nanomolecules and nanodevices. Due to the limited resolution and sensitivity of present methods, it remains a challenge to determine the accurate configuration of photoresponsive oligonucleotides, much less a precise description of their photoconversion process. Here, we used an aerolysin (AeL) nanopore-based confined space for real-time determination and quantification of the absolute cis/ trans configuration of each azobenzene-modified oligonucleotide (Azo-ODN) with a single molecule resolution. The two completely separated current distributions with narrow peak widths at half height (<0.62 pA) are assigned to cis/ trans-Azo-ODN isomers, respectively. Due to the high current sensitivity, each isomer of Azo-ODN could be undoubtedly identified, which gives the accurate photostationary conversion values of 82.7% for trans-to- cis under UV irradiation and 82.5% for cis-to- trans under vis irradiation. Further real-time kinetic evaluation reveals that the photoresponsive rate constants of Azo-ODN from trans-to- cis and cis-to -trans are 0.43 and 0.20 min -1 , respectively. This study will promote the sophisticated design of photoresponsive ODN to achieve an efficient and applicable photocontrollable process.

Clarifying the hidden but intrinsic feature of single nanoparticles by nanoelectrochemistry could help understand its potential for diverse applications. The uncontrolled interface and bandwidth limitation in the electrochemical measurement put the obstacle in single particle collision. Here, we demonstrate a well-defined 30 nm nanopore electrode with a rapid chemical-electrochemical fabrication method which provides a high reproducibility in both size and performance. A capacitance-based detection mechanism is demonstrated to achieve a high current resolution of 0.6 pA ±0.1 pA (RMS) and a high the temporal resolution of 0.01 ms. By utilizing this electrode, the dynamic interactions of every single particle in the mixture could be directly read during the collision process. The collision frequency is two orders of magnitude higher than previous reports, which helps reveal the hidden features of nanoparticles during the complex and multidimensional interaction processes., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Informational masking (IM) can greatly reduce speech intelligibility, but the neural mechanisms underlying IM are not understood. Binaural differences between target and masker can improve speech perception. In general, improvement in masked speech intelligibility due to provision of spatial cues is called spatial release from masking. Here, we focused on an aspect of spatial release from masking, specifically, the role of spatial attention. We hypothesized that in a situation with IM background sound (a) attention to speech recruits lateral frontal cortex (LFCx) and (b) LFCx activity varies with direction of spatial attention. Using functional near infrared spectroscopy, we assessed LFCx activity bilaterally in normal-hearing listeners. In Experiment 1, two talkers were simultaneously presented. Listeners either attended to the target talker (speech task) or they listened passively to an unintelligible, scrambled version of the acoustic mixture (control task). Target and masker differed in pitch and interaural time difference (ITD). Relative to the passive control, LFCx activity increased during attentive listening. Experiment 2 measured how LFCx activity varied with ITD, by testing listeners on the speech task in Experiment 1, except that talkers either were spatially separated by ITD or colocated. Results show that directing of auditory attention activates LFCx bilaterally. Moreover, right LFCx is recruited more strongly in the spatially separated as compared with colocated configurations. Findings hint that LFCx function contributes to spatial release from masking in situations with IM.

Here, we show a designed solid-state nanopore sensor for the direct sensing and quantification of prostate-specific antigen (PSA) as cancer biomarker in serum without any pretreatment. This nanopore technique provides a convenient, fast, and low-cost quantification of cancer biomarkers in clinical samples.

Nanopore is an ultra-sensitive electrochemical technique for single molecular detection in confined space. To suppress the noise in detection of the weak current of nanopore, we investigated the influence of membrane capacitance and applied voltage on the noise of the current signal by model analysis, simulation and experiment. The obtained results demonstrated that membrane capacitance affects the noise by amplifying the noise of the applied voltage. Therefore, suppression of applied voltage noise is an efficient approach for reducing the noise in nanopore detection. Here, we developed an ultra-low noise instrument system for detecting the single molecule signal in nanopores. As demonstrated by nanopore experiments, the p-p noise of the developed system during the recording is reduced to 3.26pA using the filter of 5kHz. Therefore, the developed system could be applied in highly sensitive nanopore detection., (Copyright © 2017 Science China Press. Published by Elsevier B.V. All rights reserved.)

To achieve accurate detection of cancer biomarkers with nanopore sensors, the precise recognition of multi-level current blockage events (signature) is a pivotal problem. However, it remains rather a challenge to identify the multi-level current blockages of target biomarkers in nanopore experiments, especially for the nanopore analysis of serum samples. In this work, we combined a modified DBSCAN (Density-Based Spatial Clustering of Applications with Noise) algorithm with the Viterbi training algorithm of the hidden Markov model (HMM) to achieve intelligent retrieval of multi-level current signatures from microRNA in serum samples. The results showed that the developed intelligent data analysis method is highly efficient for processing the large-scale nanopore data, which facilitates future application of nanopores to the clinical detection of cancer biomarkers.

Herein, the structural stability of single azobenzene modified DNA duplexes, including the trans form and cis form, has been examined separately based on their distinguishable unzipping kinetics from the mixture by an α-hemolysin nanopore. Furthermore, the accurate isomerization efficiency between the trans and cis form was obtained with single molecule resolution.

The nanopipette has been employed for the single molecule analysis due to its advantage of easy fabrication and controllable diameter. Herein, we present that the single molecule immunoreaction could be monitored by using the quartz nanopipette through the discrimination of characteristic blockade current, which reflect the intrinsic character of the individual unlabeled protein molecules due to its heterogeneous motion in solution. Our methods show the ability to monitor the immunoreaction between single α-fetal protein (AFP) and its specific antibody in aqueous solution without any labeling. Our studies may open a new door to comprehensively understand the single molecule immunoreaction, which gain more insight into the molecular dynamic of elementary steps.

Understanding the single molecular protein-protein interaction has great significance in evaluating the affinity of a specific antibody. Herein, the interaction between single α-fetal protein (AFP) and its antibody was monitored via transient ionic current recording by using the antibody functionalized nanopore sensors. More importantly, the kinetic evaluation was performed at the single molecule level to determine the dissociation constant of this interaction. This method enables the monitoring of the kinetic antigen-antibody interaction in their heterogenetic state without any labelling. Our results provided new insights into the evaluation of the antibody's binding affinity and more into the development of immunoassays for diagnostics.

Solid-state nanopore-based techniques have become a promising strategy for diverse single molecule detections. Owing to the challenge in well and rapid fabrication of solid-state nanopores with the diameter less than 2 nm, small molecule detection is hard to be addressed by existing label-free nanopore methods. In this work, we for the first time propose a metal-coated wireless nanopore electrode (WNE) which offers a novel and generally accessible detection method for analyzing small molecules and ions at the single molecule/ion level. Here, a silver-coated WNE is developed as a proof-of-principle model which achieves the detection the self-generated H 2 , the smallest known molecule, and Ag + at single molecule/ion level by monitoring the enhanced ionic signatures. Under a bias potential of -800 mV, the WNE could accomplish the distinction of as low as 14 H 2 molecules and 28 Ag + from one spike signal. The finite element simulation is introduced to suggest that the generation of H 2 at the orifice of the WNE results in the enhanced spike of ionic current. As a proof-of-concept experiment, the WNE is further utilized to directly detect Hg 2+ from 100 pM to 100 nM by monitoring the frequency of the spike signals. This novel nanoelectrode provides a brand new label-free, ultrasensitive, and simple detection mechanism for various small molecules/ions detection, especially for redox analytes.

The self-assembly of nanoparticles is a challenging process for organizing precise structures with complicated and ingenious structures. In the past decades, a simple, high-efficiency, and reproducible self-assembly method from nanoscale to microscale has been pursued because of the promising and extensive application prospects in bioanalysis, catalysis, photonics, and energy storage. However, microscale self-assembly still faces big challenges including improving the stability and homogeneity as well as pursuing new assembly methods and templates for the uniform self-assembly. To address these obstacles, here, a novel silver-coated nanopore is developed which serves as a template for electrochemically generating microcyclic structures of gold nanoparticles at micrometers with highly homogenous size and remarkable reproducibility. Nanopore-induced microcyclic structures are further applied to visualize the diffusion profile of ionic flux. Based on this novel strategy, a nanopore could potentially facilitate the delivery of assembled structures for many practical applications including drug delivery, cellular detection, catalysis, and plasmonic sensing., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)