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We developed a bipolar SiNx nanopore for the observation of single-molecule heterogeneous enzymatic dynamics.

This review highlights the important historical and technological developments of in situ ToF-SIEMS technique, especially, the challenges encountered when extending ToF-SIMS from a high vacuum condition to a liquid electrochemical environment.

Octameric Aep1 shows unique suitability for the gamma-cyclodextrin (γ-CD) adapter with 8 subunits, enabling the discrimination for β-nicotinamide mononucleotide.

Inspired by the biological processes of molecular recognition and transportation across membranes, nanopore techniques have evolved in recent decades as ultrasensitive analytical tools for individual molecules. In particular, nanopore-based single-molecule DNA/RNA sequencing has advanced genomic and transcriptomic research due to the portability, lower costs and long reads of these methods. Nanopore applications, however, extend far beyond nucleic acid sequencing. In this Review, we present an overview of the broad applications of nanopores in molecular sensing and sequencing, chemical catalysis and biophysical characterization. We highlight the prospects of applying nanopores for single-protein analysis and sequencing, single-molecule covalent chemistry, clinical sensing applications for single-molecule liquid biopsy, and the use of synthetic biomimetic nanopores as experimental models for natural systems. We suggest that nanopore technologies will continue to be explored to address a number of scientific challenges as control over pore design improves.

The precise manipulation of single cells plays a fundamental role for single cell measurement, which is crucial for understanding the diverse cellular mechanisms. Unusual single cell behavior could thus be identified by integrating with advanced analytical methods such as single cell omics, unraveling the intrinsic cellular heterogeneity hidden in ensemble measurements. Herein, this technical note reports a nanopipet-based versatile method for manipulation of an ultrasmall volume of liquid, which further enables the precise manipulation of single cells. Femtoliter volumes of cytoplasm were extracted from single living cells and analyzed by time-of-flight secondary ion mass spectrometry. Moreover, several kinds of exogenous components were injected simultaneously into a cell, offering a delicate tool for multi-imaging in single living cells.

Chirality is essential in nearly all biological organization and chemical reaction but is rarely considered due to technical limitations in identifying L/D isomerization. Using OmpF, a membrane channel from E. coli with an electrostatically asymmetric constriction zone, allows discriminating chiral amino acids in a single peptide. The heterogeneous distribution of charged residues in OmpF causes a strong asymmetric electrostatic field at the constriction. This asymmetry forces the sidechains of the peptides to specific orientations within OmpF, causing distinct ion current fluctuations. Using a statistical analysis of the respective ion current variations allows distinguishing the presence and position of each chiral amino acid. To explore potential applications, the disease-related peptide β-Amyloid, and its D-Asp1 isoform, as well as a mixture of the Icatibant peptide drug (HOE 140) and its D-Ser7 mutant, have been discriminated. Both chiral isomers were not applicable to be distinguished by mass spectrum approaches. These findings highlight a novel sensing mechanism for identifying single amino acids in single peptides and even for achieving single-molecule protein sequencing.

RNA modifications modulate essential cellular functions. However, it is challenging to quantitatively identify the differences in RNA modifications. To further improve the single-molecule sensing ability of nanopores, we propose a machine-learning algorithm called SmartImage for identifying and classifying nanopore electrochemical signals based on a combination of improved graph conversion methods and deep neural networks. SmartImage is effective for nearly all ranges of signal duration, which breaks the limitation of the current nanopore algorithm. The overall accuracy (OA) of our proposed recognition strategy exceeded 90% for identifying three types of RNAs. Prediction experiments show that the SmartImage owns the ability to recognize one modified RNA molecule from 1000 normal RNAs with OA90%. Thus our proposed model and algorithm hold the potential application in clinical applications.

A biological nanopore is one of the predominant single-molecule approaches as a result of its controllable single-biomolecule interface, which could reflect the "intrinsic" information on an individual molecule in a label-free way. Because the current blockage is normally treated as the most important parameter for nanopore identification of every single molecule, the fluctuation of current blockage for certain types of molecules, defined as full width at half maximum (fwhm) of current blockage, actually owns a dominant influence on nanopore resolution. Therefore, controlling the fwhm of current blockage of molecules is critical for the sensing capability of the nanopore. Here, taking an aerolysin nanopore as a model, by precisely controlling the functional group in this single-biomolecule interface, we could narrow the fwhm of nanopore current blockage for DNA identification and prolong the duration inside the nanopore. Moreover, a substantial correlation between fwhm of current blockage and duration is established, showing a non-monotonic variation. Besides, the mechanism is also clarified with studying the detailed current blockage events. This proposed correlation is further demonstrated to be applied uniformly across different mutant aerolysins for a certain DNA. This study proposes a new strategy for regulating molecular sensing from the duration of the analyte, which could guide the resolution of heterogeneity analysis using nanopores.

Protein-protein interactions occur in a wide range of biological processes and are of great significance to life function. Characterization of transient protein-protein interactions remains a significant barrier to our understanding of cellular processes. Nanopores provide unique nanoscale environments that accommodate single molecules from the surrounding bulk solution. This method permits label-free sensing at the single-molecule level with extremely high sensitivity. Herein, the interaction between a single P450cam monooxygenase and its redox partner putidaredoxin (Pdx) was monitored via transient ionic current by using functionalized glass nanopores. Results show that the volume of P450cam determines the blockage current while the interactions between the P450cam and Pdx give a long blockage duration. Our glass nanopore sensor with adjustable diameter could be applied for real-time sensing of protein-protein interactions between individual proteins with a wide range of molecular weight.

The transport of molecules and ions through biological nanopores is governed by interaction networks among restricted ions, transported molecules, and residue moieties at pore inner walls. However, identification of such weak ion fluctuations from only few tens of ions inside nanopore is hard to achieve owing to electrochemical measurement limitations. Here, we developed an advanced frequency method to achieve qualitative and spectral analysis of ion interaction networks inside a nanopore. The peak frequency fm reveals the dissociation rate between nanopore and ions; the peak amplitude am depicts the amount of combined ions with the nanopore after interaction equilibrium. A mathematical model for single-molecule frequency fingerprint achieved the prediction of interaction characteristics of mutant nanopores. This single-molecule frequency fingerprint is important for classification, characterization, and prediction of synergetic interaction networks inside nanoconfinement.

The nanopore approach holds the possibility for achieving single-molecule protein sequencing. However, ongoing challenges still remain in the biological nanopore technology, which aims to identify 20 natural amino acids by reading the ionic current difference with the traditional current-sensing model. In this paper, taking aerolysin nanopores as an example, we calculate and compare the current blockage of each of 20 natural amino acids, which are all far from producing a detectable current blockage difference. Then, we propose a modified solution conductivity of σ' in the traditional volume exclusion model for nanopore sensing of a peptide. The σ' value describes the comprehensive result of ion mobility inside a nanopore, which is related to but not limited to nanopore-peptide interactions, and the positions, orientations, and conformations of peptides inside the nanopore. The nanopore experiments of a short peptide (VQIVYK) in wild type and mutant nanopores further demonstrate that the traditional volume exclusion model is not enough to fully explain the current blockage contribution and that many other factors such as enhanced nanopore-peptide interactions could contribute to a dominant part of the current change. This modified sensing model provides insights into the further development of nanopore protein sequencing methods.

The potential distribution at the electrode interface is a core factor in electrochemistry, and it is usually treated by the classic Gouy-Chapman-Stern (G-C-S) model. Yet the G-C-S model is not applicable to nanosized particles collision electrochemistry as it describes steady-state electrode potential distribution. Additionally, the effect of single nanoparticles (NPs) on potential should not be neglected because the size of a NP is comparable to that of an electrode. Herein, a theoretical model termed as Metal-Solution-Metal Nanoparticle (M-S-MNP) is proposed to reveal the dynamic electrode potential distribution at the single-nanoparticle level. An explicit equation is provided to describe the size/distance-dependent potential distribution in single NPs stochastic collision electrochemistry, showing the potential distribution is influenced by the NPs. Agreement between experiments and simulations indicates the potential roles of the M-S-MNP model in understanding the charge transfer process at the nanoscale.

Accurate discrimination of amyloid-β (Aβ) peptides containing familial point mutations would advance the knowledge of their roles in early-onset Alzheimer's disease. Herein, we simultaneously identified the mutant A21G, E22G, E22Q, and the wild-type (WT) Aβ

Single-entity electrochemistry (SEE) provides powerful means to measure single cells, single particles, and even single molecules at the nanoscale by diverse well-defined interfaces. The nanoconfined electrode interface has significantly enhanced structural, electrical, and compositional characteristics that have great effects on the assay limitation and selectivity of single-entity measurement. In this Perspective, after introducing the dynamic chemistry interactions of the target and electrode interface, we present a fundamental understanding of how these dynamic interactions control the features of the electrode interface and thus the stochastic and discrete electrochemical responses of single entities under nanoconfinement. Both stochastic single-entity collision electrochemistry and nanopore electrochemistry as examples in this Perspective explore how these interactions alter the transient charge transfer and mass transport. Finally, we discuss the further challenges and opportunities in SEE, from the design of sensing interfaces to hybrid spectro-electrochemical methods, theoretical models, and advanced data processing.