Heparin(HP) and heparan sulfate(HS) are highly anionic glycosaminoglycans that play essential roles in diverse biological processes through the metal ion-mediated interactions with proteins. However, direct characterization of HS-metal ion interactions at the single-molecule level in solution remains challenging. Nanopore electrochemistry is a label-free and single-molecule technique that enables direct analysis of individual molecular interactions. In this study, a T232K/K238Q Aerolysin nanopore featuring an enhanced electrostatic repelling barrier was utilized to probe the interactions between HS and different metal ions. By systematically varying the electrolyte cations(Na+, K+, and Ca2+), we have found that the metal ions significantly regulate HS translocation behavior by modulating its conformation, charge screening, and HS-nanopore interactions. Notably, in addition to Ca2+, which exhibits strong binding affinity to HS, the monovalent cations Na+ and K+ with similar physicochemical properties and weaker binding also induce distinct single-molecule signal signatures. Our results demonstrate that the nanopore-based single-molecule analysis holds strong potential to resolve the fine structural features of HS, enabling the characterization of sulfation site distributions, repeat-unit lengths, and related sequence features, and thereby providing a new avenue for high-resolution analysis of complex glycans.

Conventional DNA encryption methods often require additional noncoding strands as physical keys or covering media, leading to a density decrease from redundancy. Here, we present a nanopore-based photoresponsive DNA information steganography system (NAPDISS) that encodes 26 encrypted English letters by using only five isomerizable azobenzene-modified coding DNAs, thereby eliminating the need for extra synthesis. Encoding was implemented through two complementary schemes comprising a letter code with sequence-defined photoresponsive DNAs representing letters and an address code with poly(dA)(3) (A3) concentrations defining the letters' positions. Utilizing light as secret keys, NAPDISS conceals messages by obscuring the nanopore readouts, allowing recovery only through combined pre- and post-irradiation analyses. This approach achieves a logical storage density of 0.2-1.0 bits per nucleotide-about one order of magnitude higher than those of existing DNA-structure-based methods. Moreover, simplified sample processing reduces the readout time from hours to <10 minutes. Collectively, this work provides fresh insights into balancing density, security and efficiency, advancing DNA steganography toward secure and instantaneous messaging applications.

Multiplex biomolecule profiling provides comprehensive molecular insights essential for precision health diagnosis. Yet, achieving simultaneous analysis of diverse biomolecule types, such as DNAs, RNAs, peptides, oligosaccharides, and metabolites, remains challenging due to their fundamentally diversity including size, charge, structure, and chemical composition. We present a nanopore multiplex sensor (NMS) based on a bioelectronic microchip that enables concurrent profiling of multiple biomolecules in a single experiment, which was previously unattainable. Cross-validation using multiple types of orthogonal nanopores with complementary recognition abilities offered the simultaneous detection and relative quantification of five major types of biomolecules relevant to non-small-cell lung cancer. Our work establishes a robust and generalizable nanopore platform for rapid and parallel biomarker sensing, providing an important step toward addressing the vital challenge of single-molecule multiomics.

Wireless nanopore electrodes (WNEs) exploit bipolar electrochemistry to provide stable and reproducible nanoscale electrochemical interfaces. The conductive material at the nanopore tip serves as a well-defined sensing interface. This feature enables high spatial and temporal resolution while eliminating the need for traditional wire sealing and thereby avoiding noise or instability arising from the contacted electrode. Therefore, the WNE has become a powerful tool in nanoelectrochemistry with broad applications across electroanalysis and electrocatalysis. This perspective outlines the fabrication of closed-type and open-type WNEs and discusses their applications in monitoring the growth of catalytic materials, assessing electrocatalytic activity, and performing intracellular measurements. Special emphasis is placed on their integration with mass spectrometry and optical spectroscopy to achieve multidimensional insights. Finally, we highlight future research directions, focusing on enhancing stability, precise surface functionalization, and the development of miniaturized, portable devices for practical use.

Posttranslational modifications (PTMs) are crucial regulators of protein functions, localization, and interactions, contributing to nearly all aspects of cellular physiology. Conventional analytical approaches, such as mass spectrometry (MS), have achieved high sensitivity and coverage, yet they often require complex sample preparation, enrichment, and high-end instrumentation. Nanopore has emerged as a promising single-molecule approach for PTM detection, offering label-free, high-throughput, and potentially portable analysis capabilities. Despite remarkable progress in nucleic acid sequencing with nanopores, its application to PTM detection in proteins remains at an early stage, hindered by challenges in protein capture, controlled unfolding, the interplay of sequence and structural heterogeneity, and diverse PTM chemistries. In this chapter, we summarize the current landscape of nanopore-based PTM identification and describe two experimental strategies that represent the most feasible near-term workflows for translating nanopore technology into practical protein PTM analysis.

Abstract The direct detection of small hydrophobic biomolecules remains a major challenge for biological nanopores. This is due to the weak interactions provided by the hydrophilic lumens, which result in ultrafast translocation and insufficient molecular recognition. Here, to reconcile the hydrophilicity for ionic flow and hydrophobic interactions for molecule recognition, we engineer a hydrophobic confinement stabilized by an interconnected hydrogen-bond network. By introducing tyrosine and tryptophan into the primary sensing region, a localized hydrophobic environment is established while maintaining nanopore structural stability and low current noise. Molecular dynamics simulations demonstrated the formation of a localized hydrophobic environment stabilized by an interconnected hydrogen-bond network, enabling the direct identification of thyroid hormones, aromatic amino acid-derived hydrophobic biomolecules containing iodinated aromatic structures, through enhanced molecular interactions. We show that the hydrophobic confinement could discriminate single iodine atom differences within diverse thyroid hormones, of which individual events can be automatically classified using an unsupervised clustering algorithm without prior model training. Furthermore, the isobaric triiodothyronine and reverse triiodothyronine could be directly resolved in a mixture. This work establishes hydrophobic confinement engineering as an effective strategy for extending biological nanopores to the direct single-molecule measurement of clinically relevant hydrophobic biomolecules.

ABSTRACT Protein oxidation generates diverse chemical states that regulate cellular signaling yet progressively accumulate as molecular damage during aging. However, characterizing oxidation‐state heterogeneity of individual proteins or peptides remains challenging, as conventional ensemble methods average over coexisting species. Here, we report a nanopore strategy based on a confined hydrogen‐bond network to decode oxidation states within individual native peptides. By engineering a constricted recognition region enriched with hydrogen‐bond interactions, the changes induced by diverse oxidation states were specifically recognized. This enables discrimination of closely related oxidative modifications, including proline hydroxylation, methionine oxidation, and tryptophan oxidation, even at ultra‐low abundance. We show that the reversible and regulated oxidation state could be clearly distinguished from irreversible and damaged oxidation with the same mass without separation or purification. Furthermore, we quantified heterogeneous oxidation distributions generated under controlled oxidative conditions. This approach provides a molecular‐level strategy for characterizing oxidation‐state heterogeneity in individual peptides, with potential applications in studying oxidative modifications associated with aging and disease in future biological investigations.

Reliable identification of proteins and their post-translationally modified variants remains a formidable analytical challenge due to charge heterogeneity, sequence similarity, and comparable molecular weights. In this study, we demonstrate distinctive nanopore current fingerprints for clear identification of neurodegenerative disease-associated Tau protein and its phosphorylated variants using an asymmetric-electrolyte sensing system composed of different salts. The asymmetric configuration facilitates simultaneous detection of positively, neutrally, and negatively charged peptide fragments, resulting in 3.2-16-fold higher capture frequencies and 2.1-5.3-fold longer event durations, thereby yielding information-rich fingerprints that enhance protein recognition. Protein profiling was achieved within 1 min through integration with a droplet nanopore platform, which reduces sample consumption to the nanogram level while increasing throughput to >1800 events per minute. This work advances the nanopore fingerprinting approach for rapid, high-throughput, and low-sample protein biomarker detection, offering strong potential for clinical proteomics and early disease diagnosis.

Abstract The experimentally observable dynamical landscape of biomolecules is fundamentally shaped by rapid thermal motions of the surrounding aqueous environment. Although lowering temperature could expand this observable landscape, the liquid-solid phase transition of water has long prevented real-time single-molecule measurements into deeply subzero aqueous environments. Here we show that nanoconfinement within a solid-state nanopore overcomes the fundamental limitation imposed by bulk water freezing, spontaneously stabilizing a persistent liquid-in-ice environment that remains electrically accessible despite surrounding electrolyte crystallization. This aqueous environment creates a time-stretched dynamical regime, extending molecular translocation timescales by up to ∼400-fold and revealing previously inaccessible single-molecule dynamics. These findings establish a new low-temperature aqueous regime for real-time single-molecule measurements, opening new opportunities to investigate biomolecular dynamics across previously inaccessible timescales and extreme aqueous environments.

Despite substantial progress in nanopore sensing, residue-by-residue peptide sequencing remains a major challenge. Herein, we present EANPSeq, an exopeptidase-assisted nanopore peptide identification strategy based on peptide libraries to decode the peptide sequence. By continuously recognizing the resulting fragments from digesting peptides stepwise through a nanopore, this approach could achieve the identification of peptide sequence based on the comparison of fragment data with libraries of shortened and mutated peptides, with the assistance of machine learning. Notably, compared with previously reported nanopore peptide sensing strategies, EANPSeq shows sufficient resolution to recognize the continuous sequence of peptides containing adjacent identical residues and to precisely localize post-translational modification (PTM) sites within consecutive residues. These proof-of-concept results highlight our nanopore-based strategy as a new avenue for single-molecule protein sequencing.

Reactive oxygen species (ROS) play central roles in neuroinflammatory signaling and neuroimmune function. However, quantitative detection of ROS in living neuroimmune cells remains challenging due to their low abundance and high sensitivity to electrical and chemical perturbations. While conventional nanoelectrodes can monitor ROS in cancer cells and macrophages, they require high operation potentials that may disrupt membrane integrity and redox homeostasis, limiting their use in sensitive neuroimmune cells. Here, we report a photo-driven asymmetric nanopore electrode (PNE) that enables zero-bias (0 mV) detection of intracellular H2O2. By integrating photo-responsive g-C3N4 quantum dots within an asymmetric quartz nanopipette, pulsed light excitation generates an ionic photocurrent that decreases quantitatively with increasing H2O2 concentration. The PNE delivers a linear detection range from 10 nM to 5 µM with a detection limit down to 10 nM. Further cellular imaging characterization confirms that this platform minimizes cellular perturbation, enabling in situ monitoring of intracellular H2O2 dynamics in single microglial cells under oxidative stress stimulation. By virtue of this minimal perturbation, this study represents the first real-time observation of a concentration-dependent transition in ROS scavenging dynamics in single microglial cells, providing a previously inaccessible view of neuroinflammatory redox regulation under undisturbed physiological conditions.

Glycans, unlike uniformly charged DNA and compositionally diverse peptides, are typically uncharged and possess rich stereoisomeric diversity in the glycosidic bonds between two monosaccharide units. These unique features, including charge heterogeneity and structural complexity, pose significant challenges for accurate analysis. Herein, we developed a novel single-molecule oligosaccharide sensor, OmpF nanopore. The natural electroosmotic flow within OmpF generates a robust driving force for unlabeled neutral oligosaccharides, enabling detection at a concentration as low as 6.4 μM. Furthermore, the asymmetric constriction zone of OmpF was employed to construct a stereoselective recognition site, enabling sensitive identification of glycosidic bond differences in cell lysate samples. With the assistance of machine learning algorithms, the OmpF nanopore achieved a recognition accuracy of 99.9 % for tetrasaccharides differing in only one glycosidic bond was achieved. This nanopore sensor provides a highly sensitive analytical tool with a broad dynamic range. It enables chiral recognition of oligosaccharides at low concentrations and is suitable for analysing both low-abundance and practical samples.

Enzymatic reactions in cells control the diversity of biomolecular composition, structure, and function, by virtue of their dynamics and heterogeneity. Here, we describe the use of a protein nanopore to monitor, in real time, the action of Exonuclease I (Exo I) on its substrate (homogeneous and heterogeneous short single-stranded DNA, ssDNA) on a single-reactant molecule basis. The nanopore-based single-molecule measurement, combined with a transition kinetic analysis, determines the temporal dynamics and heterogeneous cleavage and release pathways of ssDNA by Exo I. The results demonstrate a stepwise cleavage that is sequence-dependent on short ssDNA molecules (<15 nt), which differs from the kinetic model based on bulk measurements. In addition, we show that damaged DNA irreversibly changes the enzymatic reaction processes by Exo I. Thus, nanopores might prove to be useful for studying multienzyme cascade reactions at the single-molecule level.

Nanopipette electrochemistry has emerged as a versatile platform for nanoscale analytical measurements, functional device fabrication, and biomimetic interface construction. The confined geometry at the nanopipette orifice can be a powerful tool for ultrasensitive measurement through analyzing the ionic current across the nanopore. Chemists can rationally engineer the surface properties of nanopipettes by modifying the glass orifice through physical deposition or chemical reactions, enabling dynamic tuning of ion transport based on the interaction between the analyte and the interface. Such interfacial modulation governs the ionic flux and provides insights into local molecular processes. We first discuss ion current rectification (ICR) sensing, which enables surface-state probing via asymmetric ionic flux. Facilitated by the development of low-noise, high-bandwidth instruments, label-free and high-throughput detection and characterization of nanoparticles, single molecules, and real-time biological interactions could be achieved through resistive-pulse sensing. Furthermore, we highlight the role of wireless nanopore electrodes (WNEs) in studying electron transfer processes at the single-entity level, including redox processes in molecules, nanomaterials, and cellular metabolism. Nanopipettes also offer precise spatial control for the bottom-up electrochemical construction of functional nanostructures. Looking ahead, the integration of nanopipette arrays, hybrid analytical techniques, and adaptive interfaces and nanopipette electrochemistry is expected to enable the development of intelligent ionic circuits, neuromorphic systems, and next-generation molecular-scale computing platforms.

Nanopore sequencing, a third-generation sequencing technology, widely uses biological nanopores due to their high reproducibility. To effectively capture signals generated as target molecules pass through the nanopores, the readout circuit should be characterized with high bandwidth, high gain, low noise, and high throughput. However, parasitic capacitance limits the circuit stability and bandwidth. This paper presents a nanopore front-end readout integrated chip, composed of a nanopore microelectrode chip and analog front-end chips, adopting heterogeneous packaging technology and on-chip circuit to minimize parasitic capacitance. The nanopore microelectrode chip is manufactured by micromanufacturing technology, where nanopores can be incorporated on it. The analog front-end chips adopt resistive feedback trans-impedance amplifiers and are taped out in 0.18-mu m CMOS. Ultimately, a 16-channel nanopore front-end readout integrated chip is applied for biological nanopore sensing. After biological nanopores are incorporated, each channel of the nanopore front-end readout integrated chip has bandwidth exceeding 11 kHz and equivalent input noise less than 3 pA. These characteristics enable effective detection of signals generated by target analytes passing through the biological nanopores.