Biological nanopores are powerful tools for biomolecular detection, but conventional platforms often suffer from low efficiency in manual fluid exchange and low throughput. Here, we present a probe-driven biological nanopore (PBN) sensing platform, which integrates three core components: a 3D nanopositioning system, a functionalized glass pore probe, and a multi-well sample chamber. These components synergistically overcome critical limitations of conventional systems. These core components significantly improves measurement accuracy, throughput, and operational flexibility in nanopore-based biosensing applications. Optimized fabrication and silanization of glass pores, combined with a bubble-blowing method for lipid bilayer formation, significantly enhanced membrane stability with bilayers remaining intact for over 1 h under experimental conditions, and minimized leakage currents. Systematic studies revealed that bilayer integrity and sensing performance depend on pore diameter and internal structure. By embedding multiple alpha-HL channels, the system achieved enhanced capture rates and improved signal resolution. The sensor exhibited excellent linear response (R2=0.993) for DNA concentrations from 100 to 500 nM and successfully distinguished nucleic acids of different lengths based on event charge deficits. Furthermore, the platform demonstrated high recovery rates (103%-113%) in detecting DNA spiked into human serum, underscoring its applicability to complex biological samples. Enabled by the 3D nanopositioning system and multi-well chamber, the platform allows rapid sample switching and realtime analysis, supporting efficient multi-sample detection. This robust, high-precision system provides a versatile solution for sensitive and high efficient nucleic acid detection, with promising applications in clinical diagnostics, genetic analysis, and advancing next-generation biosensing technologies.

Sub-15 nm line structures are key building blocks for advanced device prototyping, nanoscale electrodes, and lithography templates such as etch/deposition masks. Although ultrahigh-voltage (>= 100 kV) electron-beam lithography (EBL) can more readily achieve extremely small critical dimensions, its tool and infrastructure requirements limit widespread adoption in many laboratories. In contrast, 30 kV field-emission SEM platforms are far more accessible; however, resolution-limit patterning at 30 kV is more sensitive to beam current, exposure dose, and development conditions, motivating the establishment of a reproducible process flow and a well-defined process window. Here, we investigate the resolution limit of isolated lines using a Zeiss Gemini 460 system operated at 30 kV and an in-house pattern generator with 950 k PMMA C2 resist. To demonstrate device-level applicability, we develop a stable lift-off process, and all critical dimensions are evaluated on metal lines after e-beam evaporation and lift-off. By screening beam current and scanning dose to build the dose-to-size relationship, we show that reducing beam current significantly improves the achievable minimum line width. Under 35 pA, using CD <= 15 nm as the criterion for sub-15 nm window extraction, the usable dose range is [700, 804.3] mu C/cm(2), corresponding to a dose latitude of similar to 14.9%. The best performance is obtained at 700 mu C/cm(2), yielding a transferred metal line width of 13.85 nm after lift-off. This work provides a practical resolution-limit process flow and a quantitative process window for performing sub-15 nm patterning on accessible 30 kV platforms, supported by product-level lift-off validation.

Electron beam lithography (EBL) systems rely on highly precise and responsive deflection fields to ensure nanoscale pattern fidelity. However, various physical factors-such as nonuniformity in the deflection field, nonlinearity in driver output current, and other internal and external system effects-collectively introduce complex nonlinear distortions. These distortions degrade writing accuracy and increase stitching and overlay errors. Conventional linear or global polynomial correction models are limited in their capacity to accurately capture localized, irregular distortions. To address this limitation, this study presents a distortion correction approach based on 2D tensor product B-spline interpolation. The method involves acquiring secondary electron images from standard grid samples, extracting sub-pixel feature coordinates. A smooth and continuous deformation field is then reconstructed via B-spline fitting. Subsequently, an inverse mapping compensation model is derived to enable real-time correction of the electron beam trajectory. Simulation results and experimental validation confirms the effectiveness of the method. With only an 11 & times; 11 twisted grid and a 6 & times; 6 sampling set, fourth-order B-spline correction achieves a root mean square error (RMSE) of 0.132 pixels (approximate to 13.7 nm), outperforming the optimal fifth-order polynomial model. For dense 41 & times; 41 twisted grids, the third-order B-spline model under a 21 & times; 21 sampling set yields an RMSE similar to 14.9% lower than that of the fifth-order polynomial model. Compared with global polynomial models, the B-spline-based approach offers superior accuracy and enhanced robustness to noise at lower model orders. This method provides a practical solution for hardware-level electron beam deflection control and establishes a foundation for future adaptive correction strategies.

The multi-beam electron optical system (MBEOS) is critical for high-throughput electron microscopy and electron beam lithography. However, their performance is constrained by both on-axis and off-axis aberrations, which significantly degrade the final spot size. A systematic approach to control these aberrations and to optimize the overall system design has not yet been fully addressed. In this work, we extend the analytical model proposed by M. J. van Bruggen to develop a simplified method for rapidly determining the optimal operating conditions-specifically, the beam deflection angle at the field lens (an intermediate accelerating lens) and the corresponding field lens strength-that minimize off-axis aberrations in a two-lens demagnification system. Our results reveal that the minimum total aberration occurs when the beamlets pass very close to, rather than exactly through, the center of the second lens. Furthermore, we observe a non‑monotonic relationship between off‑axis aberration and the beamlet's radial distance to the optical axis at the field lens. As this radial distance increases, the aberration first decreases and then increases. After geometric aberrations are minimized, spherical and chromatic aberrations become the dominant contributors to the final spot size. Based on these insights, we propose a practical optimization routine applicable to MBEOS designs employing two-lens demagnification system. Using this method, we demonstrate that a total usable beamlet generation area of up to 480 µm × 480 µm can be achieved while maintaining a spot size near 10 nm. This work provides a practical analytical framework for designing high-performance MBEOS with minimized off-axis aberrations.

Nanopipettes hold great promise for single molecule sensing owing of their tunable geometry and superior physicochemical properties, yet a systematic experimental understanding of how intrinsic geometric parameters govern sensing performance remains incomplete. Herein, we fabricated nanopipette with well-defined, diameter, cone angle, and wall thickness via a laser-assisted pulling method, and quantitatively investigated their impacts on resistive pulse DNA detection and ion current rectification (ICR) using λ-DNA as a model analyte. Our results show that reducing nanopipette diameter elevated current blockade amplitude and signal to noise ratio, but lowered DNA capture rate due to strengthened. Enlarging cone angles significantly enhanced capture efficiency by reinforcing electrophoretic driving forces, while thinning wall thickness optimized local electric field distributions, boosting both capture rates and rectification sensitivity. ICR is most pronounced in nanopipette with small diameters, thin walls, and narrow cone angles, arising from enhanced electric double layers and amplified surface charge effects. These results quantitatively elucidate the coupled roles of geometry and surface interactions in regulating molecular transport and ionic flux, establishing a systematic framework for designing high-performance nanopipette sensors. This work provides direct guidance for optimizing nanopipette geometry toward high resolution single-molecule detection and ion selective nanofluidic applications.

DNA nanotechnology has advanced beyond sequence design toward precise control of local substructures, such as single-stranded gaps and branched motifs, whose configuration governs mechanical stability and function. However, quantitative interrogation of these dynamic elements at the single-molecule level under native solution conditions remains challenging. Here, we present a quasi-static nanopore scanning strategy that enables deterministic electrical imaging of DNA substructures. Using surface-tethered dual-gap DNA scaffolds, we demonstrate that ionic blockade amplitudes from unstructured single-stranded branches scale with high linearity (R2 = 0.998) over nearly an order of magnitude in length (10-81 nt), achieving 5-nucleotide resolution. In contrast, base-paired architectures (hairpins and aptamers) exhibit pronounced nonlinear amplification. This work establishes nanopore scanning as a quantitative electrical imaging modality for simultaneous readout of branch length and topology, providing a foundation for quality control, structural validation, and real-time monitoring of complex DNA nanodevices.

The controllable fabrication of tapered three-dimensional (3D) microstructures by ion-beam processing remains challenging, especially when both profile fidelity and geometric controllability are required. Tapered conical microstructures are of interest because they are relevant to a variety of applications, including micro-optical elements, functional textured surfaces, biomimetic interfaces, and field-enhancing emitter-related structures, where taper angle, aspect ratio, and structural uniformity strongly influence the resulting performance. In this work, a longitudinal layer-wise strategy is proposed for tapered micro-cone fabrication by ion-beam etching. The core idea is to discretize a continuous cone profile along the vertical direction into a sequence of annular layers whose dimensions are determined by the local geometry of the target three-dimensional structure. After this geometric discretization step, each individual layer is executed using a conventional multi-pass strategy, thereby combining longitudinal profile construction with stabilized local material removal. A dedicated pattern-design software, EBWriter, was developed to automatically generate annular patterns and process files from user-defined geometric parameters. Experimental validation was carried out on single-crystal silicon substrates using a dual-beam microscope platform operated at 30 kV. The results show that increasing the longitudinal layer number effectively weakens the staircase effect and improves the continuity of the reconstructed cone profile. For positive micro-cones fabricated using annular patterns with a nominal outer processing diameter of 3 mu m, the increasing-inner-radius strategy enables preservation of the cone apex and reconstruction of tapered morphologies with improved fidelity. Under the present processing conditions, an empirical correspondence between the target geometric ratio and the recommended layer number was further summarized: layer numbers of approximately 50, 100, and 300 support cone structures with base-diameter-to-height ratios close to 1:2, 1:3, and 1:4, respectively. In addition, a 3 & times; 3 positive micro-cone array was successfully fabricated, with a total processing time of about 80 s. The measured cone base diameter and height were 0.886 +/- 0.005 mu m and 2.354 +/- 0.023 mu m, respectively, with dimensional variations controlled within +/- 2%. These results demonstrate that the proposed method provides a feasible layer-wise ion-beam fabrication route for tapered microstructures and offers a useful process basis for future studies on micro-optical surfaces, functional textured interfaces, and emitter-related microstructures.

DNA nanotechnology has advanced beyond sequence design toward precise control of local substructures, such as single-stranded gaps and branched motifs, whose configuration governs mechanical stability and function. However, quantitative interrogation of these dynamic elements at the single-molecule level under native solution conditions remains challenging. Here, we present a quasi-static nanopore scanning strategy that enables deterministic electrical imaging of DNA substructures. Using surface-tethered dual-gap DNA scaffolds, we demonstrate that ionic blockade amplitudes from unstructured single-stranded branches scale with high linearity (R-2 = 0.998) over nearly an order of magnitude in length (10-81 nt), achieving 5-nucleotide resolution. In contrast, base-paired architectures (hairpins and aptamers) exhibit pronounced nonlinear amplification. This work establishes nanopore scanning as a quantitative electrical imaging modality for simultaneous readout of branch length and topology, providing a foundation for quality control, structural validation, and real-time monitoring of complex DNA nanodevices.

Currently, domestically produced X-ray tube high-voltage (HV) power supplies have exhibited excessive volume, making them unsuitable for portable applications. To meet the portability requirements of micro X-ray tubes, a compact, low-ripple, low-power HV power supply was designed, capable of generating an adjustable negative HV ranging from 0 to −50 kV with a power output of 5 W. The inverter section employed a push-pull converter circuit, while the voltage boost topology consisted of a high-frequency transformer and a voltage doubling rectifier circuit, to achieve the desired output through two-stage boosting. A PWM (Pulse Width Modulation) method was adopted, utilizing the SG3525 PWM chip to regulate the output voltage. A resistive voltage divider was used to sample the output HV for feedback, to enable closed-loop voltage stabilization control. Testing of the HV power supply showed an output ripple coefficient below 0.22

High accuracy and precision overlay and stitching in electron beam lithography demand rapid and robust detection of alignment marks. Here, we implement an autocorrelation-based adaptive alignment mark detection method that processes one-dimensional backscattered electron signals using first-order differences and autocorrelation to detect marks. Adaptive filter parameters and window sizing are employed to suppress noise while maintaining high computational efficiency, in contrast to conventional two-dimensional approaches. Experiments show that the method achieves sub-2 nm precision (99.99% confidence interval) in both X and Y directions with only 10 line scans at a sampling resolution of 512, corresponding to low positioning uncertainty and a detection time of just 15 ms per mark. These results confirm the method’s suitability for real-time applications (e.g., drift compensation and automatic alignment) and suggest its further integration into key processes in Gaussian beam electron beam lithography, including both linear and nonlinear corrections as well as stitching and overlay processes. A contingency scheme employing robust two-dimensional template matching is proposed to handle potential failures caused by specific conditions (e.g., mark defects or extremely low SNR).

Mycotoxins are widely found in agricultural products that poses serious threats to human health. Therefore, the development of precise techniques for detecting mycotoxins is critical. We functionalized Fe3O4 with strepstrands and incubated it with varying concentrations of aflatoxin B1 and swing arms. After adding MgCl2, we obtained short-strand DNA. C4 was modified on the electrodeposition of COF/MoS2/AuPt electrode. The shortstrand DNA could bind to C4 and trigger A1 and A2 to create MoS2/Au/A1/A2/four-way, which absorbed numerous MB for electrochemical analysis of aflatoxin B1. This approach achieved ultrasensitive detection of aflatoxin B1 from 0.01 to 1000 ng mL-1 (detection limit: 0.0062 ng mL-1). Notably, our electrochemical aptasensor showed highly sensitive and rapid detection of toxins in peanut samples. The successful detection indicates a new model for the detection of various aptamer-recognized mycotoxins in food safety analysis.

Solid-state nanopore technology has emerged as a transformative tool for single-molecule detection, facilitating label-free, and real-time analysis of biomolecules, including DNA, RNA, and proteins. Understanding the capture mechanism and dynamic processes involved in biomolecule capture and translocation through solid-state nanopores is crucial for advancing fundamental research in life sciences and clinical applications. This review focuses on the factors that affect the single-molecule capture efficiency of solid-state nanopores, highlighting significant progress in enhancing this efficiency. Advanced approaches are explored for fabricating high-precision, structurally robust nanopores, along with strategies for enhancing capture efficiency through nanopore functionalization and modulation of driving mechanisms. Moreover, this review discusses state-of-the-art single-molecule nanopore capture validation with translocation monitoring techniques, addressing existing challenges in the field. Finally, prospective directions are outlined for improving the performance and scalability of nanopore capture efficiency, emphasizing the potential for interdisciplinary collaboration to drive further innovation in solid-state nanopore technology. By addressing both advances and challenges, this review aims to provide insights into the preferred technique for enhancing nanopore capture efficiency, thereby advancing fundamental research in life sciences and improving clinical diagnostics applications.

To enhance the throughput of single-beam devices,such as scanning electron microscopes,a multi-beam electron source system utilizing a Schottky gun was developed.This study encompasses the design methodologies and fabrication processes for the collimator lens,aperture array,and micro-arrayed electrostatic lenses.The electrostatic collimator lens was designed based on the emission characteristics of the Schottky cathode,and the performance of the collimated beam was subsequently calculated.Aperture arrays,including configurations of 3×3 and 10×10 for beam splitting,were fabricated utilizing MEMS technology,and a high-precision assembly system was established to enable the assembly of micro-arrayed electrostatic lenses.Experiments concerning beam collimation,splitting,and focusing were conducted on a dedicated multi-beam electron source experimental platform,validating the performance of the 3×3 mul-tibeam electron source.Experimental results indicate that the collimated spot size was measured to be 600 μm with a beam current density of 4.11 A/m² and a uniformity of 6.06%.The average diameter of the fo-cused beamlet is recorded at 5.32 μm,accompanied by size uniformity of 5.91%,intensity uniformity of 4.36%,and pitch uniformity of 3.06%,all of which meet the design criteria of the multi-beam setup.

The stainless steel microporous structure has important application value in biomedicine. The filter membranes prepared by electrochemical microfabrication enable precise adjustment of pore size adjustment from 10.7 to 15.4 mu m by precisely controlling etching voltage and time. The pore size is uniform, the surface is smooth and free of burrs, meeting the strict requirements of biomedicine for filter membrane accuracy. This study presents three core innovations: Firstly, the novel combination of linear sweep voltammetry (LSV) and electrochemical impedance spectroscopy (EIS) for quantitative evaluation of mask exposure quality. This overcomes the limitations of traditional optical characterization. Secondly, a critical potential control strategy for oxygen evolution is established, strictly limit the voltage within 1.60 V vs. SCE by controlling the oxygen evolution reaction. This avoid bubble interference with mass transfer, and ensure pore size uniformity with a d(90)-d(50) value of 1.5-1.6 mu m. Thirdly, innovatively using electrochemical etched filter membranes in dual membrane filtration for Giant Unilamellar Vesicles (GUVs), the GUVs size range was narrowed from 5.0 similar to 46.0 mu m to 5.0 similar to 26.0 mu m, and the d(90)-d(50) value was reduced from 12.2 mu m to 6.5 mu m. The research boasts enormous practical application potential, capable of advancing development of fields such as biofilm simulation, cell separation, and preparation of nano drug carriers. Moreover, the corrosion resistance and repeatability of stainless steel materials meet the stability and safety requirements of biomedical devices.

The COVID-19 pandemic caused by the SARS-CoV-2 virus has exposed the urgency of research on rapid and efficient virus detection and strategies to inhibit its replication. Previous studies have mostly focused on traditional immunoassay or optical methods, but they have limitations in terms of sensitivity, timeliness, and in-depth analysis of molecular interaction mechanisms. Solid-state nanopore single-molecule detection methods, which can monitor molecular conditions in real time at the single-molecule level, bring new opportunities to solve this problem. The nucleocapsid protein (N protein) of SARS-CoV-2 was systematically investigated under different conditions, such as external drive voltage, pH, nanopore size, and N protein concentration. The translocation of the N protein through the nanopore was then analyzed. Subsequently, we analyzed the translocation characteristics of the N protein, RNA, and N protein–RNA complexes. With the aid of EMSA experiments, we conclusively confirmed that RNA binds to the N protein. Building on this finding, we further explored small molecules that could affect the nanopore translocation of N protein–RNA complexes, such as gallocatechin gallate (GCG), epigallocatechin gallate (EGCG), and the influenza A viral inhibitor Nucleozin. The results show that GCG can disrupt the liquid-phase condensation of the N protein–RNA complex and inhibit the replication of the N protein. Meanwhile, the structural isomer EGCG of GCG and the small molecule Nucleozin can also block RNA-triggered N protein liquid–liquid phase separation (LLPS). Our results confirmed that GCG, EGCG, and Nucleozin exhibit antagonistic effects on the N protein, with differences in their effective concentrations and the potency of their antagonism. Herein, using solid-state nanopore single-molecule detection technology, we developed an experimental method that can effectively detect RNA-induced changes in N protein properties and the regulatory effects of small molecules on the LLPS of N protein–RNA complexes. These findings not only provide highly valuable insights for in-depth research on the molecular interactions involved in viral replication, but also open up promising new avenues for future responses to similar viral outbreaks, the development of a rapid and effective detection method based on solid-state nanopores and single-molecule detection, and antiviral therapies targeting N protein–RNA interactions.

Solid-state nanopores have emerged as transformative tools for single-molecule detection and analysis of DNA, RNA, and proteins in the field of biotechnology. This review focuses on controlled dielectric breakdown (CBD), an in-situ fabrication technique that utilizes electric fields to induce membrane dielectric breakdown, offering low-cost, scalable nanopore fabrication in insulating materials. The principles of dielectric breakdown mechanisms, integrating thermal, electrical, and chemical mechanisms, are analyzed, highlighting the critical role of parameters such as electric field intensity, material dielectric properties, and solid-liquid interface dynamics in enabling precise control over nanopore fabrication. Unlike expensive lithography methods, CBD avoids complex ex situ processes, enabling real-time monitoring via leakage currents. Key advancements in strategies for localized area thinning and laser-assisted pre-damage, micropipette-based localized confined electrolyte, and atomic force microscope tip-induced localized electric field have addressed the traditional CBD's stochasticity. These strategies enable deterministic sub-2 nm nanopore formation with tunable morphology. Advanced CBD techniques have evolved from a probabilistic method to a versatile platform for scalable and rapid nanopore fabrication. Future directions emphasize microfluidic integration with novel dielectric materials, positioning CBD as a versatile platform for next-generation single-molecule biosensing and sequencing applications.

Polymer translocation through solid-state nanopores has attracted widespread attention in many biological processes. However, the dynamics of capture and translocation through nanopores are highly correlated with polymer conformation. Using plasmid pBR322 DNA with supercoiled and linear structures, this work explores the effects of polymer conformation on capture and translocation through nanopores. The polymer translocation dynamics are discussed with the relationship between current blockage characteristics and applied voltages across various pore sizes. Subsequently, linear plasmids are used to clarify the effect of polymer conformation on nanopore capture and translocation. The nonlinear relationships between current blockage amplitudes and voltages demonstrate that the linear conformation affects the plasmid DNA capture process. A decrease in the ratio of supercoiled plasmids improved the frequency of folding translocation and decreased the standard deviation of blockage current amplitudes. As the proportion of supercoiled plasmids rises, the impact of partially folding linear conformation on mixed plasmids translocation is enhanced with increasing applied voltage. Furthermore, the results reveal a polymer conformation-dependent bias in capture and translocation processes, named the "crowding effect". This study provides valuable insights into the dynamics of polymer conformational transitions through solid-state nanopores, that have significant implications for improving sequencing and sensing technologies.

Despite significant advances in nanopore nucleic acid sequencing and sensing, protein detection remains challenging due to the inherent complexity of protein molecular properties (i.e., net charges, polarity, molecular conformation & dimension) and sophisticated environmental parameters (i.e., biofluids), resulting in unsatisfactory electrical signal resolution for protein detection such as poor accessibility, selectivity and sensitivity. The selection of an appropriate electroanalytical approach is strongly desired which should be capable of offering easily detectable and readable signals regarding proteins particularly depending on the practical application. Herein, a molecular sandwich-based cooperative DNAzyme catalytic reaction nanopore detecting approach was designed. Specifically, this approach uses Mg2+ catalyzed DNAzyme (10-23) toward nucleic acids digestion for efficient antigen protein examination. The proposed strategy operates by initial formation of a molecular sandwich containing capture antibody-antigen-detection antibody for efficient entrapment of target proteins (herein taking the HIV p24 antigen for example) and immobilization on magnetic beads surfaces. After that, the DNAzyme was linked to the detection antibody via a biotin-streptavidin interaction. In the presence of Mg2+, the DNAzyme catalytic reaction was triggered to digest nucleic acid substrates and release unique cleavage fragments as reporters capable of transducing more easily detectable nucleic acids as a substitute for the complicated and hard to yield protein signals, in a nanopore. Notably, experimental validation confirms the detecting stability and sensitivity for the target antigen referenced with other antigen proteins, meanwhile it demonstrates a detection efficacy in a human serum environment at very low concentration (LoD ∼1.24 pM). This cooperative DNAzyme nanopore electroanalytical approach denotes an advance in protein examination, and may benefit in vitro testing of proteinic biomarkers for disease diagnosis and prognosis assessment.

The Scanning Electron Microscope (SEM) is essential for its exceptional resolution, especially in medical imaging, semiconductor technology, and nanomaterial characterization. This paper presents an optical imaging system that measures the SEM electron beam spot size using a magnification lens and a Complementary Metal Oxide Semiconductor (CMOS) sensor. The system acquires image data of a steel ruler and analyzes the correlation between unit length and pixel dimensions, the uncertainty of system is studied also. By analyzing the experimental data, a correction curve for the imaging system can be derived, and the corrected error is kept within +/- 0.043 mu m.