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Nanopores fabricated from synthetic materials (solid-state nanopores), platforms for characterizing biological molecules, have been widely studied among researchers. Compared with biological nanopores, solid-state nanopores are mechanically robust and durable with a tunable pore size and geometry. Solid-state nanopores with sizes as small as 1.3 nm have been fabricated in various films using engraving techniques, such as focused ion beam (FIB) and focused electron beam (FEB) drilling methods. With the demand of massively parallel sensing, many scalable fabrication strategies have been proposed. In this review, typical fabrication technologies for solid-state nanopores reported to date are summarized, with the advantages and limitations of each technology discussed in detail. Advanced shrinking strategies to prepare nanopores with desired shapes and sizes down to sub-1 nm are concluded. Finally, applications of solid-state nanopores in DNA sequencing, single molecule detection, ion-selective transport, and nanopatterning are outlined.

Solid‐state nanopore arrays are emerging as powerful tools for label‐free, ultrasensitive biosensing, yet their implementation has been constrained by inter‐pore crosstalk and limited fabrication uniformity. A multilayer Al2O3/Au/Si3N4 nanopore architecture, produced via helium ion beam lithography, is introduced to address these limitations through structural and materials‐level innovation. Finite‐element analysis identifies a critical inter‐pore spacing approximately 20 times the pore radius as necessary to minimize electric field coupling, enabling rational array design. The membrane structure incorporates a dielectric Al2O3 layer for electrical isolation and an intermediate gold layer for site‐specific aptamer immobilization, confining molecular recognition to the nanopore interior. Arrays with ∼30 nm pores and <5% size variation achieve 300 nm spacing and support statistically independent, parallel signal acquisition. Diverse nanopore arrays with 75 nm pores and 800 nm spacing are utilized for the specific detection of alpha‐fetoprotein. Detection of alpha‐fetoprotein demonstrates label‐free sensing at concentrations down to ∼3 fM across six orders of magnitude in dynamic range. This platform defines a closed‐loop pathway from theoretical modeling to scalable fabrication, establishing a foundation for rational design and high‐throughput deployment of solid‐state nanopore biosensors. The critical pore spacing—approximately twenty times the pore radius—required to minimize electric field coupling is determined via finite element analysis. A multi‐layered Al2O3/Au/Si3N4 nanopore structure fabricated by helium ion beam lithography is proposed, enabling quantitative analysis of the target analyte while mitigating inter‐pore crosstalk.

Solid‐state nanopore sensors, a type of resistive pulse sensing, achieve optimal signal‐to‐noise performance with a single nanopore. However, the processes involved in solid‐state nanopore fabrication and subsequent measurements frequently lead to the formation of multiple nanopores, posing a challenge for precise detection. To address this issue, here, a novel and expedient technique to verify the presence of a single nanopore on a chip is developed. The methodology includes measuring the nanopore's conductance in solutions of various salt conditions, followed by a comparison of these results against a theoretical conductance model. This comparison is instrumental in distinguishing between single and multiple nanopores. Additionally, the study delves into various factors that influence the conductance curve, such as deviations in pore shape from the standard circle and inconsistencies in pore diameter. This approach significantly enhances the practical application of low‐cost nanopore preparation techniques, particularly in scenarios like controlled breakdown nanopore fabrication, where the formation of multiple nanopores is a common concern. The formation of multiple nanopores involved in the processes of solid‐state nanopore fabrication poses a challenge for precise detection. To address this issue, a novel and expedient technique to verify the presence of a single nanopore on a chip is developed by measuring the nanopore's conductance in solutions of various salt conditions.

Nanopore-based DNA analysis is a single-molecule technique with revolutionary potential. It promises to carry out a range of analyses, orders of magnitude faster than current methods, including length measurement, specific sequence detection, single-molecule dynamics and even sequencing. The concept involves using an applied voltage to drive DNA molecules through a narrow pore that separates chambers of electrolyte solution. This voltage also drives a flow of electrolyte ions through the pore, measured as an electric current. When molecules pass through the pore, they block the flow of ions and, thus, their structure and length can be determined based on the degree and duration of the resulting current reductions. In this review, I explain the nanopore-based DNA analysis concept and briefly explore its historical foundations, before discussing and summarizing all experimental results reported to date. I conclude with a summary of the obstacles that must be overcome for it to realize its promised potential.

Nanopore-based sensing has emerged as a transformative approach for biomarker detection, offering label-free, single-molecule analysis with exceptional sensitivity and specificity. By monitoring ionic current modulations as individual molecules traverse biological or solid-state nanopores, these platforms provide direct insights into molecular size, structure, charge, and interactions. Over the past five years, advances in nanopore fabrication, surface engineering, and signal interpretation have expanded their analytical scope, from nucleic acids and proteins to peptides and small metabolites, while enabling operation in complex biological matrices. This review focuses on recent advancements (from last 5 years) that enhance detection performance and specificity through recognition strategies, engineered pore chemistries, and data analysis. Applications are discussed across key disease areas, including cancer, cardiovascular, neurological, metabolic, and infectious diseases, emphasizing early detection, multiplexed measurements, clinical adaptability and translational diagnostics, highlighting progress toward portable, point-of-care systems. Collectively, these developments underscore the potential of nanopore biosensing to bridge fundamental research and real-world diagnostics, paving the way for rapid, sensitive, and accessible health monitoring tools in precision medicine. Graphical Abstract Schematic illustration of sensing of various health biomarkers (protein, peptides, miRNA, small molecules etc.) through nanopores.

Many small proteins move across cellular compartments through narrow pores. In order to thread a protein through a constriction, free energy must be overcome to either deform or completely unfold the protein. In principle, the diameter of the pore, along with the effective driving force for unfolding the protein, as well as its barrier to translocation, should be critical factors that govern whether the process proceeds via squeezing, unfolding/threading, or both. To probe this for a well-established protein system, we studied the electric-field–driven translocation behavior of cytochrome c (cyt c) through ultrathin silicon nitride (SiNₓ) solid-state nanopores of diameters ranging from 1.5 to 5.5 nm. For a 2.5-nm-diameter pore, we find that, in a threshold electric-field regime of ∼30 to 100 MV/m, cyt c is able to squeeze through the pore. As electric fields inside the pore are increased, the unfolded state of cyt c is thermodynamically stabilized, facilitating its translocation. In contrast, for 1.5- and 2.0-nm-diameter pores, translocation occurs only by threading of the fully unfolded protein after it transitions through a higher energy unfolding intermediate state at the mouth of the pore. The relative energies between the metastable, intermediate, and unfolded protein states are extracted using a simple thermodynamic model that is dictated by the relatively slow (∼ms) protein translocation times for passing through the nanopore. These experiments map the various modes of protein translocation through a constriction, which opens avenues for exploring protein folding structures, internal contacts, and electric-field–induced deformability.

Abstract Background Circulating tumor DNAs (ctDNAs) are highly promising cancer biomarkers, potentially applicable for noninvasive liquid biopsy and disease monitoring. However, to date, sequencing of ctDNAs has proven to be challenging primarily due to small sample size and high background of fragmented cell-free DNAs (cfDNAs) derived from normal cells in the circulation, specifically in early stage cancer. Methods Solid-state nanopores (ssNPs) have recently emerged as a highly efficient tool for single-DNA sensing and analysis. Herein, we present a rapid nanopore genotyping strategy to enable an amplification-free identification and classification of ctDNA mutations. A biochemical ligation detection assay was used for the creation of specific fluorescently-labelled short DNA reporter molecules. Color conjugation with multiple fluorophores enabled a unique multi-color signature for different mutations, offering multiplexing potency. Single-molecule readout of the fluorescent labels was carried out by electro-optical sensing via solid-state nanopores drilled in titanium oxide membranes. Results As proof of concept, we utilized our method to detect the presence of low-quantity ERBB2 F310S and PIK3Ca H1047R breast cancer mutations from both plasmids and xenograft mice blood samples. We demonstrated an ability to distinguish between a wild type and a mutated sample, and between the different mutations in the same sample. Conclusions Our method can potentially enable rapid and low cost ctDNA analysis that completely circumvents PCR amplification and library preparation. This approach will thus meet a currently unmet demand in terms of sensitivity, multiplexing and cost, opening new avenues for early diagnosis of cancer.

The application of solid-state (SS) nanopore devices to single-molecule nucleic acid sequencing has been challenging. Thus, the early successes in applying SS nanopore devices to the more difficult class of biopolymer, glycosaminoglycans (GAGs), have been surprising, motivating us to examine the potential use of an SS nanopore to analyze synthetic heparan sulfate GAG chains of controlled composition and sequence prepared through a promising, recently developed chemoenzymatic route. A minimal representation of the nanopore data, using only signal magnitude and duration, revealed, by eye and image recognition algorithms, clear differences between the signals generated by four synthetic GAGs. By subsequent machine learning, it was possible to determine disaccharide and even monosaccharide composition of these four synthetic GAGs using as few as 500 events, corresponding to a zeptomole of sample. These data suggest that ultrasensitive GAG analysis may be possible using SS nanopore detection and well-characterized molecular training sets.

Compared with the status of bio-nanopores, there are still several challenges that need to be overcome before solid-state nanopores can be applied in commercial DNA sequencing. Low spatial and low temporal resolution are the two major challenges. Owing to restrictions on nanopore length and the solid-state nanopores’ surface properties, there is still room for improving the spatial resolution. Meanwhile, DNA translocation is too fast under an electrical force, which results in the acquisition of few valid data points. The temporal resolution of solid-state nanopores could thus be enhanced if the DNA translocation speed is well controlled. In this mini-review, we briefly summarize the methods of improving spatial resolution and concentrate on controllable methods to promote the resolution of nanopore detection. In addition, we provide a perspective on the development of DNA sequencing by nanopores.

Controlled breakdown has recently emerged as a highly accessible technique to fabricate solid-state nanopores. However, in its most common form, controlled breakdown creates a single nanopore at an arbitrary location in the membrane. Here, we introduce a new strategy whereby breakdown is performed by applying the electric field between an on-chip electrode and an electrolyte solution in contact with the opposite side of the membrane. We demonstrate two advantages of this method. First, we can independently fabricate multiple nanopores at given positions in the membrane by localising the applied field to the electrode. Second, we can create nanopores that are self-aligned with complementary nanoelectrodes by applying voltages to the on-chip electrodes to locally heat the membrane during controlled breakdown. This new controlled breakdown method provides a path towards the affordable, rapid, and automatable fabrication of arrays of nanopores self-aligned with complementary on-chip nanostructures.