DNA Origami Incorporated into Solid-State Nanopores Enables Enhanced Sensitivity for Precise Analysis of Protein Translocations.

The rapidly advancing field of nanotechnology is driving the development of precise sensing methods at the nanoscale, with solid-state nanopores emerging as promising tools for biomolecular sensing. This study investigates the increased sensitivity of solid-state nanopores achieved by integrating DNA origami structures, leading to the improved analysis of protein translocations. Using holo human serum transferrin (holo-hSTf) as a model protein, we compared hybrid nanopores incorporating DNA origami with open solid-state nanopores. Results show a significant enhancement in holo-hSTf detection sensitivity with DNA origami integration, suggesting a unique role of DNA interactions beyond confinement. This approach holds potential for ultrasensitive protein detection in biosensing applications, offering advancements in biomedical research and diagnostic tool development for diseases with low-abundance protein biomarkers. Further exploration of origami designs and nanopore configurations promises even greater sensitivity and versatility in the detection of a wider range of proteins, paving the way for advanced biosensing technologies.

Communicative competence of generative artificial intelligence in responding to patient queries about colorectal cancer surgery.

PURPOSE: To examine the ability of generative artificial intelligence (GAI) to answer patients' questions regarding colorectal cancer (CRC). METHODS: Ten clinically relevant questions about CRC were selected from top-rated hospitals' websites and patient surveys and presented to three GAI tools (Chatbot Generative Pre-Trained Transformer [GPT-4], Google Bard, and CLOVA X). Their responses were compared with answers from the CRC information book. Response evaluation was performed by two groups, each consisting of five healthcare professionals (HCP) and patients. Each question was scored on a 1-5 Likert scale based on four evaluation criteria (maximum score, 20 points/question). RESULTS: In an analysis including only HCPs, the information book scored 11.8 ± 1.2, GPT-4 scored 13.5 ± 1.1, Google Bard scored 11.5 ± 0.7, and CLOVA X scored 12.2 ± 1.4 (P = 0.001). The score of GPT-4 was significantly higher than those of the information book (P = 0.020) and Google Bard (P = 0.001). In an analysis including only patients, the information book scored 14.1 ± 1.4, GPT-4 scored 15.2 ± 1.8, Google Bard scored 15.5 ± 1.8, and CLOVA X scored 14.4 ± 1.8, without significant differences (P = 0.234). When both groups of evaluators were included, the information book scored 13.0 ± 0.9, GPT-4 scored 14.4 ± 1.2, Google Bard scored 13.5 ± 1.0, and CLOVA X scored 13.3 ± 1.5 (P = 0.070). CONCLUSION: The three GAIs demonstrated similar or better communicative competence than the information book regarding questions related to CRC surgery in Korean. If high-quality medical information provided by GAI is supervised properly by HCPs and published as an information book, it could be helpful for patients to obtain accurate information and make informed decisions.

Real-time monitoring of Ti(IV) metal ion binding of transferrin using a solid-state nanopore.

Transferrin, a central player in iron transport, has been recognized not only for its role in binding iron but also for its interaction with other metals, including titanium. This study employs solid-state nanopores to investigate the binding of titanium ions [Ti(IV)] to transferrin in a single-molecule and label-free manner. We demonstrate the novel application of solid-state nanopores for single-molecule discrimination between apo-transferrin (metal-free) and Ti(IV)-transferrin. Despite their similar sizes, Ti(IV)-transferrin exhibits a reduced current drop, attributed to differences in translocation times and filter characteristics. Single-molecule analysis reveals Ti(IV)-transferrin's enhanced stability and faster translocations due to its distinct conformational flexibility compared to apo-transferrin. Furthermore, our study showcases solid-state nanopores as real-time monitors of biochemical reactions, tracking the gradual conversion of apo-transferrin to Ti(IV)-transferrin upon the addition of titanium citrate. This work offers insights into Ti(IV) binding to transferrin, promising applications for single-molecule analysis and expanding our comprehension of metal-protein interactions at the molecular level.

Korean Black Goat Extract Exerts Estrogen-like Osteoprotective Effects by Stimulating Osteoblast Differentiation in MC3T3-E1 Cells and Suppressing Osteoclastogenesis in RAW 264.7 Cells.

Postmenopausal osteoporosis, characterized by an imbalance between osteoclast-mediated bone resorption and osteoblast-driven bone formation, presents substantial health implications. In this study, we investigated the role of black goat extract (BGE), derived from a domesticated native Korean goat, estrogen-like activity, and osteoprotective effects in vitro. BGE's mineral and fatty acid compositions were analyzed via the ICP-AES method and gas chromatography-mass spectrometry, respectively. In vitro experiments were conducted using MCF-7 breast cancer cells, MC3T3-E1 osteoblasts, and RAW264.7 osteoclasts. BGE exhibits a favorable amount of mineral and fatty acid content. It displayed antimenopausal activity by stimulating MCF-7 cell proliferation and augmenting estrogen-related gene expression (ERα, ERß, and pS2). Moreover, BGE positively impacted osteogenesis and mineralization in MC3T3-E1 cells through Wnt/ß-catenin pathway modulation, leading to heightened expression of Runt-related transcription factor 2, osteoprotegerin, and collagen type 1. Significantly, BGE effectively suppressed osteoclastogenesis by curtailing osteoclast formation and activity in RAW264.7 cells, concurrently downregulating pivotal signaling molecules, including receptor activator of nuclear factor κ B and tumor necrosis factor receptor-associated factor 6. This study offers a shred of preliminary evidence for the prospective use of BGE as an effective postmenopausal osteoporosis treatment.

Over One Million DNA and Protein Events Through Ultra-Stable Chemically-Tuned Solid-State Nanopores.

Stability, long lifetime, resilience against clogging, low noise, and low cost are five critical cornerstones of solid-state nanopore technology. Here, a fabrication protocol is described wherein >1 million events are obtained from a single solid-state nanopore with both DNA and protein at the highest available lowpass filter (LPF, 100 kHz) of the Axopatch 200B-the highest event count mentioned in literature. Moreover, a total of ≈8.1 million events are reported in this work encompassing the two analyte classes. With the 100 kHz LPF, the temporally attenuated population is negligible while with the more ubiquitous 10 kHz, ≈91% of the events are attenuated. With DNA experiments, the pores are operational for hours (typically >7 h) while the average pore growth is merely ≈0.16 ± 0.1 nm h-1 . The current noise is exceptionally stable with traces typically showing <10 pA h-1 increase in noise. Furthermore, a real-time method to clean and revive pores clogged with analyte with the added benefit of minimal pore growth during cleaning (< 5% of the original diameter) is showcased. The enormity of the data collected herein presents a significant advancement to solid-state pore performance and will be useful for future ventures such as machine learning where large amounts of pristine data are a prerequisite.

Use of a solid-state nanopore for profiling the transferrin receptor protein and distinguishing between transferrin receptor and its ligand protein.

A nanopore device is capable of providing single-molecule level information of an analyte as they translocate through the sensing aperture-a nanometer-sized through-hole-under the influence of an applied electric field. In this study, a silicon nitride (Six Ny )-based nanopore was used to characterize the human serum transferrin receptor protein (TfR) under various applied voltages. The presence of dimeric forms of TfR was found to decrease exponentially as the applied electric field increased. Further analysis of monomeric TfR also revealed that its unfolding behaviors were positively dependent on the applied voltage. Furthermore, a comparison between the data of monomeric TfR and its ligand protein, human serum transferrin (hSTf), showed that these two protein populations, despite their nearly identical molecular weights, could be distinguished from each other by means of a solid-state nanopore (SSN). Lastly, the excluded volumes of TfR were experimentally determined at each voltage and were found to be within error of their theoretical values. The results herein demonstrate the successful application of an SSN for accurately classifying monomeric and dimeric molecules while the two populations coexist in a heterogeneous mixture.

Kynurenine pathway can be a potential biomarker of fatigue in primary Sjögren's syndrome.

OBJECTIVES: Primary Sjögren's syndrome (pSS) is a chronic autoimmune disease with low quality of life caused by various constitutional symptoms and glandular dysfunction. Although fatigue is one of the most frequent symptoms in pSS, its aetiology or biomarkers are poorly elucidated. We investigated potential relationship between severity of fatigue and the kynurenine pathway in pSS. METHODS: Clinical data and blood samples of 81 patients were obtained from a prospective cohort for pSS and compared with age- and sex-matched healthy controls (HC). Severity of fatigue was defined according to the fatigue domain scores in the ESSPRI. Potential biomarkers related to the kynurenine pathway were determined using ELISA. RESULTS: Of the total, 44 patients were defined as the "severe fatigue (ESSPRI fatigue ≥ 5)" group, whereas 37 as the "less fatigue (ESSPRI fatigue < 5)". Serum tryptophan levels in the severe fatigue group were significantly lower while those of kynurenine were higher. Serum interferon gamma, IDO1, and quinolinic acid levels were mostly higher in the less fatigue group. Kynurenine/tryptophan ratios were distinctly higher in the severe fatigue group than both HC and the less fatigue group (p < 0.001). This ratio showed a strong degree of positive correlation (r = 0.624, p < 0.001) with severity of fatigue in pSS while the other markers showed fair degrees of correlation. CONCLUSIONS: Serum markers related to the kynurenine pathway, especially the kynurenine/tryptophan ratio, may be associated with severity of fatigue in pSS. These results can provide guidance for further investigations on fatigue in pSS.

AtMAD: Arabidopsis thaliana multi-omics association database.

Integration analysis of multi-omics data provides a comprehensive landscape for understanding biological systems and mechanisms. The abundance of high-quality multi-omics data (genomics, transcriptomics, methylomics and phenomics) for the model organism Arabidopsis thaliana enables scientists to study the genetic mechanism of many biological processes. However, no resource is available to provide comprehensive and systematic multi-omics associations for Arabidopsis. Here, we developed an Arabidopsis thaliana Multi-omics Association Database (AtMAD, http://www.megabionet.org/atmad), a public repository for large-scale measurements of associations between genome, transcriptome, methylome, pathway and phenotype in Arabidopsis, designed for facilitating identification of eQTL, emQTL, Pathway-mQTL, Phenotype-pathway, GWAS, TWAS and EWAS. Candidate variants/methylations/genes were identified in AtMAD for specific phenotypes or biological processes, many of them are supported by experimental evidence. Based on the multi-omics association strategy, we have identified 11 796 cis-eQTLs and 10 119 trans-eQTLs. Among them, 68 837 environment-eQTL associations and 149 622 GWAS-eQTL associations were identified and stored in AtMAD. For expression-methylation quantitative trait loci (emQTL), we identified 265 776 emQTLs and 122 344 pathway-mQTLs. For TWAS and EWAS, we obtained 62 754 significant phenotype-gene associations and 3 993 379 significant phenotype-methylation associations, respectively. Overall, the multi-omics associated network in AtMAD will provide new insights into exploring biological mechanisms of plants at multi-omics levels.

Investigating protein translocation in the presence of an electrolyte concentration gradient across a solid-state nanopore.

Electrolyte chemistry plays an important role in the transport properties of analytes through nanopores. Here, we report the translocation properties of the protein human serum transferrin (hSTf) in asymmetric LiCl salt concentrations with either positive (Ctrans /Ccis < 1) or negative chemical gradients (Ctrans /Ccis > 1). The cis side concentration was fixed at 4 M for positive chemical gradients and at 0.5 M LiCl for negative chemical gradients, while the trans side concentration varied between 0.5 to 4 M which resulted in six different configurations, respectively, for both positive and negative gradient types. For positive chemical gradient conditions, translocations were observed in all six configurations for at least one voltage polarity whereas with negative gradient conditions, dead concentrations where no events at either polarity were observed. The flux of Li+ and Cl- ions and their resultant cation or anion enrichment zones, as well as the interplay of electrophoretic and electroosmotic transport directions, would determine whether hSTf can traverse across the pore.

Nanopore Data Analysis: Baseline Construction and Abrupt Change-Based Multilevel Fitting.

Solid-state nanopore technology delivers single-molecule resolution information, and the quality of the deliverables hinges on the capability of the analysis platform to extract maximum possible events and fit them appropriately. In this work, we present an analysis platform with four baseline fitting methods adaptive to a wide range of nanopore traces (including those with a step or abrupt changes where pre-existing platforms fail) to maximize extractable events (2× improvement in some cases) and multilevel event fitting capability. The baseline fitting methods, in the increasing order of robustness and computational cost, include arithmetic mean, linear fit, Gaussian smoothing, and Gaussian smoothing and regressed mixing. The performance was tested with ultra-stable to vigorously fluctuating current profiles, and the event count increased with increasing fitting robustness prominently for vigorously fluctuating profiles. Turning points of events were clustered using the dbscan method, followed by segmentation into preliminary levels based on abrupt changes in the signal level, which were then iteratively refined to deduce the final levels of the event. Finally, we show the utility of clustering for multilevel DNA data analysis, followed by the assessment of protein translocation profiles.

Niclosamide suppresses the expansion of follicular helper T cells and alleviates disease severity in two murine models of lupus via STAT3.

BACKGROUND: Autoantibody production against endogenous cellular components is pathogenic feature of systemic lupus erythematosus (SLE). Follicular helper T (TFH) cells aid in B cell differentiation into autoantibody-producing plasma cells (PCs). The IL-6 and IL-21 cytokine-mediated STAT3 signaling are crucial for the differentiation to TFH cells. Niclosamide is an anti-helminthic drug used to treat parasitic infections but also exhibits a therapeutic effect on autoimmune diseases due to its potential immune regulatory effects. In this study, we examined whether niclosamide treatment could relieve lupus-like autoimmunity by modulating the differentiation of TFH cells in two murine models of lupus. METHODS: 10-week-old MRL/lpr mice were orally administered with 100 mg/kg of niclosamide or with 0.5% methylcellulose (MC, vehicle) daily for 7 weeks. TLR7 agonist, resiquimod was topically applied to an ear of 8-week-old C57BL/6 mice 3 times a week for 5 weeks. And they were orally administered with 100 mg/kg of niclosamide or with 0.5% MC daily for 5 weeks. Every mouse was analyzed for lupus nephritis, proteinuria, autoantibodies, immune complex, immune cell subsets at the time of the euthanization. RESULTS: Niclosamide treatment greatly improved proteinuria, anti-dsDNA antibody levels, immunoglobulin subclass titers, histology of lupus nephritis, and C3 deposition in MRL/lpr and R848-induced mice. In addition, niclosamide inhibited the proportion of TFH cells and PCs in the spleens of these animals, and effectively suppressed differentiation of TFH-like cells and expression of associated genes in vitro. CONCLUSIONS: Niclosamide exerted therapeutic effects on murine lupus models by suppressing TFH cells and plasma cells through STAT3 inhibition.

Assessment of 1/f noise associated with nanopores fabricated through chemically tuned controlled dielectric breakdown.

Recently, we developed a fabrication method-chemically-tuned controlled dielectric breakdown (CT-CDB)-that produces nanopores (through thin silicon nitride membranes) surpassing legacy drawbacks associated with solid-state nanopores (SSNs). However, the noise characteristics of CT-CDB nanopores are largely unexplored. In this work, we investigated the 1/f noise of CT-CDB nanopores of varying solution pH, electrolyte type, electrolyte concentration, applied voltage, and pore diameter. Our findings indicate that the bulk Hooge parameter (αb ) is about an order of magnitude greater than SSNs fabricated by transmission electron microscopy (TEM) while the surface Hooge parameter (αs ) is ∼3 order magnitude greater. Theαs of CT-CDB nanopores was ∼5 orders of magnitude greater than theirαb , which suggests that the surface contribution plays a dominant role in 1/f noise. Experiments with DNA exhibited increasing capture rates with pH up to pH ∼8 followed by a drop at pH ∼9 perhaps due to the onset of electroosmotic force acting against the electrophoretic force. The1/f noise was also measured for several electrolytes and LiCl was found to outperform NaCl, KCl, RbCl, and CsCl. The 1/f noise was found to increase with the increasing electrolyte concentration and pore diameter. Taken together, the findings of this work suggest the pH approximate 7-8 range to be optimal for DNA sensing with CT-CDB nanopores.

Detection of nucleotides in hydrated ssDNA via 2D h-BN nanopore with ionic-liquid/salt-water interface.

Accomplishing slow translocation speed with high sensitivity has been the most critical mission for solid-state nanopore (SSN) device to electrically detect nucleobases in ssDNA. In this study, a method to detect nucleobases of ssDNA using a 2D SSN is introduced by considerably reducing the translocation speed and effectively increasing its sensitivity. The ultra-thin titanium dioxide coated hexagonal boron nitride nanopore was fabricated, along with an ionic-liquid 1-butyl-3-methylimidazolium hexafluorophosphate/2.0 M KCl aqueous (cis/trans) interface, for increasing both the spatial and the temporal resolutions. As the ssDNA molecules entered the nanopore, a brief surge of electrical conductivity occurred, which was followed by multiple resistive pulses from nucleobases during the translocation of ssDNA and another brief current surge flagging the exit of the molecule. The continuous detection of nucleobases using a 2D SSN device is a novel achievement: the water molecules bound to ssDNA increased the molecular conductivity and amplified electrical signals during the translocation. Along with the experiment, computational simulations using COMSOL Multiphysics are presented to explain the pivotal role of water molecules bound to ssDNA to detect nucleobases using a 2D SSN.

Quantification of low affinity binding interactions between natural killer cell inhibitory receptors and targeting ligands with a self-induced back-action actuated nanopore electrophoresis (SANE) sensor.

A plasmonic nanopore sensor enabling detection of bimodal optical and electrical molecular signatures was fabricated and tested for its ability to characterize low affinity ligand-receptor interactions. This plasmonic nanosensor uses self-induced back-action (SIBA) for optical trapping to enable SIBA-actuated nanopore electrophoresis (SANE) through a nanopore located immediately below the optical trap volume. A natural killer (NK) cell inhibitory receptor heterodimer molecule CD94/NKG2A was synthesized to target a specific peptide-presenting Qa-1b Qdm ligand as a simplified model of low-affinity interactions between immune cells and peptide-presenting cancer cells that occurs during cancer immunotherapy. A cancer-irrelevant Qa-1b GroEL ligand was also targeted by the same receptor as a control experiment to test for non-specific binding. The analysis of different pairs of bimodal SANE sensor signatures enabled discrimination of ligand, receptor and their complexes and enabled differentiating between specific and non-specific ligand interactions. We were able to detect ligand-receptor complex binding at concentrations over 500 times lower than the free solution equilibrium binding constant (K D ). Additionally, SANE sensor measurements enabled estimation of the fast dissociation rate (k off) for this low-affinity specific ligand-receptor system, previously shown to be challenging to quantify with commercial technologies. The k off value of targeted peptide-presenting ligands is known to correlate with the subsequent activation of immune cells in vivo, suggesting the potential utility of the SANE senor as a screening tool in cancer immunotherapy.

Multimodal Imaging Probe Development for Pancreatic ß Cells: From Fluorescence to PET.

Pancreatic ß cells are responsible for insulin secretion and are important for glucose regulation in a healthy body and diabetic disease patient without prelabeling of islets. While the conventional biomarkers for diabetes have been glucose and insulin concentrations in the blood, the direct determination of the pancreatic ß cell mass would provide critical information for the disease status and progression. By combining fluorination and diversity-oriented fluorescence library strategy, we have developed a multimodal pancreatic ß cell probe PiF for both fluorescence and for PET (positron emission tomography). By simple tail vein injection, PiF stains pancreatic ß cells specifically and allows intraoperative fluorescent imaging of pancreatic islets. PiF-injected pancreatic tissue even facilitated an antibody-free islet analysis within 2 h, dramatically accelerating the day-long histological procedure without any fixing and dehydration step. Not only islets in the pancreas but also the low background of PiF in the liver allowed us to monitor the intraportal transplanted islets, which is the first in vivo visualization of transplanted human islets without a prelabeling of the islets. Finally, we could replace the built-in fluorine atom in PiF with radioactive 18F and successfully demonstrate in situ PET imaging for pancreatic islets.

UHRF1 silences gelsolin to inhibit cell death in early stage cervical cancer.

Persistent infection with high-risk strains of human papillomavirus (HPV) is the primary cause of cervical cancer, the fourth most common cancer among women worldwide. Two oncoproteins encoded by the HPV genome, E6 and E7, are required for epigenetic modifications that promote cervical cancer development. We found that knockdown of HPV E6/E7 by siRNA reduced the levels of ubiquitin-like containing PHD and RING finger domain 1 (UHRF1) but increased the levels of gelsolin (GSN) in early stage cervical cancer cells. In addition, we found that UHRF1 levels were increased and GSN levels were decreased in early stage cervical cancer compared with those in normal cervical tissues, as shown by Western blot analysis, immunohistochemistry, and analysis of the Oncomine database. Moreover, knockdown of UHRF1 resulted in increased cell death in cervical cancer cell lines. Treatment of E6/E7-transformed HaCaT (HEK001) cells and HeLa cells with the DNA-hypomethylating agent 5-aza-2'-deoxycytidine and the histone deacetylase inhibitor Trichostatin A increased GSN expression levels. UHRF1 knockdown in HEK001 cells by siRNA or the UHRF1 antagonist thymoquinone increased GSN levels, induced cell cycle arrest and apoptosis, and increased the levels of p27 and cleaved PARP. Those results indicate that upregulation of UHRF1 by HPV E6/E7 causes GSN silencing and a reduction of cell death in early stage cervical cancer, suggesting that GSN might be a useful therapeutic target in early stage cervical cancer.

Mechanical characterization of vesicles and cells: A review.

Vesicles perform many essential functions in all living organisms. They respond like a transducer to mechanical stress in converting the applied force into mechanical and biological responses. At the same time, both biochemical and biophysical signals influence the vesicular response in bearing mechanical loads. In recent years, liposomes, artificial lipid vesicles, have gained substantial attention from the pharmaceutical industry as a prospective drug carrier which can also serve as an artificial cell-mimetic system. The ability of these vesicles to enter through pores of even smaller size makes them ideal candidates for therapeutic agents to reach the infected sites effectively. Engineering of vesicles with desired mechanical properties that can encapsulate drugs and release as required is the prime challenge in this field. This requirement has led to the modifications of the composition of the bilayer membrane by adding cholesterol, sphingomyelin, etc. In this article, we review the manufacturing and characterization techniques of various artificial/synthetic vesicles. We particularly focus on the electric field-driven characterization techniques to determine different properties of vesicle and its membranes, such as bending rigidity, viscosity, capacitance, conductance, etc., which are indicators of their content and mobility. Similarities and differences between artificial vesicles, natural vesicles, and cells are highlighted throughout the manuscript since most of these artificial vesicles are intended for cell mimetic functions.

Fabrication of hexagonal boron nitride based 2D nanopore sensor for the assessment of electro-chemical responsiveness of human serum transferrin protein.

In this work, we present a step-by-step workflow for the fabrication of 2D hexagonal boron nitride (h-BN) nanopores which are then used to sense holo-human serum transferrin (hSTf) protein at pH ∼8 under applied voltages ranging from +100 mV to +800 mV. 2D nanopores are often used for DNA, however, there is a great void in the literature for single-molecule protein sensing and this, to the best of our knowledge, is the first time where h-BN-a material with large band-gap, low dielectric constant, reduced parasitic capacitance and minimal charge transfer induced noise-is used for protein profiling. The corresponding ΔG (change in pore conductance due to analyte translocation) profiles showed a bimodal Gaussian distribution where the lower and higher ΔG distributions were attributed to (pseudo-) folded and unfolded conformations respectively. With increasing voltage, the voltage induced unfolding increased (evident by decrease in ΔG) and plateaued after ∼400 mV of applied voltage. From the ΔG versus voltage profile corresponding to the pseudo-folded state, we calculated the molecular radius of hSTf, and was found to be ∼3.1 nm which is in close concordance with the literature reported value of ∼3.25 nm.

Highly efficient production of diverse rare ginsenosides using combinatorial biotechnology.

The rare ginsenosides are recognized as the functionalized molecules after the oral administration of Panax ginseng and its products. The sources of rare ginsenosides are extremely limited because of low ginsenoside contents in wild plants, hindering their application in functional foods and drugs. We developed an effective combinatorial biotechnology approach including tissue culture, immobilization, and hydrolyzation methods. Rh2 and nine other rare ginsenosides were produced by methyl jasmonate-induced culture of adventitious roots in a 10 L bioreactor associated with enzymatic hydrolysis using six ß-glycosidases and their combination with yields ranging from 5.54 to 32.66 mg L-1 . The yield of Rh2 was furthermore increased by 7% by using immobilized BglPm and Bgp1 in optimized pH and temperature conditions, with the highest yield reaching 51.17 mg L-1 (17.06% of protopanaxadiol-type ginsenosides mixture). Our combinatorial biotechnology method provides a highly efficient approach to acquiring diverse rare ginsenosides, replacing direct extraction from Panax plants, and can also be used to supplement yeast cell factories.

Beyond nanopore sizing: improving solid-state single-molecule sensing performance, lifetime, and analyte scope for omics by targeting surface chemistry during fabrication.

Solid-state nanopores (SSNs) are single-molecule resolution sensors with a growing footprint in real-time bio-polymer profiling-most prominently, but far from exclusively, DNA sequencing. SSNs accessibility has increased with the advent of controlled dielectric breakdown (CDB), but severe fundamental challenges remain: drifts in open-pore current and (irreversible) analyte sticking. These behaviors impede basic research and device development for commercial applications and can be dramatically exacerbated by the chemical complexity and physical property diversity of different analytes. We demonstrate a SSN fabrication approach attentive to nanopore surface chemistry during pore formation, and thus create nanopores in silicon nitride (SiNx) capable of sensing a wide analyte scope-nucleic acid (double-stranded DNA), protein (holo-human serum transferrin) and glycan (maltodextrin). In contrast to SiNx pores fabricated without this comprehensive approach, the pores are Ohmic in electrolyte, have extremely stable open-pore current during analyte translocation (>1 h) over a broad range of pore diameters ([Formula: see text]3- ∼30 nm) with spontaneous current correction (if current deviation occurs), and higher responsiveness (i.e. inter-event frequency) to negatively charged analytes (∼6.5 × in case of DNA). These pores were fabricated by modifying CDB with a chemical additive-sodium hypochlorite-that resulted in dramatically different nanopore surface chemistry including ∼3 orders of magnitude weaker Ka (acid dissociation constant of the surface chargeable head-groups) compared to CDB pores which is inextricably linked with significant improvements in nanopore performance with respect to CDB pores.