Fast Fabrication Nanopores on a PMMA Membrane by a Local High Electric Field Controlled Breakdown.

The sensitivity and accuracy of nanopore sensors are severely hindered by the high noise associated with solid-state nanopores. To mitigate this issue, the deposition of organic polymer materials onto silicon nitride (SiNx) membranes has been effective in obtaining low-noise measurements. Nonetheless, the fabrication of nanopores sub-10 nm on thin polymer membranes remains a significant challenge. This work proposes a method for fabricating nanopores on polymethyl methacrylate (PMMA) membrane by the local high electrical field controlled breakdown, exploring the impact of voltage and current on the breakdown of PMMA membranes and discussing the mechanism underlying the breakdown voltage and current during the formation of nanopores. By improving the electric field application method, transient high electric fields that are one-seven times higher than the breakdown electric field can be utilized to fabricate nanopores. A comparative analysis was performed on the current noise levels of nanopores in PMMA-SiNx composite membranes and SiNx nanopores with a 5 nm diameter. The results demonstrated that the fast fabrication of nanopores on PMMA-SiNx membranes exhibited reduced current noise compared to SiNx nanopores. This finding provides evidence supporting the feasibility of utilizing this technology for efficiently fabricating low-noise nanopores on polymer composite membranes.

The combination of DNA nanostructures and materials for highly sensitive electrochemical detection.

Due to the wide range of electrochemical devices available, DNA nanostructures and material-based technologies have been greatly broadened. They have been actively used to create a variety of beautiful nanostructures owing to their unmatched programmability. Currently, a variety of electrochemical devices have been used for rapid sensing of biomolecules and other diagnostic applications. Here, we provide a brief overview of recent advances in DNA-based biomolecular assays. Biosensing platform such as electrochemical biosensor, nanopore biosensor, and field-effect transistor biosensors (FET), which are equipped with aptamer, DNA walker, DNAzyme, DNA origami, and nanomaterials, has been developed for amplification detection. Under the optimal conditions, the proposed biosensor has good amplification detection performance. Further, we discussed the challenges of detection strategies in clinical applications and offered the prospect of this field.

Omnidirectional Monolithic Marker for Intra-Operative MR-Based Positional Sensing in Closed MRI.

We present a design of an inductively coupled radio frequency (ICRF) marker for magnetic resonance (MR)-based positional tracking, enabling the robust increase of tracking signal at all scanning orientations in quadrature-excited closed MR imaging (MRI). The marker employs three curved resonant circuits fully covering a cylindrical surface that encloses the signal source. Each resonant circuit is a planar spiral inductor with parallel plate capacitors fabricated monolithically on flexible printed circuit board (FPC) and bent to achieve the curved structure. Size of the constructed marker is Ø3-mm ×5 -mm with quality factor > 22, and its tracking performance was validated with 1.5 T MRI scanner. As result, the marker remains as a high positive contrast spot under 360° rotations in 3 axes. The marker can be accurately localized with a maximum error of 0.56 mm under a displacement of 56 mm from the isocenter, along with an inherent standard deviation of 0.1-mm. Accrediting to the high image contrast, the presented marker enables automatic and real-time tracking in 3D without dependency on its orientation with respect to the MRI scanner receive coil. In combination with its small form-factor, the presented marker would facilitate robust and wireless MR-based tracking for intervention and clinical diagnosis. This method targets applications that can involve rotational changes in all axes (X-Y-Z).

Interactive Multi-Stage Robotic Positioner for Intra-Operative MRI-Guided Stereotactic Neurosurgery.

Magnetic resonance imaging (MRI) demonstrates clear advantages over other imaging modalities in neurosurgery with its ability to delineate critical neurovascular structures and cancerous tissue in high-resolution 3D anatomical roadmaps. However, its application has been limited to interventions performed based on static pre/post-operative imaging, where errors accrue from stereotactic frame setup, image registration, and brain shift. To leverage the powerful intra-operative functions of MRI, e.g., instrument tracking, monitoring of physiological changes and tissue temperature in MRI-guided bilateral stereotactic neurosurgery, a multi-stage robotic positioner is proposed. The system positions cannula/needle instruments using a lightweight (203 g) and compact (Ø97 × 81 mm) skull-mounted structure that fits within most standard imaging head coils. With optimized design in soft robotics, the system operates in two stages: i) manual coarse adjustment performed interactively by the surgeon (workspace of ±30°), ii) automatic fine adjustment with precise (<0.2° orientation error), responsive (1.4 Hz bandwidth), and high-resolution (0.058°) soft robotic positioning. Orientation locking provides sufficient transmission stiffness (4.07 N/mm) for instrument advancement. The system's clinical workflow and accuracy is validated with lab-based (<0.8 mm) and MRI-based testing on skull phantoms (<1.7 mm) and a cadaver subject (<2.2 mm). Custom-made wireless omni-directional tracking markers facilitated robot registration under MRI.

Highly accelerated multi-shot intravoxel incoherent motion diffusion-weighted imaging in brain enabled by parametric POCS-based multiplexed sensitivity encoding.

Recently, intravoxel incoherent motion (IVIM) diffusion-weighted imaging (DWI) has also been demonstrated as an imaging tool for applications in neurological and neurovascular diseases. However, the use of single-shot diffusion-weighted echo-planar imaging for IVIM DWI acquisition leads to suboptimal data quality: for instance, geometric distortion and deteriorated image quality at high spatial resolution. Although the recently commercialized multi-shot acquisition methods, such as multiplexed sensitivity encoding (MUSE), can attain high-resolution and high-quality DWI with signal-to-noise ratio (SNR) performance superior to that of the conventional parallel imaging method, the prolonged scan time associated with multi-shot acquisition is impractical for routine IVIM DWI. This study proposes an acquisition and reconstruction framework based on parametric-POCSMUSE to accelerate the four-shot IVIM DWI with 70% reduction of total scan time (13 min 8 s versus 4 min 8 s). First, the four-shot IVIM DWI scan with 17 b values was accelerated by acquiring only one segment per b value except for b values of 0 and 600 s/mm2 . Second, an IVIM-estimation scheme was integrated into the parametric-POCSMUSE to enable joint reconstruction of multi-b images from under-sampled four-shot IVIM DWI data. In vivo experiments on both healthy subjects and patients show that the proposed framework successfully produced multi-b DW images with significantly higher SNRs and lower reconstruction errors than did the conventional acceleration method based on parallel imaging. In addition, the IVIM quantitative maps estimated from the data produced by the proposed framework showed quality comparable to that of fully sampled MUSE-reconstructed images, suggesting that the proposed framework can enable highly accelerated multi-shot IVIM DWI without sacrificing data quality. In summary, the proposed framework can make multi-shot IVIM DWI feasible in a routine MRI examination, with reasonable scan time and improved geometric fidelity.

Enhanced Biogenic Sulfidation of Zero-Valent Iron in Columns: Implications for Promoting Dechlorination in Permeable Reactive Barriers.

Biogenic sulfidation of zero-valent iron (ZVI) using sulfate reducing bacteria (SRB) has shown enhanced dechlorination rates comparable to those produced by chemical sulfidation. However, controlling and sustaining biogenic sulfidation to enhance in situ dechlorination are poorly understood. Detailed interactions between SRB and ZVI were examined for 4 months in column experiments under enhanced biogenic sulfidation conditions. SRB proliferation and changes in ZVI surface properties were characterized along the flow paths. The results show that ZVI can stimulate SRB activity by removing excessive free sulfide (S2-), in addition to lowering reduction potential. ZVI also hinders downgradient movement of SRB via electrostatic repulsion, restricting SRB presence near the upgradient interface. Dissolved organic carbon (e.g., >2.2 mM) was essential for intense biogenic sulfidation in ZVI columns. The presence of SRB in the upgradient zone appeared to promote the formation of iron polysulfides. Biogenic FeSx deposition increased the S content on ZVI surfaces ∼3-fold, corresponding to 3-fold and 2-fold improvements in the trichloroethylene degradation rate and electron efficiency in batch tests. Elucidation of SRB and ZVI interactions enhances sustained sulfidation in ZVI permeable reactive barrier.

Single-molecule lipopolysaccharides identification and the interplay with biomolecules via nanopore readout.

Lipopolysaccharides (LPS) are the major constituent on the cell envelope of all gram-negative bacteria. They are ubiquitous in air, and are toxic inflammatory stimulators for urinary disorders and sepsis. The reported optical, thermal, and electrochemical sensors via the intermolecular interplay of LPS with proteins and aptamers are generally complicated methods. We demonstrate the single-molecule nanopore approach for LPS identification in distinct bacteria as well as the serotypes discrimination. With a 4 nm nanopore, we achieve a detection limit of 10 ng/mL. Both the antibiotic polymyxin B (PMB) and DNA aptamer display specific binding to LPS. The identification of LPS in both human serum and tap water show good performance with nanopore platforms. Our work shows a highly-sensitive and easy-to-handle scheme for clinical and environmental biomarkers determination and provides a promising screening tool for early warning of contamination in water and medical supplies.

Thymosin α1 modulated the immune landscape of COVID-19 patients revealed by single-cell RNA and TCR sequencing.

BACKGROUND: The Coronavirus disease-19 (COVID-19) pandemic has posed a serious threat to global health. Thymosin α1 (Tα1) was considered to be applied in COVID-19 therapy. However, the data remains limited. METHODS: Participants with or without Tα1 treatment were recruited. Single cell RNA-sequencing (scRNA-seq) and T cell receptor-sequencing (TCR-seq) of the peripheral blood mononuclear cell (PBMC) samples were done to analyze immune features. The differential expression analysis and functional enrichment analysis were performed to explore the mechanism of Tα1 therapy. RESULTS: 33 symptomatic SARS-CoV-2-infected individuals (COV) and 11 healthy controls (HC) were enrolled in this study. The proportion of CD3+ KLRD1+ NKT, TBX21+ CD8+ NKT was observed to increase in COVID-19 patients with Tα1 treatment (COVT) than those without Tα1 (COV) (p = 0.024; p = 0.010). These two clusters were also significantly higher in Health controls with Tα1 treatment (HCT) than those without Tα1 (HC) (p = 0.016; p = 0.031). Besides, a series of genes and pathways related to immune responses were significantly higher enriched in Tα1 groups TBX21+ CD8+ NKT, such as KLRB1, PRF1, natural killer cell-mediated cytotoxicity pathway, chemokine signaling pathway, JAK-STAT signaling pathway. The increased TRBV9-TRBJ1-1 pair existed in both HCs and COVID-19 patients after Tα1 treatment. 1389 common complementarity determining region 3 nucleotides (CDR 3 nt) were found in COV and HC, while 0 CDR 3 nt was common in COVT and HCT. CONCLUSIONS: Tα1 increased CD3+ KLRD1+ NKT, TBX21+ CD8+ NKT cell proportion and stimulated the diversity of TCR clones in COVT and HCT. And Tα1 could regulate the expression of genes associated with NKT activation or cytotoxicity to promote NKT cells. These data support the use of Tα1 in COVID-19 patients.

Low-noise and high-speed trans-impedance amplifier for nanopore sensor.

The small current detection circuit is the core component of the accurate detection of the nanopore sensor. In this paper, a compact, low-noise, and high-speed trans-impedance amplifier is built for the nanopore detection system. The amplifier consists of two amplification stages. The first stage performs low-noise trans-impedance amplification by using ADA4530-1, which is a high-performance FET operational amplifier, and a high-ohm feedback resistor of 1 GΩ. The high pass shelf filter in the second stage recovers the higher frequency above the 3 dB cutoff in the first stage to extend the maximum bandwidth up to 50 kHz. The amplifier shows a low noise below sub-2 pA rms when tuned to have a bandwidth of around 5 kHz. It also guarantees a stable frequency response in the nanopore sensor.

Phenylboronic acid-modified polyethyleneimine assisted neutral polysaccharide detection and weight-resolution analysis with a nanopipette.

In this work, an innovative method based on a nanopipette assisted with o-phenylboronic acid-modified polyethyleneimine (PEI-oBA) is proposed to detect neutral polysaccharides with different degrees of polymerization. Herein, dextran is used as the research target. Dextran, with its low molecular weight (104 < MW < 105 Da), has important applications in medicine and is one of the best plasma substitutes at present. Through the interaction between the boric acid group and a hydroxyl group, the synthesized high-charge polymer molecule PEI-oBA combines with dextran, increasing the electrophoretic force and exclusion volume of the target molecule to obtain a high signal-to-noise ratio for nanopore detection. These results show that the current amplitude increased significantly with the increase of dextran molecular weight. Furthermore, an aggregation-induced emission (AIE) molecule was introduced to adsorb onto PEI-oBA to verify that PEI-oBA combined with a polysaccharide entered the nanopipette together and was driven by electrophoresis. With the introduction of the modifiability of polymer molecules, the proposed method is conducive to improving the nanopore detection sensitivity of other important molecules with low charges and low molecular weights.

Nanopore discrimination and sensitive plasma detection of multiple natriuretic peptides: The representative biomarker of human heart failure.

Natriuretic peptides can relieve cardiovascular stress and closely related to heart failure. Besides, these peptides also have preferable interactions of binding to cellular protein receptors, and subsequently mediate various physiology actions. Hence, detection of these circulating biomarkers could be evaluated as a predictor ("Gold standard") for rapid, early diagnosis and risk stratification in heart failure. Herein, we proposed a measurement to discriminate multiple natriuretic peptides via the peptide-protein nanopore interaction. The nanopore single-molecular kinetics revealed that the strength of peptide-protein interactions was in the order of ANP > CNP > BNP, which was demonstrated by the simulated peptide structures using SWISS-MODEL. More importantly, the peptide-protein interaction analyzing also allowed us to measure the peptide linear analogs and structure damage in peptide by single-chemical bond breakup. Finally, we presented an ultra-sensitive detection of plasma natriuretic peptide using asymmetric electrolyte assay, obtaining a detection limit of ∼770 fM for BNP. At approximately, it is 1597 times lower than that of using symmetric assay (∼1.23 nM), 8 times lower than normal human level (∼6 pM), and 13 times lower than the diagnostic values (∼10.09 pM) complied in the guideline of European Society of Cardiology. That said, the designed nanopore sensor is benefit for natriuretic peptides measurement at single molecule level and demonstrates its potential for heart failure diagnosis.

Effect of sulfidation on nitrobenzene removal from groundwater by microscale zero-valent iron: Insights into reactivity, reaction sites and removal pathways.

While it has been recognized that sulfidation can effectively improve the reactivity of microscale zero valent iron (mZVI), there is limited understanding of nitrobenzene (ArNO2) removal by sulfidated mZVI. To understand the reduction capacity and pathway of ArNO2 by sulfidated mZVI, ball-milling sulfidated mZVI (S-mZVIbm) with different S/Fe molar ratios (0-0.2) was used to conduct this experiment. The results showed that sulfidation could efficiently enhance ArNO2 removal under iron-limited and iron excess conditions, which was attributed to the presence of FeSx sites that could provide higher Fe(0) utilization efficiency and stronger passivation resisting for S-mZVIbm. The optimum ArNO2 reduction could be obtained by S-mZVIbm with S/Fe molar ratio at 0.1, which could completely transform ArNO2 to aniline (ArNH2) with a rate constant of 4.36 × 10-2 min-1 during 120-min reaction. FeSx phase could act as electron transfer sites for ArNO2 reduction and it could still be reserved in S-mZVIbm after reduction reaction. The product distribution indicated that sulfidation did not change the types of reduction products, while the removal of ArNO2 by S-mZVIbm was a step-by-step reduction progress along with the adsorption of ArNH2. In addition, a faster reduction of ArNO2 in groundwater/soil system further demonstrated the feasibility of S-mZVIbm in the real field remediation.

An ultrasensitive aptasensor of SARS-CoV-2 N protein based on ion current rectification with nanopipettes.

Since the outbreak of COVID-19 in the world, it has spread rapidly all over the world. Rapid and effective detection methods have been a focus of research. The SARS-CoV-2 N protein (NP) detection methods currently in use focus on specific recognition of antibodies, but the reagents are expensive and difficult to be produced. Here, aptamer-functionalized nanopipettes utilize the unique ion current rectification (ICR) of nanopipette to achieve rapid and highly sensitive detection of trace NP, and can significantly reduce the cost of NP detection. In the presence of NP, the surface charge at the tip of the nanopipette changes, which affects ion transport and changes the degree of rectification. Quantitative detection of NP is achieved through quantitative analysis. Relying on the high sensitivity of nanopipettes to charge fluctuations, this sensor platform achieves excellent sensing performance. The sensor platform exhibited a dynamic working range from 102-106 pg/mL with a detection limit of 73.204 pg/mL, which showed great potential as a tool for rapidly detecting SARS-CoV-2. As parallel and serial testing are widely used in the clinic to avoid missed diagnosis or misdiagnosis, we hope this platform can play a role in controlling the spread and prevention of COVID-19.

Transport of nanoscale zero-valent iron in saturated porous media: Effects of grain size, surface metal oxides, and sulfidation.

Knowledge of the fate and transport of nanoscale zero-valent iron (nZVI) in saturated porous media is crucial to the development of in situ remediation technologies. This work systematically compared the retention and transport of carboxymethyl cellulose (CMC) modified nZVI (CMC-nZVI) and sulfidated nZVI (CMC-S-nZVI) particles in saturated columns packed with quartz sand of various grain sizes and different surface metal oxide coatings. Grain size reduction had an inhibitory effect on the transport of CMC-S-nZVI and CMC-nZVI due to increasing immobile zone deposition and straining in the columns. Metal oxide coatings had minor effect on the transport of CMC-S-nZVI and CMC-nZVI because the sand surface was coated by the free CMC in the suspensions, reducing the electrostatic attraction between the nZVI and surface metal oxides. CMC-S-nZVI displayed greater breakthrough (C/C0 = 0.82-0.90) and higher mass recovery (84.9%-89.3%) than CMC-nZVI (C/C0 = 0.70-0.80 and mass recovery = 70.9%-79.6%, respectively) under the same experimental conditions. A mathematical model based on the advection-dispersion equation simulated the experimental data of nZVI breakthrough curves very well. Findings of this study suggest sulfidation could enhance the transport of CMC-nZVI in saturated porous media with grain and surface heterogeneities, promoting its application in situ remediation.

DNA nanostructure-assisted detection of carcinoembryonic antigen with a solid-state nanopore.

In this paper, a novel detection technique for tumor marker carcinoembryonic antigen (CEA) has been developed by using a solid-state nanopore as a tool. The system utilizes the specific affinity between aptamer-modified magnetic Fe3O4 and CEA, rather than directly detecting the translocation of CEA through the nanopore. The aptamer-modified magnetic Fe3O4 was hybridized with tetrahedral DNA nanostructures (TDNs), and TDNs were released after CEA was added. We investigate the translocation behavior of individual TDNs through solid-state nanopores. The frequency of the blockage signals for TDNs is recorded for indirect detection of CEA. We realized the detection of CEA with a concentration as low as 0.1 nM and proved the specificity of the interaction between the aptamer. In addition, our designed nanopore sensing strategy can detect CEA in real samples.

Simultaneous Dual-Site Identification of 5mC/8oG in DNA Triplex Using a Nanopore Sensor.

DNA triplex participates in delivering site-specific epigenetic modifications critical for the regulation of gene expression. Among these marks, 5mC with 8oG functions comprehensively on gene expression. Recently, few research studies have emphasized the necessity of incorporation detection of 5mC with 8oG using one DNA triplex at the same time. Herein, DNA triplex structure was designed and tailored for the site-specific identification of 5mC with 8oG by means of nanopore electroanalysis. The identification was associated with the distinguishable current modulation types caused by DNA unzipping through the nanopore in an electrical field. Results demonstrated that the epigenetic modification proximity to the latch zone or constriction area of the nanopore enables differentiation of modification series at single nucleotide resolution in one DNA triplex, at both physiological and mildly acidic environment. In addition, our nanopore method enables the kinetic and thermodynamic studies to calculate the free energy of modified DNA triplex with applied potentials. Gibbs' energy provided the direct evidence that the DNA triplex with these epigenetic modifications is more stable in acidic environment. Considering modified DNA functions significantly in gene expression, the presented method may provide future opportunities to understand incorporating epigenetic mechanisms of many dysregulated biological processes on the basis of accurate detection.

De novo profiling of insect-resistant proteins of rice via nanopore peptide differentiation.

Nanopore is used as a single-molecule detector for proteins, peptides and amino acids identification of biomedical importance. We, on the first try, extended its profiling application for peptidome encoded by the insect-resistance gene in rice. Taking brown planthopper (Bph) for example, we previously cloned and verified Bph32 gene, which encodes SCR ("Bph32") protein as direct executor against this injurious insect. However, the homology protein expressed in susceptible line doesn't have this antibiosis resistance. Hence, profiling the structural modulars (peptide domains) of Bph32 proteins may provide essential basis to understand rice in response to insects. Herein, we combined approaches of bioinformatics, biochemistry and nanopore analysis to profile the rice peptides with diverse properties. Bph32 proteins were theoretically modeled into 24 functional peptide domains using Swiss-Model workspace. Next, 22 water-soluble peptides were identified by biuret-chemistry amplified nanopore current modulations. Among those, 16 ones were distinguished at one amino acid resolution via reading the current modulation spectrum, consequently providing the peptidome fingerprints. In addition, the current modulations were evidenced as quadratic function of peptide's molecular masses. These findings suggest that nanopore may work as a new generation of mass detector for more omics analysis, especially in agricultural field where demands strongly.

Development and characterization of biodegradable ultraviolet protective and antibacterial polylactic acid-cellulose acetate film modified by phenyl salicylate.

The polylactic acid composite films were successfully fabricated via the technique of solvent casting using cellulose acetate (20%, wt) as the reinforcing material and phenyl salicylate as the ultraviolet (UV) absorbent and antibacterial agent. Polylactic acid-cellulose acetate-phenyl salicylate composite films displayed complete absorption effect at the region of UV-C (280-100 nm) and UV-B (315-280 nm), and more than 95% UV absorption effect at the region of UV-A (400-315 nm). These results indicate that the UV shielding performance of the composite films could be significantly improve by addition of phenyl salicylate. Moreover, the addition of 20% phenyl salicylate could improve the steam resistance, mechanical properties and thermal stability of the films, and the composite films had also better antibacterial activity against Escherichia coli. The composite films could reduce the decay rate of fresh lilies and extend their storage time. The degradation characteristics of the films were explored in the natural environment and the laboratory level, which provided application prospect for the development of degradable food packaging materials with anti-ultraviolet and anti-bacteria effect.

Nanopore-based aptasensor for label-free and sensitive vanillin determination in food samples.

Dielectric breakdown technique was utlised to fabricate 5-6 nm nanopores for vanillin detection in various food samples. A highly selective aptamer (Van_74) with high binding affinity towards vanillin was used as capture probe. Under optimal conditions, aptamer/vanillin complex translocation induced deeper events than the bare aptamer. As a result, the proposed nanopore aptasensor exhibits a linear range from 0.5 to 5 nM (R2 = 0.972) and a low detection limit of 500 pM, which is significantly better than conventional platforms. Furthermore, our aptasensor showed excellent immunity against different interferons and was used to detect vanillin in different food samples. The food sample measurements were confirmed with an additional UV-Vis assay, the results of the two techniques were statistically evaluated and showed no statistically significant difference. Hence, this work represents a proof-of-concept involving the design and testing of aptamer/nanopore sensors for small molecules detection, which plays a critical role in food safety.