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.

Impact of cervical intraepithelial neoplasia and treatment on IVF/ICSI outcomes.

STUDY QUESTION: Does treatment selection for cervical lesions affect the outcome of IVF/ICSI? SUMMARY ANSWER: There was no difference in pregnancy outcome between treated and untreated groups, or between different types of IVF/ICSI treatment. WHAT IS KNOWN ALREADY: Human papillomavirus (HPV) infection and HPV-induced cervical lesions are associated with decreased fertility, and cervical intraepithelial neoplasia (CIN) treatment may increase the risk of adverse pregnancy outcomes. STUDY DESIGN, SIZE, DURATION: Between 2018 and 2020, 190 women with infertility who had abnormal HPV screening or cytology results prior to IVF/ICSI, and were diagnosed with CIN2/CIN3 by colposcopy biopsy at a tertiary hospital, were enrolled in a retrospective cohort study with follow-up until 31 December 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients with infertility who were diagnosed with CIN2/CIN3 by colposcopy biopsy were divided into the treatment and expectant management groups. The treatment group was divided into two intervention subgroups: the ablative therapy group and the surgical treatment group. The baseline data, number of oocytes retrieved, and rates of fertilization, high-quality embryos, positive serum HCG, clinical pregnancy, abortion, live birth, and cumulative pregnancy were compared among groups. MAIN RESULTS AND THE ROLE OF CHANCE: Among the 190 patients included in the study, 152 were diagnosed with CIN2, and 38 patients had CIN3. There was no significant difference in the baseline data between the treatment and expectant groups. The time from confirmed lesions to the onset of gonadotrophin administration in the surgical treatment group was significantly longer than in the ablative therapy group and the expectant group (P = 0.007 and P = 0.024, respectively). For the treatment and expectant groups, respectively, the average number of oocytes retrieved (12.95 ± 8.77; 13.32 ± 9.16), fertilization rate (71.01 ± 23.86; 64.84 ± 26.24), and high-quality embryo rate (48.93 ± 30.72; 55.17 ± 34.13) did not differ, and no differences were detected between the different treatment subgroups. There were no differences among groups in rates of HCG positivity, clinical pregnancy, miscarriage, live birth, or cumulative pregnancy. The live birth rate in the surgical treatment group was slightly higher than that in the expectant groups (77.78% versus 66.67%), but the difference was not statistically significant. The 3-year cumulative pregnancy rates in the surgical treatment and expectant groups were 58.19% and 64.00%, respectively. LIMITATIONS, REASONS FOR CAUTION: This is a retrospective study, which by nature can include selection bias, and the number of cases in the expectant group was <30, which may result in a false-negative result owing to the small sample size. WIDER IMPLICATIONS OF THE FINDINGS: For patients with CIN2/CIN3, the treatment of cervical lesions does not affect the outcome of IVF/ICSI. Patients with CIN2 can enroll for IVF/ICSI cycles, with close follow-up to prevent the progression of cervical lesions, in order to avoid further delay in starting ART. For patients with CIN3, ovulation induction and embryo cryopreservation can be initiated as soon as possible after cervical lesions are treated, and frozen-thawed embryo transfer can be carried out 9-12 months later. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by the Key Clinical Projects of the Peking University Third Hospital (to Y.W., BYSYZD2021014). The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

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.

Comparison Study on Single Nucleotide Transport Phenomena in Carbon Nanotubes.

To be considered as a promising candidate for mimicking biological nanochannels, carbon nanotubes (CNTs) have been used to explore the mass transport phenomena in recent years. In this study, the single nucleotide transport phenomena are comparatively studied using individual CNTs with a length of ∼15 µm and diameters ranging from 1.5 to 2.5 nm. In the case of CNTs with a diameter of 1.57-1.98 nm, the current traces of nucleotide transport are independent with the metallicity of CNTs and consist of single peak current pulses, whereas extraordinary stepwise current signals are observed in CNT with a diameter of 2.33 nm. It suggests that there is only one molecule in the nanochannel at a time until the diameter of CNT increases to 2.33 nm. Furthermore, it also demonstrates that the single nucleotides can be identified statistically according to their current pulses, indicating the potential application of CNT-based sensors for nucleotides identification.

Reversible photo-regulation on the folding/unfolding of telomere G-quadruplexes with solid-state nanopores.

The formation of G-quadruplexes (G4) in human telomere and other important biological regions inhibits the replication and transcription of DNA, thereby influencing further cell proliferation. The investigation of G4 formation and unfolding is vital for understanding their modulation in biological processes and life science. Photo regulation is a facile and sensitive approach for monitoring the structures of biomacromolecules and material surface properties. The nanopore-based technique is also prevalent for label-free single-molecule characterization with high accuracy. This study provides a combination of solid-state nanopore technology with light-switch as a platform for the modulation of human telomere G4 formation and splitting under switchable light exposure. The introduction of molecular switch, namely azobenzene moiety at different positions of the DNA sequence influences the formation and stability of G4. Three azobenzenes immobilized on each of the G-quartet plane (hTelo-3azo-p) or four azobenzenes on the same plane (hTelo-4azo-4p) of the human telomere G4 sequence realized the reversible control of G4 folding/unfolding at the temporal scale upon photo regulation, and the formation and splitting of G4 with hTelo-4azo-4p is slower and not thorough compared to that with hTelo-3azo-p due to the coplanar steric hindrance. Moreover, the G4 formation recorded with the combined nanopore and photo-responsive approach was also characterized with fluorescence, and the variation in the fluorescence intensity of the NMM and G4 complex exhibited a different tendency under reverse light irradiation due to the distinct interactions of NMM with the azobenzene-modified G4. Our study demonstrated a controllable and sensitive way for the manipulation of G4 structures, which will be inspiring for the intervention of G4-related cell senescence, cancer diagnosis and drug exploration.

Development of an all-sky imaging system for cloud cover assessment.

This study develops a novel automatic all-sky imaging system, namely, an all-sky camera (ASC) system, for cloud cover assessment. The proposed system does not require conventional solar occulting devices and can capture complete hemispheric sky images. Cloud detection is performed innovatively using a convolutional neural network model (i.e., the optimized U-Net model). Experiments demonstrate that the optimized U-Net model can effectively detect clouds from sky images. In terms of cloud cover, the estimation results of the ASC system exhibit a high correlation with those obtained via manual observation, thereby indicating the applicability of the ASC system in ground-based cloud observation and analysis.

Label-Free Sensitive Detection of Microcystin-LR via Aptamer-Conjugated Gold Nanoparticles Based on Solid-State Nanopores.

A versatile and highly sensitive strategy for nanopore detection of microcystin-LR (MC-LR) is proposed herein based on the aptamer and host-guest interactions by employing a gold nanoparticle (AuNP) probe. The aptamer of MC-LR and its complementary DNA (cDNA) are respectively immobilized on AuNPs with distinct sizes (5 nm AuNPs for the aptamer and 20 nm for the cDNA), and the constructed polymeric AuNP network via the hybridization of the aptamer and cDNA was disintegrated upon the addition of MC-LR. The specific interactions between the aptamer and MC-LR disrupt and release the cDNA-AuNPs that were then removed by centrifugation, leaving the MC-LR-aptamer-AuNP species in the supernatant for subsequent nanopore determination. By monitoring the current blockade of released MC-LR-aptamer-AuNPs using a specific tailored nanopore (10 and 20 nm in diameter, generated by current dielectric breakdown), we could deduce the presence of MC-LR, as the bulky NP network could not pass through a nanopore with a relatively smaller size. We realized the detection of MC-LR with a concentration as low as 0.1 nM; additionally, we have proved the specificity of the interaction between the aptamer and MC-LR by replacing MC-LR with other congener toxins (MC-RR and MC-YR), chlorophyll (a component abundantly coexists in water), and the mixture of the four.

Dual-drug loaded nanoneedles with targeting property for efficient cancer therapy.

BACKGROUND: Since the anticancer drugs have diverse inhibited mechanisms to the cancer cells, the use of two or more kinds of anticancer agents may achieve excellent therapeutic effects, especially to the drug-resistant tumors. RESULTS: In this study, we developed a kind of dual drug [methotrexate (MTX) and 10-hydroxycamptothecine (HCPT)] loaded nanoneedles (DDNDs) with pronounced targeting property, high drug loading and prolonged drug release. The anti-solvent precipitation of the HCPT and MTX modified PEG-b-PLGA (PEG-b-PLGA-MTX, PPMTX) leads to nucleation of nanoneedles with nanocrystalline HCPT as the core wrapped with PPMTX as steric stabilizers. In vitro cell uptake studies showed that the DDNDs revealed an obviously targeting property and entered the HeLa cells easier than the nanoneedles without MTX modification. The cytotoxicity tests illustrated that the DDNDs possessed better killing ability to HeLa cells than the individual drugs or their mixture in the same dose, indicating its good synergistic effect and targeting property. The in vivo studies further confirmed these conclusions. CONCLUSIONS: This approach led to a promising sustained drug delivery system for cancer diagnosis and treatment.

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.

Surface enhanced Raman scattering substrate with metallic nanogap array fabricated by etching the assembled polystyrene spheres array.

A sensitive surface enhanced Raman scattering (SERS) substrate with metallic nanogap array (MNGA) is fabricated by etching of an assembled polystyrene (PS) spheres array, followed by the coating of a metal film. The substrate is reproducible in fabrication and sensitive due to the nanogap coupling resonance (NGCR) enhancement. The NGCR is analyzed with the finite difference time domain (FDTD) method, and the relationship between the gap parameter and the field enhancement is obtained. Experimental measurements of R6G on demonstrate that the enhancement factor (EF) of the MNGA SERS substrate is increased by more than two fold compared with the control sample.

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.

Analysis of starch dissolved in ionic liquid by glass nanopore at single molecular level.

In this paper, the glass nanopore technology was proposed to detect a single molecule of starch dissolved in ionic liquid [1-butyl-3-methylimidazolium chloride (BmimCl)]. Firstly, the influence of BmimCl on nanopore detection is discussed. It is found that a certain amount of strong polar ionic liquids will disturb the charge distribution in nanopores and increase the detection noise. Then, by analysis of the characteristic current signal of the conical nanopore, the motion behaviour of starch near the entrance of the nanopore was studied and analysis the dominant ion of starch in the BmimCl dissolution process. Finally, based on nuclear magnetic resonance (NMR) and Fourier transform infrared (FTIR) spectroscopy simply discussed the mechanism of amylose and amylopectin dissolved in BmimCl. These results confirm that branched chain structure would affect the dissolution of polysaccharides in ionic liquids and the contribution of anions to the dissolution of polysaccharides are dominant. It is further proved that the current signal can be used to judge the charge and structure information of the analyte, and the dissolution mechanism can be assist analyzed at the single molecule level.

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.

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.

An aptamer-assisted nanopore strategy with a salt gradient for direct protein sensing.

Nanopore sensing is at the forefront of the technological revolution of the protein research field and has been widely used in molecular diagnosis and molecular dynamics, as well as for various sequencing applications. However, direct protein sensing with biological nanopores is still challenging owing to the large molecular size. Here, we propose an aptamer-assisted nanopore strategy for direct protein sensing and demonstrate its proof-of-concept utilities by experiments with SARS-Cov-2 nucleocapsid protein (NP), the most abundantly expressed viral protein, that is widely used in clinical diagnosis for COVID-19. NP binds with an oligonucleotide-tailed aptamer to form a protein-DNA complex which induces a discriminative two-level pattern of current blockades. We reveal the potential molecular interaction mechanism for the characteristic blockades and identify the salt gradient condition as the dominant factor of the phenomenon. Furthermore, we achieve a high sensitivity of 10 pM for NP detection within one hour and make a preliminary exploration on clinical diagnosis. This work promises a new platform for rapid and label-free protein detection.

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.

Single-Molecule Identification of the Conformations of Human C-Reactive Protein and Its Aptamer Complex with Solid-State Nanopores.

Human C-reactive protein (CRP) is an established inflammatory biomarker and was proved to be potentially relevant to disease pathology and cancer progression. A large body of methodologies have been reported for CRP analysis, including electrochemical/optical biosensors, aptamer, or antibody-based detection. Although the detection limit is rather low until pg/uL, most of which are time-consuming and relatively expensive, and few of them provided CRP single-molecule information. This work demonstrated the nanopore-based approach for the characterization of CRP conformation under versatile conditions. With an optimized pore of 14 nm in diameter, we achieved the detection limit as low as 0.3 ng/µL, voltage polarity significantly influences the electro-osmotic force and CRP translocation behavior, and the pentameric conformation of CRP may dissociate into pro-inflammatory CRP isoforms and monomeric CRP at bias potential above 300 mV. CRP tends to translocate through nanopores faster along with the increase in pH values, due to more surface charge on both CRP and pore inner wall and stronger electro-osmotic force. The CRP could specifically bind with its aptamer of different concentrations to form complexes, and the complexes exhibited distinguishable nanopore translocation behavior compared with CRP alone. The variation of the molar ratio of aptamer significantly influences the orientation of CRP translocation. The plasma test under physiological conditions displayed the ability of the nanopore system on the CRP identification with a concentration of 3 ng/µL.

Visualizing of AuNPs protection aptamer from DNase I enzyme digestion based on Nanopipette and its use for Microcystin-LR detection.

A simple and effective fluorescence platform has been established for visualizing nanomaterials' protective effect of DNA from cellular enzyme digestion based on nanopipette. In a proof-of-concept trial, gold nanoparticles (AuNPs) protect aptamer was designed, and it used for Microcystin-LR (MC-LR) sensitive detection. In the absence of MC-LR, FAM-labeled aptamers were combined on AuNPs, resulting in weak fluorescence emission. In the presence of MC-LR, aptamer bound with MC-LR. The formed complex leaves the surface of AuNPs. With the addition of the deoxyribonuclease I (DNase I) enzyme, the aptamer was selectively cleavaged, and MC-LR was released as an additional target molecule to achieve signal amplification and obtain strong fluorescence intensity. At the optimized conditions, a wide linear range (0.25 nM-20 nM) of fluorescence response for MC-LR was obtained. Further, by electrochemically manipulation MC-LR and DNase I inside confining nanopipette, which is filled with aptamer/AuNPs. The fluorescence intensity change with the aptamer and AuNPs interaction, these results directly visualize the process of DNA cleavage, and the interaction with AuNPs can effectively prevent the cleavage at the nanoscale confinement. This convenient nanoscale device provides new kinetic information about the dynamic chemical processes at a single-molecule level.

Treatment of knee osteoarthritis with acupuncture combined with Chinese herbal medicine: a systematic review and meta-analysis.

BACKGROUND: Many studies have demonstrated that acupuncture combined with Chinese herbal medicine (CHM) effectively treats knee osteoarthritis (KOA), with few side effects. However, few systematic reviews have offered evidence-based support. Here we conducted a meta-analysis on the combination of acupuncture with CHM in treating KOA. METHODS: Databases including CNKI, Wanfang, VIP, PubMed, EMBASE, and Cochrane library were systematically searched for articles on the treatment of KOA by acupuncture combined with CHM from the establishment of the database to May 2021. Three researchers independently searched, screened, extracted, and included articles that met the inclusion standards. The primary outcome measure was overall response rate (ORR), and the secondary outcome measures included Visual Analogue Scale (VAS) score, Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score, and Lysholm score. ORR was a binary variable, while other indicators were continuous variables. The quality of literature was assessed with a modified Jadad scale. The RevMan 5.3 software provided by the Cochrane Collaboration was used for statistical analysis. RESULTS: Thirty-three randomized controlled trials involving 3,954 patients were included. Meta-analysis showed that ORR [odds ratio (OR) =5.41; 95% confidence interval (CI): (4.38, 6.68); P<0.00001], VAS score [mean difference (MD) =-1.86; 95% CI: (-2.44, -1.29); P<0.00001], WOMAC score [MD =-13.05; 95% CI: (-21.70, -4.41); P=0.003], and Lysholm score [MD =10.47; 95% CI: (5.21, 15.72); P<0.0001] in the combination group were significantly superior to those in the control group. DISCUSSION: Compared with acupuncture alone or CHM/Western drug alone, acupuncture combined with CHM can effectively alleviate knee pain, improve knee function, and increase the quality of life. Thus, this combination can be used as a conservative treatment for KOA. However, due to the small number of high-quality articles and possible biases in our analysis, our conclusions need to be further verified in more and higher-quality studies.

A novel dielectric breakdown apparatus for solid-state nanopore fabrication with transient high electric field.

The dielectric breakdown used to fabricate solid-state nanopores has separated the device from capital-intensive industries and has been widely adopted by various research teams, but there are still problems with low production efficiency and uncertain location. In this work, based on the transient breakdown phenomenon of nanofilms, a new type of dielectric breakdown apparatus for nanopore fabrication is reported. It integrates both nano-manipulation technology and dielectric breakdown nanopore fabrication technology. The nanometer distance detection method and circuit are introduced in detail. The generation principle and procedures of the transient high electric field are explained step by step. The characterization of the nanopores shows that this apparatus can fabricate sub-2 nm nanopores at a pre-located position. Besides, the nanopore diameter can be easily adjusted by setting the transient high electric field value.