Directly Characterizing the Capture Radius of Tethered Double-Stranded DNA by Single-Molecule Nanopipette Manipulation.

The tethered molecule exhibits characteristics of both free and fixed states, with the electrodynamics involved in its diffusion, electrophoresis, and stretching processes still not fully understood. We developed a Single-Molecule Manipulation, Identification, and Length Examination (SMILE) system by integrating piezoelectric devices with nanopipettes. This system enabled successful capture and stretching of tethered double-stranded DNA within the nanopore. Our research unveiled distinct capture (rcapture) and stretch radii (rstretch) surrounding the DNA's anchor point. Notably, consistent ratios of capture radius for DNA of varying lengths (2k, 4k, and 6k base pairs) were observed across different capturing voltages, approximately 1:1.4:1.83, showing a resemblance to their gyration radius ratios. However, the ratios of stretch radius are consistent to their contour length (L0), with the stretching ratio (rstretch/L0) increasing from 70 to 90% as the voltage rose from 100 to 1000 mV. Additionally, through numerical simulations, we identified the origin of capture and stretch radii, determined by the entropic elasticity-induced capture barrier and the electric field-dominant escape barrier. This research introduces an innovative methodology and outlines research perspectives for a comprehensive exploration of the conformational, electrical, and diffusion characteristics of tethered molecules.

Research Progress on Saccharide Molecule Detection Based on Nanopores.

Saccharides, being one of the fundamental molecules of life, play essential roles in the physiological and pathological functions of cells. However, their intricate structures pose challenges for detection. Nanopore technology, with its high sensitivity and capability for single-molecule-level analysis, has revolutionized the identification and structural analysis of saccharide molecules. This review focuses on recent advancements in nanopore technology for carbohydrate detection, presenting an array of methods that leverage the molecular complexity of saccharides. Biological nanopore techniques utilize specific protein binding or pore modifications to trigger typical resistive pulses, enabling the high-sensitivity detection of monosaccharides and oligosaccharides. In solid-state nanopore sensing, boronic acid modification and pH gating mechanisms are employed for the specific recognition and quantitative analysis of polysaccharides. The integration of artificial intelligence algorithms can further enhance the accuracy and reliability of analyses. Serving as a crucial tool in carbohydrate detection, we foresee significant potential in the application of nanopore technology for the detection of carbohydrate molecules in disease diagnosis, drug screening, and biosensing, fostering innovative progress in related research domains.

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.

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.

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.

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.

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.

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.

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.

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.

Label-free single-molecule identification of telomere G-quadruplexes with a solid-state nanopore sensor.

Telomere sequences can spontaneously form G-quadruplexes (G4) in the presence of some cations. In view of their relevance to genetic processes and potential as therapeutic-targets, hitherto, a wealth of conventional techniques have been reported for interrogation of G4 conformation diversity and corresponding folding kinetics, most of which are limited in precision and sensitivity. This work introduces a label-free solid-state nanopore (SSN) approach for the determination of inter-, intra- and tandem molecular G4 with distinct base permutation in various cation buffers with a tailored aperture and meanwhile captures the single-molecule G4 folding process. SSN translocation properties elucidated that both inter- and intramolecular G4 generated higher current blockage with longer duration than flexible homopolymer nucleotide, and intramolecular G4 are structurally more stable with higher event frequency and longer blockage time than intermolecular ones; base arrangement played weak role in translocation behaviors; the same sequences with one, two and three G4 skeletons displayed an increase in current blockage and a gradual extension in dwell time with the increase of molecule size recorded in the same nanopore. We observed the conformation change of single-molecule G4 which indicated the existence of folding/unfolding equilibration in nanopore, and real-time test suggested a gradual formation of G4 with time. Our results could provide a continued and improved understanding of the underlying relevance of structural stability and G4 folding process by utilizing SSN platform which exhibits strategic potential advances over the other methods with high spatial and temporal resolution.

Nanopore Fabrication via Transient High Electric Field Controlled Breakdown and Detection of Single RNA Molecules.

The fabrication of nanopores through a dielectric breakdown method, achieved by simple, low-cost desktop setups, has promoted the research of solid-state nanopore sensing. This paper reports a method for fabricating nanopores. This method uses transient high electric field controlled breakdown (THCBD) to form electric-field-dependent nanopores with different diameters in the order of milliseconds. By manipulating a micropipette with a high electric field to establish the meniscus contact with the SiNx membrane, nanopores can be formed through an "auto-brake" fabrication process. Compared with the traditional dielectric breakdown, THCBD can greatly shorten the breakdown time and form pores of different sizes under higher electric fields without causing additional damage to the SiNx membrane. The nanopores formed by this method can be successfully used to detect two types of RNA molecules. One is transfer RNA from yeast extract and the other is a synthetic RNA oligonucleotide fragment (rArArArArArArArArArArArA).

Facile and Controllable Synthesis of Large-Area Monolayer WS2 Flakes Based on WO3 Precursor Drop-Casted Substrates by Chemical Vapor Deposition.

Monolayer WS2 (Tungsten Disulfide) with a direct-energy gap and excellent photoluminescence quantum yield at room temperature shows potential applications in optoelectronics. However, controllable synthesis of large-area monolayer WS2 is still challenging because of the difficulty in controlling the interrelated growth parameters. Herein, we report a facile and controllable method for synthesis of large-area monolayer WS2 flakes by direct sulfurization of powdered WO3 (Tungsten Trioxide) drop-casted on SiO2/Si substrates in a one-end sealed quartz tube. The samples were thoroughly characterized by an optical microscope, atomic force microscope, transmission electron microscope, fluorescence microscope, photoluminescence spectrometer, and Raman spectrometer. The obtained results indicate that large triangular monolayer WS2 flakes with an edge length up to 250 to 370 µm and homogeneous crystallinity were readily synthesized within 5 min of growth. We demonstrate that the as-grown monolayer WS2 flakes show distinctly size-dependent fluorescence emission, which is mainly attributed to the heterogeneous release of intrinsic tensile strain after growth.

Label-free identification of trace microcystin-LR with surface-enhanced Raman scattering spectra.

The analysis of trace microcystin-LR (MC-LR) plays important roles in environmental fields, especially in monitoring domestic water quality and safety, since it has particularly harmful effect on wild and domestic animals as well as humans at low doses. Herein, we combine confocal Raman spectroscopy with SERS-AG substrate to characterize the "fingerprint" information of MC-LR directly. High sensitivity of SERS-AG substrates was verified by utilizing the probe molecule Rhodamine 6 G. Mapping spectra demonstrated good reproducibility of MC-LR identification with label-free surface-enhanced Raman scattering (SERS) strategy. Differences between SERS spectra of MC-LR and R6G, microcystin-RR were evaluated by calculating their scores and loading weights with an unsupervised exploratory principal component analysis method. Then, relationship between Raman intensities and concentrations was preliminary analyzed with SERS spectra of MC-LR and the lowest concentration of MC-LR identification was 10-6 mg L-1 while using SERS-AG substrate. Thereafter, 68.6% quantitative recovery of 10-3 mg L-1 MC-LR in tap water samples was obtained by the proposed label-free SERS method. These results showed that confocal Raman spectroscopy with label-free surface-enhanced Raman scattering strategy can handle the identification of trace MC-LR for monitoring water quality and safety worldwide in future.

Investigation of the adsorption behavior of BSA with tethered lipid layer-modified solid-state nanopores in a wide pH range.

Nanopore technology was introduced for the study of the dynamic interactions between bovine serum albumin (BSA) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) phospholipids based on a modified nanopore. The results reveal that the interaction mechanism between DOPE and BSA is affected by the pH of the subphase. Far above the BSA isoelectric point (pH > 7), a weaker hydrophobic interaction and stronger electrostatic repulsion exist between the DOPE and BSA molecules. At pH = 7, the BSA structure nearly does not change, and the interaction is weak. At pH 5 and pH 6, BSA is marginally affected by the adsorption interaction, and below pH 5, the DOPE film becomes disordered, so there is a strong repulsive force interaction between the BSA and DOPE.

Assessment of physiological responses and growth phases of different microalgae under environmental changes by Raman spectroscopy with chemometrics.

The assessment for cell physiology and growth phases of microalgae plays important roles in ecological and environmental fields since it can be used to forecast water eutrophication level worldwidely. Herein, growth phases and environmental conditions of microalgae were assessed by combining resonance Raman mapping spectroscopy with multivariate analysis methods. And, primary Raman characteristic peaks of microalgae were mined with two-dimensional synchronous spectra. Thereafter, algal growth phases and environmental conditions of microalgae were preliminary classified with different tendencies of characteristic Raman peaks by unsupervised principal component analysis (PCA) and support vector machine (SVM) methods. Our results demonstrated that resonance Raman mapping spectroscopy with PCA and SVM classification models can be used to assess algal growth phases and preliminary predict environmental conditions with characteristic Raman spectra of microalgae in water bodies.

Preliminary identification of unicellular algal genus by using combined confocal resonance Raman spectroscopy with PCA and DPLS analysis.

The analysis of algae and dominant alga plays important roles in ecological and environmental fields since it can be used to forecast water bloom and control its potential deleterious effects. Herein, we combine in vivo confocal resonance Raman spectroscopy with multivariate analysis methods to preliminary identify the three algal genera in water blooms at unicellular scale. Statistical analysis of characteristic Raman peaks demonstrates that certain shifts and different normalized intensities, resulting from composition of different carotenoids, exist in Raman spectra of three algal cells. Principal component analysis (PCA) scores and corresponding loading weights show some differences from Raman spectral characteristics which are caused by vibrations of carotenoids in unicellular algae. Then, discriminant partial least squares (DPLS) classification method is used to verify the effectiveness of algal identification with confocal resonance Raman spectroscopy. Our results show that confocal resonance Raman spectroscopy combined with PCA and DPLS could handle the preliminary identification of dominant alga for forecasting and controlling of water blooms.