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.