An electrophoretic DNA extraction device using a nanofilter for molecular diagnosis of pathogens.

Rapid and efficient nucleic acid (NA) extraction and concentration are required for point-of-care analysis in order to prevent an epidemic/pandemic disease outbreak. Typical silica-based NA extraction methods have limitations such as being time-consuming, requiring human intervention, and resulting in a low recovery yield. In this study, we have developed a pathogenic DNA extraction device based on electrokinetic separation incorporated with a silicon nitride (SiNx) nanofilter, which expedites the DNA extraction procedure with advantages of being convenient, efficient, and inexpensive. This DNA extraction device consists of a computer numerical control (CNC) milled-Teflon gadget with a cis-chamber as a cell lysate reservoir and a trans-chamber as a elution solution reservoir, with the SiNx nanofilter being inserted between the two chambers. The SiNx nanofilter was fabricated using a photolithographic method in conjunction with nanoimprinting. Approximately 7.2 million nanopores of 220 nm diameter were located at the center of the nanofilter. When a DC electric field is applied through the nanopores, DNA is transferred from the cis-chamber to the trans-chamber to isolate the DNA from the cell debris. To demonstrate the DNA extraction performance, we measured the absorbances at 260 and 280 nm and performed a real-time polymerase chain reaction (real-time PCR) using the recovered DNA to verify its feasibility for downstream genetic analysis. Moreover, the DNA extraction device was successfully operated using a 1.5 V alkaline battery, which verifies the portability of the device for point-of-care testing. Such an advanced DNA extraction system can be utilized in various fields including clinical analysis, pathogen detection, forensic analysis, and on-site detection.

Direct electrophoretic microRNA preparation from clinical samples using nanofilter membrane.

A method to directly collect negatively charged nucleic acids, such as DNA and RNA, in the biosamples simply by applying an electric field in between the sample and collection buffer separated by the nanofilter membrane is proposed. The nanofilter membrane was made of low-stress silicon nitride with a thickness of 100 nm, and multiple pores were perforated in a highly arranged pattern using nanoimprint technology with a pore size of 200 nm and a pore density of 7.22 × 108/cm2. The electrophoretic transport of hsa-mir-93-5p across the membrane was confirmed in pure microRNA (miRNA) mimic solution using quantitative reverse transcription-polymerase chain reactions (qRT-PCR). Consistency of the collected miRNA quantity, stability of the system during the experiment, and yield and purity of the prepared sample were discussed in detail to validate the effectiveness of the electrical protocol. Finally, in order to check the applicability of this method to clinical samples, liquid biopsy process was demonstrated by evaluating the miRNA levels in sera of hepatocellular carcinoma patients and healthy controls. This efficient system proposed a simple, physical idea in preparation of nucleic acid from biosamples, and demonstrated its compatibility to biological downstream applications such as qRT-PCR as the conventional nucleic acid extraction protocols.

Surface modification of solid-state nanopore by plasma-polymerized chemical vapor deposition of poly(ethylene glycol) for stable device operation.

Biopolymer adsorption onto a membrane is a significant issue in the reliability of solid-state nanopore devices, since it degrades the device performance or promotes device failure. In this work, a poly(ethylene glycol) (PEG) layer was coated on a silicon nitride (SiNx) membrane by plasma-polymerized vapor deposition to inhibit biopolymer adsorption. From optical observations, the deposited PEG layer demonstrated increased hydrophilicity and anti-adsorption property compared to the SiNx surface. Electrical properties of the PEG/SiNx nanopore were characterized, showing Ohmic behavior and a 6.3 times higher flicker noise power due to the flexible conformation of PEG in water. Antifouling performance of each surface was analyzed by measuring the average time from voltage bias to the first adsorption during DNA translocation experiments, where the modified surface enabled two times prolonged device operation. The time to adsorption was dependent on the applied voltage, implying adsorption probability was dominated by the electrophoretic DNA approach to the nanopore. DNA translocation behaviors on each surface were identified from translocation signals, as the PEG layer promoted unfolded and fast movement of DNA through the nanopore. This work successfully analyzed the effect of the PEG layer on DNA adsorption and translocation in solid-state nanopore experiments.

Detection of metal corrosion characteristics in chlorine solution using solid state nanopore.

Nanopore structures were originally proposed for detection of biomolecule translocation through nanometer-scale pores that perforate membranes by transient changes in ionic current. In this study, these changes are utilized to detect corrosion of different metals in aqueous chlorine media. The corrosion behaviors of Cu, Al, Ti, and Cr were analyzed by monitoring the changes in ion current resulting from ion concentration variations in solutions due to corrosion of the metals. We observed that the Cu layer passivated by CuO x was severely corroded when a drastic change of ion current was induced, from 10 to 30 nS to the level of 104 nS, as soon as the bias voltage was applied. In the case of Al passivated by thin AlO x , the conductance increased from 10-30 to 166 ± 52 nS and became saturated. Highly localized pitting corrosion was observed on the periphery of the nanopore, where the electrical field was most concentrated. Finally, we observed that Ti and Cr passivated by oxide showed long-term stability without corrosion in 1 M KCl in the pH range of 4-11.

Translocation of DNA and protein through a sequentially polymerized polyurea nanopore.

Here, we investigated the translocation of biomolecules, such as DNA and protein, through a sequentially polymerized polyurea nanopore, with a thin (<10 nm) polymer membrane of uniform thickness. The polyurea membrane was synthesized by molecular layer deposition using p-phenylenediisocyanate (PDI) and p-phenylenediamine (PDA) as sequential precursors. The membrane exhibited a hydrophobic surface with a highly negative surface charge density (-51 mC m-2 at pH 8). It was particularly noted that the high surface charge density of the membrane resulted in a highly developed electro-osmotic flow which, in turn, strongly influenced the capture probability of biomolecules, depending on the balance between the electro-osmotic and electrophoretic forces. For instance, the capture frequency of negatively charged DNA was demonstrated to be quite low, since these two forces more or less cancelled each other, whereas that of positively charged MDM2 was much higher, since these two forces were additive. We also identified that the mean translocation time of MDM2 through the polyurea nanopore was 26.1 ± 3.7 µs while that of the SiN nanopore was 14.2 ± 2.0 µs, hence suggesting that the enhanced electrostatic interaction between positively charged MDM2 and the negatively charged pore surface affects the translocation speed.

The dynamics of electron beam scattering on metal membranes: nanopore formation in metal membranes using transmission electron microscopy.

The dynamics of nanopore formation in metal membranes using the highly focused and high energy electron beams (e-beams) of transmission electron microscopy instruments was investigated. Various metals such as Al, Ti, Cr, Cu, and Au were selected to investigate the effect of the atomic mass of the metal on nanopore drilling, namely, elastic versus inelastic scattering. We demonstrated that the effect of elastic scattering (pore formation by sputtering) decreased as the atomic mass of the metal increased. Furthermore, experimental cross-sections obtained from normalized drilled volume vs. electron dose curves (characteristic contrast curves) matched well the calculated atomic displacement cross-sections determined from elastic scattering data. The sputtering energies of Ti, Cr, and Cu were determined to be approximately 10, 9, and 7 eV, respectively, which were in good agreement with the reported range of sputtering energy values.

Recent Progress in Solid-State Nanopores.

The solid-state nanopore has attracted much attention as a next-generation DNA sequencing tool or a single-molecule biosensor platform with its high sensitivity of biomolecule detection. The platform has advantages of processability, robustness of the device, and flexibility in the nanopore dimensions as compared with the protein nanopore, but with the limitation of insufficient spatial and temporal resolution to be utilized in DNA sequencing. Here, the fundamental principles of the solid-state nanopore are summarized to illustrate the novelty of the device, and improvements in the performance of the platform in terms of device fabrication are explained. The efforts to reduce the electrical noise of solid-state nanopore devices, and thus to enhance the sensitivity of detection, are presented along with detailed descriptions of the noise properties of the solid-state nanopore. Applications of 2D materials including graphene, h-BN, and MoS2 as a nanopore membrane to enhance the spatial resolution of nanopore detection, and organic coatings on the nanopore membranes for the addition of chemical functionality to the nanopore are summarized. Finally, the recently reported applications of the solid-state nanopore are categorized and described according to the target biomolecules: DNA-bound proteins, modified DNA structures, proteins, and protein oligomers.

Highly reliable and low-noise solid-state nanopores with an atomic layer deposited ZnO membrane on a quartz substrate.

We present a fabrication scheme for a solid-state ZnO nanopore membrane directly deposited on top of a quartz substrate by atomic layer deposition (ALD) and investigate the characteristics of DNA translocation through the nanopores. We chose a ZnO membrane owing to its high isoelectric point (∼9.5) as well as its chemical and mechanical stability. Aside from the extremely low noise level exhibited by this device on a highly insulating and low dielectric quartz substrate, it also slows down the translocation speed of DNA by more than one order of magnitude as compared to that of a SiNx nanopore device. We propose that the electrostatic interaction between the positively charged ZnO nanopore wall, resulting from the high isoelectric point of ZnO, and the negatively charged phosphate backbone of DNA provides an additional frictional force that slows down the DNA translocation.

Enhancing the sensitivity of DNA detection by structurally modified solid-state nanopore.

Solid-state nanopore is an ionic current-based biosensing platform, which would be a top candidate for next-generation DNA sequencing and a high-throughput drug-screening tool at single-molecular-scale resolution. There have been several approaches to enhance the sensitivity and reliability of biomolecule detection using the nanopores particularly in two aspects: signal-to-noise ratio (SNR) and translocation dwell time. In this study, an additional nano-well of 100-150 nm diameter and the aspect ratio of ∼5 called 'guide structure' was inserted in conventional silicon-substrate nanopore device to increase both SNR and dwell time. First, the magnitude of signals (conductance drop (ΔG)) increased 2.5 times under applied voltage of 300 mV through the guide-inserted nanopore compared to the conventional SiN/Si nanopore in the same condition. Finite element simulation was conducted to figure out the origin of ΔG modification, which showed that the guide structure produced high ΔG due to the compartmental limitation of ion transports through the guide to the sensing nanopore. Second, the translocation velocity decreased in the guide-inserted structure to a maximum of 20% of the velocity in the conventional device at 300 mV. Electroosmotic drag formed inside the guide structure, when directly applied to the remaining segment of translocating DNA molecules in cis chamber, affected the DNA translocation velocity. This study is the first experimental report on the effect of the geometrical confinement to a remnant DNA on both SNR and dwell time of nanopore translocations.

Nanoparticle mechanics: deformation detection via nanopore resistive pulse sensing.

Solid-state nanopores have been widely used in the past for single-particle analysis of nanoparticles, liposomes, exosomes and viruses. The shape of soft particles, particularly liposomes with a bilayer membrane, can greatly differ inside the nanopore compared to bulk solution as the electric field inside the nanopores can cause liposome electrodeformation. Such deformations can compromise size measurement and characterization of particles, but are often neglected in nanopore resistive pulse sensing. In this paper, we investigated the deformation of various liposomes inside nanopores. We observed a significant difference in resistive pulse characteristics between soft liposomes and rigid polystyrene nanoparticles especially at higher applied voltages. We used theoretical simulations to demonstrate that the difference can be explained by shape deformation of liposomes as they translocate through the nanopores. Comparing our results with the findings from electrodeformation experiments, we demonstrated that the rigidity of liposomes can be qualitatively compared using resistive pulse characteristics. This application of nanopores can provide new opportunities to study the mechanics at the nanoscale, to investigate properties of great value in fundamental biophysics and cellular mechanobiology, such as virus deformability and fusogenicity, and in applied sciences for designing novel drug/gene delivery systems.

Single-Molecule Studies of Unlabeled Full-Length p53 Protein Binding to DNA.

p53 is an antitumor protein that plays an important role in apoptosis, preserving genomic stability and preventing angiogenesis, and it has been implicated in a large number of human cancers. For this reason it is an interesting target for both fundamental studies, such as the mechanism of interaction with DNA, and applications in biosensing. Here, we report a comprehensive study of label-free, full length p53 (flp53) and its interaction with engineered double-stranded DNA in vitro, at the single-molecule level, using atomic force microscopy (AFM) imaging and solid-state nanopore sensing. AFM data show that dimeric and tetrameric p53 bind to the DNA in a sequence-specific manner, confirming previously reported relative binding affinities. The statistical significance is tested using both the Grubbs test and stochastic simulations. For the first time, ultralow noise solid-state nanopore sensors are employed for the successful differentiation between bare DNA and p53/DNA complexes. Furthermore, translocation statistics reflect the binding affinities of different DNA sequences, in accordance with AFM data. Our results thus highlight the potential of solid-state nanopore sensors for single-molecule biosensing, especially when labeling is either not possible or at least not a viable option.