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 MoS 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.
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.
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 × 10 8 /cm 2 . 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.
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 SiN 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.
p53 is an anti-tumor 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, ultra-low noise solidstate 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 labelling is either not possible or at least not a viable option.
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