DNA translocation through a nanopore in an ultrathin self-assembled peptide membrane.

Here, we explore the possibility of using peptide-based materials as a membrane in solid-state nanopore devices in an effort to develop a sequence-specific, programmable biological membrane platform. We use a recently developed tyrosine-mediated self-assembly peptide sheet. At the air/water interface, the 5mer peptide YFCFY self-assembles into a uniform and robust two-dimensional (2D) structure, and the peptide sheet is easily transferred to a low-noise glass substrate. The thickness of the peptide membrane can be adjusted to approximately 5 nm (or even to 2 nm) by an etching process, and the diameters of the peptide nanopores can be precisely controlled using a focused electron beam with an attuned spot size. The ionic current noise of the peptide nanopore is comparable to those of typical silicon nitride nanopores or multilayer 2D materials. Using this membrane, we successfully observe translocation of 1000 bp double-stranded DNA with a sufficient signal-to-noise ratio of ∼30 and an elongated translocation speed of ∼1 bp µs-1. Our results suggest that the self-assembled peptide film can be used as a sensitive nanopore membrane and employed as a platform for applying biological functionalities to solid-state substrates.

Differentiation of selectively labeled peptides using solid-state nanopores.

Determination of the amino acid sequence of a protein is critical for understanding various biological processes. Mass spectrometry has mainly been used for protein identification; however, there are limitations to its sensitivity when detecting low abundance proteins. In this study, we attempted to distinguish between three similar peptide sequences (∼40 amino acids, ∼5 kDa) that differed only by the location or number of cysteine residues with solid-state nanopores. The cysteine residues are located at one end, one at the center, and at both ends for each of the three peptides. We found that differentiation of the three types of peptides by nanopore signals was difficult. However, when the cysteine residue was labeled with a negatively charged molecule, Flamma® 496, the labeled peptides showed distinct signals for each peptide. Comparing the relative current blockades of labeled peptides with applied voltages, we found that the label was able to change peptide conformations and the resulting ionic current signals from the three labeled peptides were distinguished based on the relative current blockade, full width at half-maximum of the current blockade distribution, and single-molecule level peak shape analysis. Our results suggest that solid-state nanopores combined with a targeted labeling strategy could be used to obtain characteristic peptide signatures that could ultimately be used for protein identification.

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.

Reduction of DNA Folding by Ionic Liquids and Its Effects on the Analysis of DNA-Protein Interaction Using Solid-State Nanopore.

DNA folding is not desirable for solid-state nanopore techniques when analyzing the interaction of a biomolecule with its specific binding sites on DNA since the signal derived from the binding site could be buried by a large signal from the folding of DNA nearby. To resolve the problems associated with DNA folding, ionic liquids (ILs), which are known to interact with DNA through charge-charge and hydrophobic interactions are employed. 1-n-butyl-3-methylimidazolium chloride (C4 mim) is found to be the most effective in lowering the incident of DNA folding during its translocation through solid-state nanopores (4-5 nm diameter). The rate of folding signals from the translocation of DNA-C4 mim is decreased by half in comparison to that from the control bare DNA. The conformational changes of DNA upon complexation with C4 mim are further examined using atomic force microscopy, showing that the entanglement of DNA which is common in bare DNA is not observed when treated with C4 mim. The stretching effect of C4 mim on DNA strands improves the detection accuracy of nanopore for identifying the location of zinc finger protein bound to its specific binding site in DNA by lowering the incident of DNA folding.

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.

Identifying the Location of a Single Protein along the DNA Strand Using Solid-State Nanopores.

Solid-state nanopore has been widely studied as an effective tool to detect and analyze small biomolecules, such as DNA, RNA, and proteins, at a single molecule level. In this study, we demonstrate a rapid identification of the location of zinc finger protein (ZFP), which is bound to a specific locus along the length of a double-stranded DNA (dsDNA) to a single protein resolution using a low noise solid-state nanopore. When ZFP labeled DNAs were driven through a nanopore by an externally applied electric field, characteristic ionic current signals arising from the passage of the DNA/ZFP complex and bare DNA were detected, which enabled us to identify the locations of ZFP binding site. We examined two DNAs with ZFP binding sites at different positions and found that the location of the additional current drop derived from the DNA/ZFP complex is well-matched with a theoretical one along the length of the DNA molecule. These results suggest that the protein binding site on DNA can be mapped or that genetic information can be read at a single molecule level using solid-state nanopores.

Synchronized optical and electronic detection of biomolecules using a low noise nanopore platform.

In the past two decades there has been a tremendous amount of research into the use of nanopores as single molecule sensors, which has been inspired by the Coulter counter and molecular transport across biological pores. Recently, the desire to increase structural resolution and analytical throughput has led to the integration of additional detection methods such as fluorescence spectroscopy. For structural information to be probed electronically high bandwidth measurements are crucial due to the high translocation velocity of molecules. The most commonly used solid-state nanopore sensors consist of a silicon nitride membrane and bulk silicon substrate. Unfortunately, the photoinduced noise associated with illumination of these platforms limits their applicability to high-bandwidth, high-laser-power synchronized optical and electronic measurements. Here we present a unique low-noise nanopore platform, composed of a predominately Pyrex substrate and silicon nitride membrane, for synchronized optical and electronic detection of biomolecules. Proof of principle experiments are conducted showing that the Pyrex substrates have substantially lowers ionic current noise arising from both laser illumination and platform capacitance. Furthermore, using confocal microscopy and a partially metallic pore we demonstrate high signal-to-noise synchronized optical and electronic detection of dsDNA.