Solid-state Nanopore Devices Research Articles

Toward protein fingerprinting with a solid-state nanopore.

Solid-state nanopores have shown great potential for single-molecule protein identification in recent years. As a first step toward single-molecule protein fingerprinting using a solid-state nanopore device, thorough characterization of the passage of proteins under the different conformational states is required. For this purpose, we use green fluorescent protein (GFP), antibiotin immunoglobulin G (IgG), and cytochrome c (cyt c) as model proteins, and show results from field-driven translocations through nanopores with diameters ranging from 2 to 10 nm in SiN membranes of thicknesses from 10 to 20 nm. As expected, the distinguishable current blockades observed during individual translocations allow the identification of different protein conformations adopted throughout its passage, ranging from partially unfolded to completely unfolded conformations. Here we show how different temperatures and voltages facilitate protein unfolding and alter their folding kinetics. This is achieved by varying voltages from 0.1 V to 1 V and by varying the temperature from room temperature to 60 oC while maintaining low-noise ionic current recording at 1 MHz bandwidth using a custom instrument with thermal control offering ±0.2 oC precision and ±1 oC repeatability. These results will serve to better understand the accessible states in the protein energy landscape and to benchmark future studies furthering protein fingerprinting.

Characterization of Avidin and Case9 single protein molecules by a solid-state nanopore device.

Solid-state nanopores made of silicon nitride have emerged as novel platforms for biosensing such as real-time single-molecule characterization of protein molecules. Solid-state nanopore detection is based on measuring the variations in ionic conductance as charged biomolecules translocate through nanometer-sized channels driven by an external voltage applied across the membrane. In this study, we characterize Avidin (MW: ∼67 kDa) and Case9 (MW: ∼160 kDa) proteins with nanopore current signal. We study how voltage and glycerol, impact on the time durations and current blockage amplitudes produced by the proteins passing through a nanopore.

DNA Origami in the Quest for Membrane Piercing

The tool kit for label-free single-molecule sensing, nucleic acid sequencing (DNA, RNA and protein) and other biotechnological applications has been significantly broadened due to the wide range of available nanopore-based technologies. Currently, various sources of nanopores, including biological, fabricated solid-state, hybrid and recently de novo chemically synthesized ion-like channels have put in use for rapid single-molecule sensing of biomolecules and other diagnostic applications. At length scales of hundreds of nanometers, DNA nanotechnology, particularly DNA origami-based devices, enables the assembly of complex and dynamic 3-dimensional nanostructures, including nanopores with precise control over the size/shape. DNA origami technology has enabled to construct nanopores by DNA alone or hybrid architects with solid-state nanopore devices or nanocapillaries. In this review, we briefly discuss the nanopore technique that uses DNA nanotechnology to construct such individual pores in lipid-based systems or coupled with other solid-state devices, nanocapillaries for enhanced biosensing function. We summarize various DNA-based design nanopores and explore the sensing properties of such DNA channels. Apart from DNA origami channels we also pointed the design principles of RNA nanopores for peptide sensing applications.

Electronic Conductance and Current Modulation through Graphdiyne Nanopores for DNA Sequencing

Solid-state nanopore devices have emerged as one of the most promising technologies that may decode the sequence of DNA nucleobases by reading electronic conductance and electric current signals. Herein we study the potential of atomically thick graphdiyne (GDY) to act as an all-electronic nanopore (NP)-based DNA sequencing device. The first-principles density functional theory method is used to study the structural properties, interaction energy (Ei) values, translocation time (τ), charge transfer [Q(e)] values, charge density difference [Δρ(r)], molecular orbital (MO) positions, and electronic density of states (DOS) properties of the GDYNP and GDYNP + nucleobase (nucleobases: adenine (A), guanine (G), thymine (T), and cytosine (C)) systems. Our results suggest that the GDYNP system could be a suitable device for the detection of nucleobases due to their specific translocation times inside the GDYNP device. The Δρ(r) results reveal that the charge fluctuates around the GDYNP edges close to the target nucleobase. This charge reorganization causes the modulation of charge transport in the GDYNP device. Furthermore, the GDYNP device is used to read-out the conductance and current signals of each DNA nucleobase while located inside the GDYNP by using nonequilibrium Green’s functions formalism. Our findings show a change in conductance with respect to the reference bare “GDYNP” device for energy values below/above the Fermi energy. The conductance as well as current sensitivity (%) values (C > G > T > A) indicates that the nucleobases could possibly be sequenced through the GDYNP device. Further, each nucleobase transmits unique current signals when transported through the GDYNP device. A distinction between purine- and pyrimidine-type nucleobases seems possible due to their different current signals. Thus, our study highlights the potentials that would motivate research toward the development of the GDYNP device, which may be even a better DNA nucleobase detector compared to graphene-based devices.

Slowing DNA translocation through a solid-state nanopore by applying hydrophobic microchannel-guided walls

Here, the authors present a novel solid-state nanopore device, fabricated by a transfer of a nanopore device to a dielectric polydimethylsiloxane (PDMS) microchannel-guided structure, and investigate the characteristics of DNA translocation through the nanopore. Apart from the extremely low noise level exhibited by this device on a highly insulating and low dielectric PDMS microchannel, this device also shows a slow translocation speed of 48 kbp Lambda DNA (λ-DNA), with a reduction of more than one order of magnitude than that in previously reported results on SiNx nanopores. The authors propose that the electrostatic interaction between the PDMS microchannel and DNA, resulting from the hydrophobicity of both PDMS and DNA, provides an additional attraction force that slows down DNA translocation. Such advanced solid-state nanopore devices can be utilized in various fields, including DNA or protein sequencing, drug screening, and microRNA detection.

Portable solid-state nanopore device is used for sensing molecules in Antarctic samples

A solid-state nanopore device, perfect for sensing biomolecules, holds potential for use on space missions and in extreme environments.

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.

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.

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.

A Novel Diagnostic System for Infectious Diseases Using Solid-State Nanopore Devices.

Nanopore-based diagnostic systems are a promising tool for counting viruses in a specimen one by one. However, despite intensive R&D efforts, it remains difficult to recognize virus subtypes by nanopore devices. We thus propose a novel diagnostic system that combines a specialized virus recognition procedure with a nanopore detection procedure. This recognition procedure consists of three steps: 1) capture target viruses using specific probes for recognition; 2) release captured targets; and 3) detect released targets by nanopore. Proof-of-concept tests are conducted using avidin-modified fluorescent particles (as a model for viruses) and biotin-modified alkane thiol (as a model for probes). The avidin-modified particles are confirmed to be captured on electrode by biotin-modified probes and then, the particles are electrochemically released from the electrode. Consequently, the released particles are successfully detected by nanopore devices. Furthermore, the concept is also proved by using human influenza viruses (H1N1, A/PR/8/34) and sugar chain (6'-sialyllactose)-modified probes. This suggests that our concept is applicable to various infectious diseases by changing probes (ligands).

Photo-Induced Ionic Noise in Si and Quartz Based Solid-State Nanopore Device

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 translocate on 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 investigate the influence of light illumination in the solid-state nanopore device fabricated either on Si substrate and quartz substrate. As has been well known, the noise signal in the solid state nanopore structure has been one of the major concerns. And the strong increase of ionic noise upon laser light illumination has been investigated before. Without light illumination, the noise signal which is characterized either by RMS value or more importantly by the power spectrum (pA2/Hz versus frequency Hz) typically shows about 40 pA in a Si substrate and 5.3 pA in a quartz substrate in RMS values and shows much improved noise characteristics on quartz substrate both in low frequency and high frequency. In general, it has been known that there are four different types of noise sources of ionic current obtained from the patch clamp system which shows different frequency dependency; 1. Voltage noise including RC noise showing f2 relationship, 2. dielectric noise which is proportional to f, 3. Johnson noise (thermal noise or Nyquist noise) and shot noise which are independent to f, and 4. Flicker noise related to a combination of noise showing 1/fa characteristics. However, upon irradiation with laser light in Si device, a completely different PSD was observed. PSD has the corner frequency, which is located at few hundreds Hz. This frequency dependence of this noise source is inconsistent with that of surface charge protonation noise or temperature-dependent thermal and dielectric noise. And, this peaks absence within the quartz-SiNx platforms power spectrum suggests the source of noise is related to the Si substrate. The optical transparency of the SiNx membrane and photon energy (2.54 eV) is sufficient for electron-hole pair generation in the Si substrate (band gap ∼1.1 eV), reported to promote photoreduction of H+ at p-type Si interfaces. Therefore, the increase in noise is via electrochemical reaction at the silicon/electrolyte surface by the excitation electron-hole pairs. In this sense, quartz device has only insignificant increase with laser illumination. Increase in thermal and dielectric noise term by localized heating can explain the small noise increase. This observation may also be quite important since there are many researchers who would like to utilize spectroscopic way of sequencing the DNA in a nanopore system.

Slowing the translocation of single-stranded DNA by using nano-cylindrical passage self-assembled by amphiphilic block copolymers

We report a novel approach to slow the translocation of single-stranded DNA (ssDNA) by employing polyethylene oxide (PEO) filled nano-cylindrical domains as transportation channels. DNA strands were demonstrated to electrophoretically translocate through PEO filled cylindrical domains with diameters of 2 and 9 nm, which were self-assembled by amphiphilic liquid crystalline block copolymers. The average translocation rate of ssDNA strands was effectively reduced to an order of 10 μs per nucleotide, which was 1-2 orders slower than that attained by utilizing conventional solid-state nanopore devices.

Novel PMMA Polymer-Based Nanopores Capable of Detection and Discrimination Between Structurally Different Biomolecules

We report, for the first time, the use of single poly (methyl-methacrylate) (PMMA) polymer-based nanopores to not only detect the translocating biomolecules of dsDNA and BSA in individual populations, but also distinguish between them in a mixed population, based on the obtained depths ( i Block) and durations (t D ) of the ionic current blockade events. Quantitative analyzes of current blockade events induced by separate populations of dsDNA and BSA molecules translocating through single PMMA-based nanopores revealed a mean i Block value of 22.4 ± 0.69 pA with a t D of 0.202 ± 0.009 ms, and a mean i Block value of 60.14 ± 0.19 pA with a t D of 0.59 ± 0.08 ms for dsDNA and BSA molecules, respectively. Evaluation of translocation events by dsDNA and BSA molecules in a mixed population through single nanopores indicated two distinct groups of current blockade events, extrapolation of which could perfectly correlate the group demonstrating i Block values of 13-38 pA to the dsDNA molecules, whereas the group demonstrating i Block values of 52-73 pA to the BSA protein molecules transporting through the nanopores. Analysis of the data suggests that the majority of dsDNA or BSA protein molecules translocating through the nanopores are in their partially folded conformation. This paper is an important milestone in the development of polymer-based solid-state nanopore devices for fast and accurate biomolecule detection, differentiation/discrimination, and future structural characterization.

Integrated solid-state nanopore devices for third generation DNA sequencing

Third generation DNA sequencing relies on monitoring the ionic current blockage during the DNA molecule’s threading through a nanoscale pore. It is still really tough to attain the single base discrimination on a DNA strand by merely analyzing the ionic current due to speedy DNA translocation and low spatial resolution. More integrated configurations are pursued to present versatile comparative dissimilarities of the four bases by enhancing the spatial resolution within a DNA molecule translocation event, such as transverse tunneling current, local potential change, and capacitance oriented voltage resonance. In this mini review, the insight is provided into the status quo on several functionalized techniques and methodologies for DNA sequencing and furthermore concluding remark and outlook are presented. DNA sequencing, solid-state nanopore, spatial resolution, integrated sensors

Characterization of Protein Unfolding with Solid-state Nanopores

In this work, we review the process of protein unfolding characterized by a solid-state nanopore based device. The occupied or excluded volume of a protein molecule in a nanopore depends on the protein's conformation or shape. A folded protein has a larger excluded volume in a nanopore thus it blocks more ionic current flow than its unfolded form and produces a greater current blockage amplitude. The time duration a protein stays in a pore also depends on the protein's folding state. We use Bovine Serum Albumin (BSA) as a model protein to discuss this current blockage amplitude and the time duration associated with the protein unfolding process. BSA molecules were measured in folded, partially unfolded, and completely unfolded conformations in solid-state nanopores. We discuss experimental results, data analysis, and theoretical considerations of BSA protein unfolding measured with silicon nitride nanopores. We show this nanopore method is capable of characterizing a protein's unfolding process at single molecule level. Problems and future studies in characterization of protein unfolding using a solid-state nanopore device will also be discussed.

Gold nanorod translocation through a solid-state nanopore

Recently, nanopores have been used in an essential technique for detecting single molecule with high sensitivity. The initial application of nanopores to DNA and RNA sequencing has been expanded to sensing proteins and nanoparticles, including Bovine serum albumin, silica nanoparticles, polystyrene beads, and others. In our study, for the first time, a positively charged gold nanorod was investigated using a solid-state nanopore device. Various gold nanorods passed through the nanopore with different current blockages and duration times, providing a measurement of the nanorod diameter, length, and charge. Our findings indicate that nanopore sensing might be a new method for characterizing the size, shape, and charge of nanoparticles.

Synchronous Optical and Electrical Measurements of Single DNA Molecules Translocating Through a Solid State Nanopore

Nucleic acids and proteins are increasingly being analyzed using solid-state nanopores. The nanopore method relies on the measurement of transient conductance changes of the nanopores as charged biomolecules are being electrophoretically driven into it. Although electrical detection is highly appealing for the label-free analysis of biological samples, our understanding of the dynamics of charged biopolymers through such nanoconfined geometries is limited by the information captured by the electrical signal alone.We report the development of an instrument capable of interrogating solid-state nanopore devices by simultaneous electrical and optical measurements. Instead of the conventional TEM-drilled nanopores, we used a method pioneered by our group, and fabricate nanopores in a thin SiNx membrane via dielectric breakdown in solution . In this presentation, we show how a range of odd behaviors of the ionic current can be explained with parallel fluorescence imaging of the nanopore. Our custom instrument allows us to: (i) study these anomalous events; (ii) probe the dynamics of DNA capture; (iii) monitor, for the first time, the creation process of a nanopore; and (iv) determine its location on the dielectric membrane;.A better understanding of these anomalous single-molecule events during DNA passage through solid-state nanopores is essential to increase reliability of nanopore-based sensing.

Graphene quantum point contact transistor for DNA sensing

By using the nonequilibrium Green's function technique, we show that the shape of the edge, the carrier concentration, and the position and size of a nanopore in graphene nanoribbons can strongly affect its electronic conductance as well as its sensitivity to external charges. This technique, combined with a self-consistent Poisson-Boltzmann formalism to account for ion charge screening in solution, is able to detect the rotational and positional conformation of a DNA strand inside the nanopore. In particular, we show that a graphene membrane with quantum point contact geometry exhibits greater electrical sensitivity than a uniform armchair geometry provided that the carrier concentration is tuned to enhance charge detection. We propose a membrane design that contains an electrical gate in a configuration similar to a field-effect transistor for a graphene-based DNA sensing device.

A Solid State Nanopore Device for Investigating the Magnetic Properties of Magnetic Nanoparticles

In this study, we explored magnetic nanoparticles translocating through a nanopore in the presence of an inhomogeneous magnetic field. By detecting the ionic current blockade signals with a silicon nitride nanopore, we found that the translocation velocity that is driven by magnetic and hydrodynamic forces on a single magnetic nanoparticle can be accurately determined and is linearly proportional to the magnetization of the magnetic nanoparticle. Thus, we obtained the magneto-susceptibility of an individual nanoparticle and the average susceptibility over one hundred particles within a few minutes.

Integration of solid-state nanopores in a 0.5 μm CMOS foundry process

High-bandwidth and low-noise nanopore sensor and detection electronics are crucial in achieving single-DNA-base resolution. A potential way to accomplish this goal is to integrate solid-state nanopores within a CMOS platform, in close proximity to the biasing electrodes and custom-designed amplifier electronics. Here we report the integration of solid-state nanopore devices in a commercial complementary metal–oxide–semiconductor (CMOS) potentiostat chip implemented in On-Semiconductor’s 0.5 μm technology. Nanopore membranes incorporating electrodes are fabricated by post-CMOS micromachining utilizing the n+ polysilicon/SiO2/n+ polysilicon capacitor structure available in the aforementioned process. Nanopores are created in the CMOS process by drilling in a transmission electron microscope and shrinking by atomic layer deposition. We also describe a batch fabrication method to process a large of number of electrode-embedded nanopores with sub-10 nm diameter across CMOS-compatible wafers by electron beam lithography and atomic layer deposition. The CMOS-compatibility of our fabrication process is verified by testing the electrical functionality of on-chip circuitry. We observe high current leakage with the CMOS nanopore devices due to the ionic diffusion through the SiO2 membrane. To prevent this leakage, we coat the membrane with Al2O3, which acts as an efficient diffusion barrier against alkali ions. The resulting nanopore devices also exhibit higher robustness and lower 1/f noise as compared to SiO2 and SiNx. Furthermore, we propose a theoretical model for our low-capacitance CMOS nanopore devices, showing good agreement with the experimental value. In addition, experiments and theoretical models of translocation studies are presented using 48.5 kbp λ-DNA in order to prove the functionality of on-chip pores coated with Al2O3.