Fabrication Of Solid-state Nanopores Research Articles

Practical guide for in-house solid-state nanopore fabrication and characterization

Solid-state nanopores are considered a better alternative to biological nanopores for several sensing applications due to their better chemical, mechanical, and temperature stability. In addition to sequencing, nanopores currently also find applications in education, biomarker identification, quantification, single-molecule chemistry, and DNA computing. Nanopore technology’s simplicity and wide interdisciplinary applications have raised further interest among industry and scientific community worldwide. However, further development in solid-state nanopore technology and exploring its applications presents the need to have the capability to fabricate them in-house. This will be a more financially viable and flexible approach, especially in resource-limited situations. In order to do an in-house fabrication of solid-state nanopores, two key steps are involved. The first step is to fabricate suspended thin films, and the second one is the drilling of pores in these suspended thin membranes. Successful implementation of these two steps involves tedious optimization and characterization of the fabricated chips and nanopores. In this work, we describe the nanopore fabrication process in a ready-to-follow step-by-step guide and present solutions for several practical difficulties faced during the silicon nitride pore fabrication process. This work will help anyone new to this field and make the pore fabrication process more accessible.

Localised solid-state nanopore fabrication via controlled breakdown using on-chip electrodes

Controlled breakdown has recently emerged as a highly accessible technique to fabricate solid-state nanopores. However, in its most common form, controlled breakdown creates a single nanopore at an arbitrary location in the membrane. Here, we introduce a new strategy whereby breakdown is performed by applying the electric field between an on-chip electrode and an electrolyte solution in contact with the opposite side of the membrane. We demonstrate two advantages of this method. First, we can independently fabricate multiple nanopores at given positions in the membrane by localising the applied field to the electrode. Second, we can create nanopores that are self-aligned with complementary nanoelectrodes by applying voltages to the on-chip electrodes to locally heat the membrane during controlled breakdown. This new controlled breakdown method provides a path towards the affordable, rapid, and automatable fabrication of arrays of nanopores self-aligned with complementary on-chip nanostructures.

Controllable Shrinking Fabrication of Solid-State Nanopores.

Nanopores have attracted widespread attention in DNA sequencing and protein or biomarker detection, owning to the single-molecule-scale detection accuracy. Despite the most use of naturally biological nanopores before, solid-state nanopores are widely developed with strong robustness, controllable sizes and geometries, a wide range of materials available, as well as flexible manufacturing. Therefore, various techniques typically based on focused ion beam or electron beam have been explored to drill nanopores directly on free-standing nanofilms. To further reduce and sculpt the pore size and shape for nano or sub-nano space-time sensing precision, various controllable shrinking technologies have been employed. Correspondingly, high-energy-beam-induced contraction with direct visual feedback represents the most widely used. The ability to change the pore diameter was attributed to surface tension induced original material migration into the nanopore center or new material deposition on the nanopore surface. This paper reviews typical solid-state nanopore shrinkage technologies, based on the careful summary of their principles and characteristics in particularly size and morphology changes. Furthermore, the advantages and disadvantages of different methods have also been compared completely. Finally, this review concludes with an optimistic outlook on the future of solid-state nanopores.

Simple Fabrication of Solid-State Nanopores on a Carbon Film.

Solid-state nanopores are widely used as a platform for stochastic nanopore sensing because they can provide better robustness, controllable pore size, and higher integrability than biological nanopores. However, the fabrication procedures, including thin film preparation and nanopore formation, require advanced micro-and nano-fabrication techniques. Here, we describe the simple fabrication of solid-state nanopores in a commercially available material: a flat thin carbon film-coated micro-grid for a transmission electron microscope (TEM). We attempted two general methods for nanopore fabrication in the carbon film. The first method was a scanning TEM (STEM) electron beam method. Nanopores were fabricated by irradiating a focused electron beam on the carbon membrane on micro-grids, resulting in the production of nanopores with pore diameters ranging from 2 to 135 nm. The second attempt was a dielectric breakdown method. In this method, nanopores were fabricated by applying a transmembrane voltage of 10 or 30 V through the carbon film on micro-grids. As a result, nanopores with pore diameters ranging from 3.7 to 1345 nm were obtained. Since these nanopores were successfully fabricated in the commercially available carbon thin film using readily available devices, we believe that these solid-state nanopores offer great utility in the field of nanopore research.

On Stochastic Reduction in Laser-Assisted Dielectric Breakdown for Programmable Nanopore Fabrication.

The controlled dielectric breakdown emerged as a promising alternative toward accessible solid-state nanopore fabrication. Several prior studies have shown that laser-assisted dielectric breakdown could help control the nanopore position and reduce the possibility of forming multiple pores. Here, we developed a physical model to estimate the probability of forming a single nanopore under different combinations of the laser power and the electric field. This model relies on the material- and experiment-specific parameters: the Weibull statistical parameters and the laser-induced photothermal etching rate. Both the model and our experimental data suggest that a combination of a high laser power and a low electric field is statistically favorable for forming a single nanopore at a programmed location. While this model relies on experiment-specific parameters, we anticipate it could provide the experimental insights for nanopore fabrication by the laser-assisted dielectric breakdown method, enabling broader access to solid-state nanopores and their sensing applications.

A novel dielectric breakdown apparatus for solid-state nanopore fabrication with transient high electric field.

The dielectric breakdown used to fabricate solid-state nanopores has separated the device from capital-intensive industries and has been widely adopted by various research teams, but there are still problems with low production efficiency and uncertain location. In this work, based on the transient breakdown phenomenon of nanofilms, a new type of dielectric breakdown apparatus for nanopore fabrication is reported. It integrates both nano-manipulation technology and dielectric breakdown nanopore fabrication technology. The nanometer distance detection method and circuit are introduced in detail. The generation principle and procedures of the transient high electric field are explained step by step. The characterization of the nanopores shows that this apparatus can fabricate sub-2 nm nanopores at a pre-located position. Besides, the nanopore diameter can be easily adjusted by setting the transient high electric field value.

Direct Observation of Redox-Induced Bubble Generation and Nanopore Formation Dynamics in Controlled Dielectric Breakdown

While controlled dielectric breakdown (CBD) emerged as a promising method for accessible solid-state nanopore fabrication, there are still significant challenges in understanding the fabrication dynamics due to the lack of in situ cross-reference characterization beyond current monitoring. In this work, we developed a multimodal method for characterizing the dielectric breakdown-based nanopore formation dynamics. With this capability, we observed for the first time the redox-induced bubble generation at the electrolyte–membrane interface. The randomly generated gas bubble would significantly alter the electric field distribution on the membrane surfaces and is an overlooked factor that can contribute to the random distribution of the nanopores. Besides, we also studied the impact of electric field strength on the number and location of nanopore(s) initially formed and after enlargement. We believe that the direct evidence of redox-induced bubble formation and the impact of the electric field on nanopore formation dynamics presented in this work would provide significant experimental insight for further improving the breakdown-based solid-state nanopore fabrication.

Solid-state nanopore fabrication by automated controlled breakdown.

Solid-state nanopores are now well established as single-biomolecule sensors that hold great promise as sensing elements in diagnostic and sequencing applications. However, until recently this promise has been limited by the expensive, labor-intensive, and low-yield methods used to fabricate low-noise and precisely sized pores. To address this problem, we pioneered a low-cost and scalable solid-state nanopore fabrication method, termed controlled breakdown (CBD), which is rapidly becoming the method of choice for fabricating solid-state nanopores. Since its initial development, nanopore research groups around the world have applied and adapted the CBD method in a variety of ways, with varying levels of success. In this work, we present our accumulated knowledge of nanopore fabrication by CBD, including a detailed description of the instrumentation, software, and procedures required to reliably fabricate low-noise and precisely sized solid-state nanopores with a yield of >85% in less than 1 h. The assembly instructions for the various custom instruments can be found in the Supplementary Manual, and take approximately a day to complete, depending on the unit that the user is building and their level of skill with mechanical and electrical assembly. Unlike traditional beam-based nanopore fabrication technologies, the methods presented here are accessible to non-experts, lowering the cost of, and technical barriers to, fabricating nanoscale pores in thin solid-state membranes.

An apparatus based on an atomic force microscope for implementing tip-controlled local breakdown.

Solid-state nanopores are powerful tools for sensing of single biomolecules in solution. Fabrication of solid-state nanopores is still challenging, however; in particular, new methods are needed to facilitate the integration of pores with larger nanofluidic and electronic device architectures. We have developed the tip-controlled local breakdown (TCLB) approach, in which an atomic force microscope (AFM) tip is brought into contact with a silicon nitride membrane that is placed onto an electrolyte reservoir. The application of a voltage bias at the AFM tip induces a dielectric breakdown that leads to the formation of a nanopore at the tip position. In this work, we report on the details of the apparatus used to fabricate nanopores using the TCLB method, and we demonstrate the formation of nanopores with smaller, more controlled diameters using a current limiting circuit that zeroes the voltage upon pore formation. Additionally, we demonstrate the capability of TCLB to fabricate pores aligned to embedded topographical features on the membranes.

High fidelity moving Z-score based controlled breakdown fabrication of solid-state nanopore

We investigate the current transport characteristics in the electrolyte-dielectric-electrolyte structure commonly used in the in situ controlled breakdown (CBD) fabrication of solid-state nanopores. It is found that the stochastic breakdown process could lead to fidelity issues of false positives (an incorrect indication of a true nanopore formation) and false negatives (inability to detect initial nanopore formation). Robust and deterministic detection of initial physical breakdown to alleviate false positives and false negatives is critical for precise nanopore size control. To this end, we report a high fidelity moving Z-score method based CBD fabrication of solid-state nanopore. We demonstrate 100% success rate of realizing the initial nanopore conductance of 3 ± 1 nS (corresponds to size of 1.7 ± 0.6 nm) regardless of the dielectric membrane characteristics. Our study also elucidates the Joule heating is the dominant mechanism for electric field-based nanopore enlargement. Single DNA molecule sensing using nanopores fabricated by this method was successfully demonstrated. We anticipate the moving Z-score based CBD method could enable broader access to the solid state nanopore-based single molecule analysis.

Solid-state nanopore fabrication in LiCl by controlled dielectric breakdown.

Nanopore fabrication via the controlled dielectric breakdown (CDB) method offers an opportunity to create solid-state nanopores directly in salt solution with sub-nanometer precision. Driven by trap assisted current tunneling, the method uses localized defects, or traps, in the dielectric material to isolate a breakdown point and fabricate a single pore in less than 10minutes. Here we present an approach to controlled dielectric breakdown of SiNx in which the nanopore is fabricated in LiCl buffer instead of the traditional KCl buffer. Direct fabrication in LiCl buffer promotes a uniform, symmetric, cylindrical nanopore structure that is fully wet and can be used for experiments in situ. We have shown that fabrication in LiCl reduces the necessity for overnight pore stabilization and allows for the desired analyte to be added in significantly less time than it would take if fabrication was performed in KCl. Pores created by this approach can be used for biosensing applications, including the detection of double-stranded DNA. DNA translocation experiments were conducted in both LiCl and KCl buffer. Experiments conducted in LiCl buffer resulted in about a 2-fold increase in dsDNA transport duration when compared to experiments conducted in KCl buffer of the same concentration. An increase in transport duration of over 10-fold in comparison to KCl was observed when the concentration of the LiCl buffer was increased by a factor of 3.

Rapid fabrication of solid-state nanopores with high reproducibility over a large area using a helium ion microscope.

The fabrication of solid-state nanopores in an insulating membrane has attracted much attention for biomolecule analysis such as DNA sequencing and detection in recent years. For practical applications and device integration, the challenges include precise size control for sub 10 nm nanopores, excellent repeatability and rapid fabrication over a large area to reduce the cost for mass production. A helium ion beam could provide an effective fabrication approach to produce such solid-state nanopores. It is easy to control the nanopore size and reach sub 10 nm pore size with a simple change in the milling time with an appropriate ion beam current. Here we report new results in a set of experiments demonstrating that with a small range of stage automatized motions and equal mill times one can obtain good fabrication reproducibility in nanopore sizes (<10% variation in size). The automation in the stage motion and milling time opens a door for the rapid mass production of nanopore chips over a wafer size of several inches.

Wireless Bipolar Nanopore Electrode for Single Small Molecule Detection.

Solid-state nanopore-based techniques have become a promising strategy for diverse single molecule detections. Owing to the challenge in well and rapid fabrication of solid-state nanopores with the diameter less than 2 nm, small molecule detection is hard to be addressed by existing label-free nanopore methods. In this work, we for the first time propose a metal-coated wireless nanopore electrode (WNE) which offers a novel and generally accessible detection method for analyzing small molecules and ions at the single molecule/ion level. Here, a silver-coated WNE is developed as a proof-of-principle model which achieves the detection the self-generated H2, the smallest known molecule, and Ag+ at single molecule/ion level by monitoring the enhanced ionic signatures. Under a bias potential of -800 mV, the WNE could accomplish the distinction of as low as 14 H2 molecules and 28 Ag+ from one spike signal. The finite element simulation is introduced to suggest that the generation of H2 at the orifice of the WNE results in the enhanced spike of ionic current. As a proof-of-concept experiment, the WNE is further utilized to directly detect Hg2+ from 100 pM to 100 nM by monitoring the frequency of the spike signals. This novel nanoelectrode provides a brand new label-free, ultrasensitive, and simple detection mechanism for various small molecules/ions detection, especially for redox analytes.

Precise fabrication of a 5 nm graphene nanopore with a helium ion microscope for biomolecule detection

We report a scalable method to fabricate high-quality graphene nanopores for biomolecule detection using a helium ion microscope (HIM). HIM milling shows promising capabilities for precisely controlling the size and shape, and may allow for the potential production of nanopores at wafer scale. Nanopores could be fabricated at different sizes ranging from 5 to 30 nm in diameter in few minutes. Compared with the current solid-state nanopore fabrication techniques, e.g. transmission electron microscopy, HIM is fast. Furthermore, we investigated the exposure-time dependence of graphene nanopore formation: the rate of pore expansion did not follow a simple linear relationship with exposure time, but a fast expansion rate at short exposure time and a slow rate at long exposure time. In addition, we performed biomolecule detection with our patterned graphene nanopore. The ionic current signals induced by 20-base single-stranded DNA homopolymers could be used as a basis for homopolymer differentiation. However, the charge interaction of homopolymer chains with graphene nanopores, and the conformations of homopolymer chains need to be further considered to improve the accuracy of discrimination.

Three-dimensional structures of magnesium nanopores

The optimization of nanopore-based devices is closely related to the nanopore three-dimensional (3D) structures. In this paper, faceted nanopores were fabricated in magnesium (Mg) by aligning the electron beam (e-beam) along the [0001] direction. Detailed structural characterization by transmission electron microscopy reveals the existence of two 3D structures: hexagonal prism-shaped and hourglass-shaped 3D morphologies. Moreover, the 3D structures of nanopores are also found to depend on the widest nanopore diameter-to-thickness ratio (D/t). A plausible formation mechanism for different 3D structures is discussed. Our results incorporate a critical piece of information regarding the nanopore 3D structures in Mg and may serve as an important design guidance for the size- and shape-controllable fabrication of solid-state nanopores applying the e-beam sculpting technique.

Fabrication of Solid State Nanopore in Thin Silicon Membrane Using Low Cost Multistep Chemical Etching.

Nanopore-based analysis is currently an area of great interest in many disciplines with the potential for exceptionally versatile applications in medicine. This work presents a novel step towards fabrication of a single solid-state nanopore (SSSN) in a thin silicon membrane. Silicon nanopores are realized using multistep processes on both sides of n-type silicon-on-insulator (SOI) <100> wafer with resistivity 1–4 Ω·cm. An electrochemical HF etch with low current density (0.47 mA/cm2) is employed to produce SSSN. Blue LED is considered to emit light in a narrow band region which facilitates the etching procedure in a unilateral direction. This helps in production of straight nanopores in n-type Si. Additionally, a variety of pore diameters are demonstrated using different HF concentrations. Atomic force microscopy is used to demonstrate the surface morphology of the fabricated pores in non-contact mode. Pore edges exhibit a pronounced rounded shape and can offer high stability to fluidic artificial lipid bilayer to study membrane proteins. Electrochemically-fabricated SSSN has excellent smoothness and potential applications in diagnostics and pharmaceutical research on transmembrane proteins and label free detection.

Solid-state nanopore-based DNA single molecule detection and sequencing

Nanopores have been used as ultrasensitive tools in sequencing of DNA, in RNA and protein conformation studies, diagnosis, drug screening, and in environmental monitoring. This review (with 128 refs.) summarizes recent progress on solid-state nanopore-based DNA single-molecule detection and sequencing. Following a description of the principles of the technique, we focus on the fabrication of solid-state nanopores. We then treat the spatial and temporal resolution that can be accomplished with them in terms of DNA translocation and sequencing. Finally, we summarize novel methods for detection such as tunneling current sensing and multichannel sensing, and then end up with a conclusion on developments and prospects of future nanopore technology.

Fine-tuning the Size and Minimizing the Noise of Solid-state Nanopores

Solid-state nanopores have emerged as a versatile tool for the characterization of single biomolecules such as nucleic acids and proteins1. However, the creation of a nanopore in a thin insulating membrane remains challenging. Fabrication methods involving specialized focused electron beam systems can produce well-defined nanopores, but yield of reliable and low-noise nanopores in commercially available membranes remains low2,3 and size control is nontrivial4,5. Here, the application of high electric fields to fine-tune the size of the nanopore while ensuring optimal low-noise performance is demonstrated. These short pulses of high electric field are used to produce a pristine electrical signal and allow for enlarging of nanopores with subnanometer precision upon prolonged exposure. This method is performed in situ in an aqueous environment using standard laboratory equipment, improving the yield and reproducibility of solid-state nanopore fabrication.

Fine-tuning the Size and Minimizing the Noise of Solid-state Nanopores

Solid-state nanopores have emerged as a versatile tool for the characterization of single biomolecules such as nucleic acids and proteins1. However, the creation of a nanopore in a thin insulating membrane remains challenging. Fabrication methods involving specialized focused electron beam systems can produce well-defined nanopores, but yield of reliable and low-noise nanopores in commercially available membranes remains low2,3 and size control is nontrivial4,5. Here, the application of high electric fields to fine-tune the size of the nanopore while ensuring optimal low-noise performance is demonstrated. These short pulses of high electric field are used to produce a pristine electrical signal and allow for enlarging of nanopores with subnanometer precision upon prolonged exposure. This method is performed in situ in an aqueous environment using standard laboratory equipment, improving the yield and reproducibility of solid-state nanopore fabrication.

(Keynote) Solid-State Nanopore Fabrication with Conventional Process

In this paper, the progress of solid-state nanopores fabrication is reviewed after a brief summary of the basic principle of the new DNA sequencing technology based on solid-state nanopores. The pore-size shrinking technique for the pores with a diameter below 10 nm is highlighted. Based on an analysis on the current works, it can be concluded that the conventional micro-nano processing technology combination with the pore-size shrinking technique are an effective way for mass fabrication of the solid-state nanopores.