Discovered the fabrication process: HK. Nanofabrication techniques for achieving dimensional control at the nanometer scale are generally equipment-intensive and time-consuming. The use of energetic beams of electrons or ions has placed the fabrication of nanopores in thin solid-state membranes within reach of some academic laboratories, yet these tools are not accessible to many researchers and are poorly suited for mass-production. Here we describe a fast and simple approach for fabricating a single nanopore down to 2-nm in size with sub-nm precision, directly in solution, by controlling dielectric breakdown at the nanoscale.
The method relies on applying a voltage across an insulating membrane to generate a high electric field, while monitoring the induced leakage current. We show that nanopores fabricated by this method produce clear electrical signals from translocating DNA molecules. Considering the tremendous reduction in complexity and cost, we envision this fabrication strategy would not only benefit researchers from the physical and life sciences interested in gaining reliable access to solid-state nanopores, but may provide a path towards manufacturing of nanopore-based biotechnologies.
Nanopore sensing relies on the electrophoretically driven translocation of biomolecules through nanometer-scale holes embedded in thin insulating membranes to confine, detect and characterize the properties or the activity of individual biomolecules electrically, by monitoring transient changes in ionic current  — .
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The field was initially shaped by the ability of researchers to exploit biological channels to translocate single molecules  , . It rapidly expanded when new techniques to fabricate individual molecular-sized holes in thin solid-state materials were developed over the last decade  — . These techniques, based on beams of high-energy particles, either produced by a dedicated ion beam machine i. Since then, a host of applications for DNA, RNA and proteins analysis using both biological and solid-state nanopores have been demonstrated  ,  , . Compared to their organic counterparts, solid-state nanopores were expected to emerge as an essential component of any practical nanopore-based instrumentation due to the size control, increased robustness of the membrane, and their natural propensity for integration with wafer-scale technologies, including CMOS and microfluidics  , .
Yet, this prospect is significantly hindered due to the constraints and limitations imposed by ion beam sculpting and transmission electron microscopy-based drilling, which, to this date, remain the only viable tools for achieving nanopores fabrication with dimensional control at the 1-nm scale. The complexity, low-throughput, and high-cost associated with these techniques restrict accessibility of the field to many researchers, greatly limit the productivity of the community, and prevent mass production of nanopore-based devices.
Alternative nanofabrication strategies are therefore needed for the field to continue to thrive, and for the promised health-related applications to be successfully commercialized including single-molecule DNA sequencing. Here, we introduce a fabrication technique based on the use of high electric fields to control dielectric breakdown in solution.
The method is automated, simple, and low-cost, allowing nanopores to be created directly in aqueous solution with sub-nm precision, greatly facilitating use and improving yield of functional solid-state nanopore devices. We envision this fabrication strategy will not only provide a path towards nanomanufacturing of nanopore-based devices for a wide range of biotechnology applications, but will democratize the use of solid-state nanopores, while offering researchers new strategies for designing nanofluidics devices, as well as integrating nanopores with CMOS and microfluidics technologies.
We fabricate individual nanopores on thin insulating solid-state membranes directly in solution. A thin silicon nitride SiN x membrane, supported by a silicon frame, is mounted in a liquid cell and separates two reservoirs containing an aqueous solution of 1M KCl.
The setup shown in Figure 1 is otherwise identical to what is commonly used for biomolecular detection  , which greatly facilitates the transition to sensing experiments, eliminating further handling of membranes. See Material and Methods section and Section S1 for more detail. It is noteworthy to realize that this experimental setup is identical with the exception of the custom current amplifier replacing the commonly used Axopatch B to the instrumentation used to study DNA or proteins translocation through nanopores.
At these high field strengths, a sustainable leakage current, I leakage , is observed through the membrane, which remains otherwise insulating at low fields. I leakage rapidly increases with electric field strength, but is typically tens of nanoamperes for our operating conditions. We attribute the dominant conduction mechanism to a form of trap-assisted tunneling of electrons, supplied by ions in solution  —  Figure 2b and 2c , since the membrane is too thick for significant direct tunnelling  , and migration of impurities cannot produce lasting currents .
Direct migration of electrolyte ions is also unlikely, or negligible, since for a given electric field strength, a higher I leakage is observed in thicker membranes Figure 2e. A larger current is observed on thicker membranes since the number of charge traps defects per unit area is greater, as their number in the material increases with volume. We provide additional discussion on the characteristics of the leakage current in Section S2. Free charges electrons or holes can be produced by redox reactions at the surface or by field ionization of incorporated ions.
The number of available charged traps structural defects sets the magnitude of the observed leakage current. The slowly increase leakage current, following the capacitive spike, is a result of the accumulation of traps in the membrane. The nanopore is allowed to grow until a pre-determined threshold current is reached, at which point the voltage is turned off. The observed current fluctuations at the onset of pore formation are attributed to significant low-frequency noise at this voltage. We observe the creation of a single nanopore i.
As the current continues to increase, the nanopore further enlarges Figure 2g. We use a feedback control mechanism to rapidly terminate the trans-membrane potential when the current exceeds a pre-determined threshold, I cutoff. This allows the nanopore size to be precisely tuned, for a particular sensing application, directly in neutral KCl solution. This method, practical for nanopores fabricated in liquids, provides a reasonable first order estimate of the pore size  ,  as confirmed by DNA translocations, and compares well with actual dimensions obtained from TEM images see Sections S4 and S8.
Figure 2 h reveals an ohmic electric response in 1 M KCl. The majority of our nanopores exhibit linear I-V curves upon fabrication. The remaining nanopores that show signs of self-gating or rectification can be conditioned, by applying moderate electric field pulses  , to slightly enlarge them until an ohmic behaviour is attained in high salt.
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Such I-V characteristics imply a relatively symmetric internal electric potential pore profile  which supports the symmetrical geometry with a uniform surface charge distribution assumed by our pore conductance model. To further characterize the nanopores, we examined the noise in the ionic current by performing power spectral density measurements.
This may be attributed to the fact that nanopores are created directly in liquid rather than in vacuum. Thus far, we have successfully fabricated hundreds of individual nanopores ranging from 1 to nm in size with comparable electrical characteristics that are stable for days, 66 of which are included in Figure 2.
In order that a single, well-defined nanopore be created each time, we postulate that the leakage current must be highly localized on the insulating membrane, since for conductive substrates semiconductors or metals anodic oxidation leads to an array of nanopores  — . The leakage spot s must also modify the membrane at the nanoscale since an aqueous KCl solution at neutral pH is not an active etchant of SiN x.
To elucidate the mechanism leading to the formation of a nanopore, we investigate the fabrication process as a function of applied voltage, membrane thickness, electrolyte composition, concentration, and pH. For a given voltage, pH has a strong effect. We have also observed that lower salt concentrations increase the fabrication time see Section S6. This observation indicates that the applied electric field in the membrane is the main driving force for initiating the fabrication of a single nanopore.
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Fields in the range of 0. According to the current understanding, dielectric breakdown mechanisms proceed as follows  —  : i probabilistic accumulation of charge traps i. We propose that the process by which we fabricate a nanopore in solution is similar, though we control the damage to the nanoscale by limiting the localized leakage current, at the onset of the first, discrete breakdown event. As indicated by Figure 3 , the likelihood of defect formation within the silicon nitride membrane increases with the applied voltage and the strength of the electric field.
At low values, the accumulation of charge traps is accomplished with relatively low efficiency compared to the amount of charge carriers traversing the membrane, since a leakage current of tens of nanoamperes can be sustained for hours or days. Given the stochastic nature of the pore creation process, multiple simultaneous nanoscale breakdown events are unlikely. Termination of the applied voltage following the occurrence of the first breakdown event, observed by the sudden irreversible increase in I leakage , ensures that ultimately a single nanopore is created.
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Moreover, the fabrication of a single nanopore may result from the fact that the formation path of a nanopore experiences increased electric field strength during growth, which locally reinforces the rate of defect generation. Another possibility is shearing due to localized plasticity of the membrane as a result of heating at the breakdown spot, but the efficiency of heat dissipation at the nanoscale, resulting from high surface-area-to-volume ratios, makes this less likely . To support the general character of nanofabrication by dielectric breakdown, we created nanopores in a different material silicon dioxide and present the data in Section S7.
The number of separate nanopores each data point is averaged over is indicated in parentheses. The vast majority of nanopores plotted are subnm in size i. All 66 nanopores plotted are subnm in size i. We performed DNA translocation experiments to demonstrate that these nanopores can be leveraged for the benefit of single-molecule detection. Electrophoretically driven passage of a DNA molecule across a membrane is expected to transiently block the flow of ions in a manner that reflects the molecule length, size, charge and shape.
The scatter plot shows event duration and average current blockage of over 2, single-molecule translocations events of 5-kb dsDNA.
sofenabemab.tk The characteristic shape of the events is indistinguishable to data obtained on TEM-drilled nanopores  ,  — . The observed quantized current blockades strongly support the presence of a single nanopore spanning the membrane. This result also suggests that the membrane thickness at the vicinity of the nanopores has not been significantly altered. Data sampled at kHz and low-pass filtered at kHz. Each data point represents a single DNA translocation event. The majority of the events are unfolded. There are very few anomalously long events, indicating weak DNA-pore interactions.
The inset shows ionic current signatures of two single-molecule translocation events, passing in a linear and partially folded conformation. Quantized levels corresponding to zero, one, two dsDNA strands in the nanopore are clearly observed. Nanopore fabrication by controlled dielectric breakdown in solution represents a major reduction in complexity and cost over current fabrication methods, which will greatly facilitate accessibility to the field to many researchers, and provides a path to commercialize nanopore-based technologies.
While we attribute the nanopore creation process to an intrinsic property of the dielectric membrane, such that the nanopore can form anywhere on the surface, our current understanding strongly suggests that the position of the pore can be determined by locally controlling the electric field strength or the material dielectric strength. This could be achieved, for instance, by nanopatterning or locally thinning the membrane, by positioning of a nanoelectrode, or by confining the field to specific areas on the membrane via micro- or nanofluidic channel encapsulation see Section S9.
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The latter would also allow for the simple integration of independently addressable nanopores in an array format on a single chip. Prior to mounting into liquids, SiN x membranes can be cleaned in oxygen plasma for 30 s at 30 W to facilitate wetting of the membrane surface, though this is not a requirement.
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All solutions used were filtered and degassed prior to use. The absence of pre-existing structural damages e. The membrane side opposing the Si etch pit is the reference point for all applied voltages in this article. A schematic of the experimental setup is shown in Figure 1. A silicon chip with an intact silicon nitride membrane is sandwiched between two silicone gaskets shown in purple on the figure.
The entire system is encapsulated in a grounded faraday cage to isolate from electromagnetic interference.