Coupled nanopores for single-molecule detection.

Rapid sensing of molecules is increasingly important in many studies and applications, such as DNA sequencing and protein identification. Here, beyond atomically thin 2D nanopores, we conceptualize, simulate and experimentally demonstrate coupled, guiding and reusable bilayer nanopore platforms, enabling advanced ultrafast detection of unmodified molecules. The bottom layer can collimate and decelerate the molecule before it enters the sensing zone, and the top 2D pore (~2 nm) enables position sensing. We varied the number of pores in the bottom layer from one to nine while fixing one 2D pore in the top layer. When the number of pores in the bottom layer is reduced to one, sensing is performed by both layers, and distinct T- and W-shaped translocation signals indicate the precise position of molecules and are sensitive to fragment lengths. This is uniquely enabled by microsecond resolution capabilities and precision nanofabrication. Coupled nanopores represent configurable multifunctional systems with inter- and intralayer structures for improved electromechanical control and prolonged dwell times in a 2D sensing zone.

Ultrafast Polymer Dynamics through a Nanopore.

Ultrathin nanopore sensors allow single-molecule and polymer measurements at sub-microsecond time resolution enabled by high current signals (∼10-30 nA). We demonstrate for the first time the experimental probing of the ultrafast translocation and folded dynamics of double-stranded DNA (dsDNA) through a nanopore at 10 MHz bandwidth with acquisition of data points per 25 ns (150 MB/s). By introducing a rigorous algorithm, we are able to accurately identify each current level present within translocation events and elucidate the dynamic folded and unfolded behaviors. The remarkable sensitivity of this system reveals distortions of short-lived folded states at a lower bandwidth. This work revisits probing of dsDNA as a model polymer and develops broadly applicable methods. The combined improvements in sensor signals, instrumentation, and large data analysis methods uncover biomolecular dynamics at unprecedentedly small time scales.

Engineering adjustable two-pore devices for parallel ion transport and DNA translocations.

We report ionic current and double-stranded DNA (dsDNA) translocation measurements through solid-state membranes with two TEM-drilled ∼3-nm diameter silicon nitride nanopores in parallel. Nanopores are fabricated with similar diameters but varying in effective thicknesses (from 2.6 to 10 nm) ranging from a thickness ratio of 1:1 to 1:3.75, producing distinct conductance levels. This was made possible by locally thinning the silicon nitride membrane to shape the desired topography with nanoscale precision using electron beam lithography (EBL). Two nanopores are engineered and subsequently drilled in either the EBL-thinned or the surrounding membrane region. By designing the interpore separation a few orders of magnitude larger than the pore diameter (e.g., ∼900 vs 3 nm), we show analytically, numerically, and experimentally that the total conductance of the two pores is the sum of the individual pore conductances. For a two-pore device with similar diameters yet thicknesses in the ratio of 1:3, a ratio of ∼1:2.2 in open-pore conductances and translocation current signals is expected, as if they were measured independently. Introducing dsDNA as analytes to both pores simultaneously, we detect more than 12 000 events within 2 min and trace them back with a high likelihood to which pore the dsDNA translocated through. Moreover, we monitor translocations through one active pore only when the other pore is clogged. This work demonstrates how two-pore devices can fundamentally open up a parallel translocation reading system for solid-state nanopores. This approach could be creatively generalized to more pores with desired parameters given a sufficient signal-to-noise ratio.

Detection of single analyte and environmental samples with silicon nitride nanopores: Antarctic dirt particulates and DNA in artificial seawater.

Nanopore sensing is a powerful tool for the detection of biomolecules. Solid-state nanopores act as single-molecule sensors that can function in harsh conditions. Their resilient nature makes them attractive candidates for taking this technology into the field to measure environmental samples for life detection in space and water quality monitoring. Here, we discuss the fabrication of silicon nitride pores from ∼1.6 to 20 nm in diameter in 20-nm-thick silicon nitride membranes suspended on glass chips and their performance. We detect pure laboratory samples containing a single analyte including DNA, BSA, microRNA, TAT, and poly-D-lys-hydrobromide. We also measured an environmental (mixed-analyte) sample, containing Antarctic dirt provided by NASA Ames. For DNA measurements, in addition to using KCl and NaCl solutions, we used the artificial (synthetic) seawater, which is a mixture of different salts mimicking the composition of natural seawater. These samples were spiked with double-stranded DNA (dsDNA) fragments at different concentrations to establish the limits of nanopore sensitivity in candidate environment conditions. Nanopore chips were cleaned and reused for successive measurements. A stand-alone, 1-MHz-bandwidth Chimera amplifier was used to determine the DNA concentration in artificial seawater that we can detect in a practical time scale of a few minutes. We also designed and developed a new compact nanopore reader, a portable read-out device with miniaturized fluidic cells, which can obtain translocation data at bandwidths up to 100 kHz. Using this new instrument, we record translocations of 400 bp, 1000 bp, and 15000 bp dsDNA fragments and show discrimination by analysis of current amplitude and event duration histograms.

Lifetime and Stability of Silicon Nitride Nanopores and Nanopore Arrays for Ionic Measurements.

Nanopores are promising for many applications including DNA sequencing and molecular filtration. Solid-state nanopores are preferable over their biological counterparts for applications requiring durability and operation under a wider range of external parameters, yet few studies have focused on optimizing their robustness. We report the lifetime and durability of pores and porous arrays in 10 to 100 nm-thick, low-stress silicon nitride (SiNx) membranes. Pores are fabricated using a transmission electron microscope (TEM) and/or electron beam lithography (EBL) and reactive ion etching (RIE), with diameters from 2 to 80 nm. We store them in various electrolyte solutions (KCl, LiCl, MgCl2) and record open pore conductance over months to quantify pore stability. Pore diameters increase with time, and diameter etch rate increases with electrolyte concentration from Δd/Δt ∼ 0.2 to ∼ 3 nm/day for 0.01 to 3 M KCl, respectively. TEM confirms the range of diameter etch rates from ionic measurements. Using electron energy loss spectroscopy (EELS), we observe a N-deficient region around the edges of TEM-drilled pores. Pore expansion is caused by etching of the Si/SiO2 pore walls, which resembles the dissolution of silicon found in minerals such as silica (SiO2) in salty ocean water. The etching process occurs where the membrane was exposed to the electron beam and can result in pore formation. However, coating pores with a conformal 1 nm-thick hafnium oxide layer prevents expansion in 1 M KCl, in stark contrast to bare SiNx pores (∼ 1.7 nm/day). EELS data reveal the atomic composition of bare and HfO2-coated pores.

Centimeter-Scale Nanoporous 2D Membranes and Ion Transport: Porous MoS2 Monolayers in a Few-Layer Matrix.

Two-dimensional nanoporous membranes have received attention as catalysts for energy generation and membranes for liquid and gas purification but controlling their porosity and facilitating large-scale production is challenging. We show the growth and fabrication of centimeter-scale molybdenum disulfide (MoS2) membranes with tunable porous areas up to ∼ 10% of the membrane and average nanopore diameters as large as ∼ 30 nm, controlled by the etch time. We also measure ionic conductance between 0.1 and 16 µS per µm2 through variably etched nanoporous membranes. Ensuring the mechanical robustness and large-area of the membrane, bilayer and few-layer regions form a strong supporting matrix around monolayer regions, observed by aberration-corrected scanning transmission electron microscopy. During etching, nanopores form in thin, primarily monolayer areas whereas thicker multilayer regions remain essentially intact. Atomic-resolution imaging reveals that after exposure to the etchant, the number of V1Mo vacancies increases and nanopores form along grain boundaries in monolayers, suggesting that etching starts at intrinsic defect sites. This work provides an avenue for the scalable production of nanoporous atomically thin membranes.

Transfer of monolayer TMD WS2 and Raman study of substrate effects.

A facile transfer process for transition metal dichalcogenide WS2 flakes is reported and the effect of the underlying substrate on the flake properties is investigated using Raman spectroscopy. The flakes are transferred from their growth substrate using polymethyl methacrylate (PMMA) and a wet etch to allow the user to transfer the flakes to a final substrate using a microscope and micromanipulator combined with semi-transparent Kapton tape. The substrates used range from insulators such as industry standard high-k dielectric HfO2 and "green polymer" parylene-C, to conducting chemical vapor deposition (CVD) grown graphene. Raman spectroscopy is used first to confirm the material quality of the transferred flakes to the substrates and subsequently to analyze and separate the effects arising from material transfer from those arising from interactions with the substrate. We observe changes in the Raman spectra associated with the interactions between the substrates in the flakes. These interactions affect both in-plane and out-of-plane modes in different ways depending on their sources, for example strain or surface charge. These changes vary with final substrate, with the strongest effects being observed for WS2 transferred onto graphene and HfO2, demonstrating the importance of understanding substrate interaction for fabrication of future devices.

Monolayer WS2 Nanopores for DNA Translocation with Light-Adjustable Sizes.

Two-dimensional materials are promising for a range of applications, as well as testbeds for probing the physics of low-dimensional systems. Tungsten disulfide (WS2) monolayers exhibit a direct band gap and strong photoluminescence (PL) in the visible range, opening possibilities for advanced optoelectronic applications. Here, we report the realization of two-dimensional nanometer-size pores in suspended monolayer WS2 membranes, allowing for electrical and optical response in ionic current measurements. A focused electron beam was used to fabricate nanopores in WS2 membranes suspended on silicon-based chips and characterized using PL spectroscopy and aberration-corrected high-resolution scanning transmission electron microscopy. It was observed that the PL intensity of suspended WS2 monolayers is ∼10-15 times stronger when compared to that of substrate-supported monolayers, and low-dose scanning transmission electron microscope viewing and drilling preserves the PL signal of WS2 around the pore. We establish that such nanopores allow ionic conductance and DNA translocations. We also demonstrate that under low-power laser illumination in solution, WS2 nanopores grow slowly in size at an effective rate of ∼0.2-0.4 nm/s, thus allowing for atomically controlled nanopore size using short light pulses.