Characterizing the Conformational Free-Energy Landscape of RNA Stem-Loops Using Single-Molecule Field-Effect Transistors.

We have developed and used single-molecule field-effect transistors (smFETs) to characterize the conformational free-energy landscape of RNA stem-loops. Stem-loops are one of the most common RNA structural motifs and serve as building blocks for the formation of complex RNA structures. Given their prevalence and integral role in RNA folding, the kinetics of stem-loop (un)folding has been extensively characterized using both experimental and computational approaches. Interestingly, these studies have reported vastly disparate timescales of (un)folding, which has been interpreted as evidence that (un)folding of even simple stem-loops occurs on a highly rugged conformational energy landscape. Because smFETs do not rely on fluorophore reporters of conformation or mechanical (un)folding forces, they provide a unique approach that has allowed us to directly monitor tens of thousands of (un)folding events of individual stem-loops at a 200 µs time resolution. Our results show that under our experimental conditions, stem-loops (un)fold over a 1-200 ms timescale during which they transition between ensembles of unfolded and folded conformations, the latter of which is composed of at least two sub-populations. The 1-200 ms timescale of (un)folding we observe here indicates that smFETs report on complete (un)folding trajectories in which unfolded conformations of the RNA spend long periods of time wandering the free-energy landscape before sampling one of several misfolded conformations or the natively folded conformation. Our findings highlight the extremely rugged landscape on which even the simplest RNA structural elements fold and demonstrate that smFETs are a unique and powerful approach for characterizing the conformational free-energy of RNA.

Charge Mapping of Pseudomonas aeruginosa Using a Hopping Mode Scanning Ion Conductance Microscopy Technique.

Scanning ion conductance microscopy (SICM) is a topographic imaging technique capable of probing biological samples in electrolyte conditions. SICM enhancements have enabled surface charge detection based on voltage-dependent signals. Here, we show how the hopping mode SICM method (HP-SICM) can be used for rapid and minimally invasive surface charge mapping. We validate our method usingPseudomonas aeruginosaPA14 (PA) cells and observe a surface charge density of σPA = -2.0 ± 0.45 mC/m2 that is homogeneous within the ∼80 nm lateral scan resolution. This biological surface charge is detected from at least 1.7 µm above the membrane (395× the Debye length), and the long-range charge detection is attributed to electroosmotic amplification. We show that imaging with a nanobubble-plugged probe reduces perturbation of the underlying sample. We extend the technique to PA biofilms and observe a charge density exceeding -20 mC/m2. We use a solid-state calibration to quantify surface charge density and show that HP-SICM cannot be quantitatively described by a steady-state finite element model. This work contributes to the body of scanning probe methods that can uniquely contribute to microbiology and cellular biology.

mr-EBL: ultra-high sensitivity negative-tone electron beam resist for highly selective silicon etching and large-scale direct patterning of permanent structures.

Electron beam lithography (EBL) is the state-of-the-art technique for rapid prototyping of nanometer-scale devices. Even so, processing speeds remain limited for the highest resolution patterning. Here, we establish Mr-EBL as the highest throughput negative tone electron-beam-sensitive resist. The 10µC cm-2dose requirement enables fabricating a 100 mm2photonic diffraction grating in a ten minute EBL process. Optimized processing conditions achieve a critical resolution of 75 nm with 3× faster write speeds than SU-8 and 1-2 orders of magnitude faster write speeds than maN-2400 and hydrogen silsesquioxane. Notably, these conditions significantly differ from the manufacturers' recommendations for the recently commercialized Mr-EBL resist. We demonstrate Mr-EBL to be a robust negative etch mask by etching silicon trenches with aspect ratios of 10 and near-vertical sidewalls. Furthermore, our optimized processing conditions are suitable to direct patterning on integrated circuits or delicate nanofabrication stacks, in contrast to other negative tone EBL resists. In conclusion, Mr-EBL is a highly attractive EBL resist for rapid prototyping in nanophotonics, MEMS, and fluidics.

Single-channel recordings of RyR1 at microsecond resolution in CMOS-suspended membranes.

Single-channel recordings are widely used to explore functional properties of ion channels. Typically, such recordings are performed at bandwidths of less than 10 kHz because of signal-to-noise considerations, limiting the temporal resolution available for studying fast gating dynamics to greater than 100 µs. Here we present experimental methods that directly integrate suspended lipid bilayers with high-bandwidth, low-noise transimpedance amplifiers based on complementary metal-oxide-semiconductor (CMOS) integrated circuits (IC) technology to achieve bandwidths in excess of 500 kHz and microsecond temporal resolution. We use this CMOS-integrated bilayer system to study the type 1 ryanodine receptor (RyR1), a Ca2+-activated intracellular Ca2+-release channel located on the sarcoplasmic reticulum. We are able to distinguish multiple closed states not evident with lower bandwidth recordings, suggesting the presence of an additional Ca2+ binding site, distinct from the site responsible for activation. An extended beta distribution analysis of our high-bandwidth data can be used to infer closed state flicker events as fast as 35 ns. These events are in the range of single-file ion translocations.

An Electrically Actuated, Carbon-Nanotube-Based Biomimetic Ion Pump.

Single-walled carbon nanotubes (SWCNTs) are well-established transporters of electronic current, electrolyte, and ions. In this work, we demonstrate an electrically actuated biomimetic ion pump by combining these electronic and nanofluidic transport capabilities within an individual SWCNT device. Ion pumping is driven by a solid-state electronic input, as Coulomb drag coupling transduces electrical energy from solid-state charge along the SWCNT shell to electrolyte inside the SWCNT core. Short-circuit ionic currents, measured without an electrolyte potential difference, exceed 1 nA and scale larger with increasing ion concentrations through 1 M, demonstrating applicability under physiological (∼140 mM) and saltwater (∼600 mM) conditions. The interlayer coupling allows ionic currents to be tuned with the source-drain potential difference and electronic currents to be tuned with the electrolyte potential difference. This combined electronic-nanofluidic SWCNT device presents intriguing applications as a biomimetic ion pump or component of an artificial membrane.

Wavelet Denoising of High-Bandwidth Nanopore and Ion-Channel Signals.

Recent work has pushed the noise-limited bandwidths of solid-state nanopore conductance recordings to more than 5 MHz and of ion channel conductance recordings to more than 500 kHz through the use of integrated complementary metal-oxide-semiconductor (CMOS) integrated circuits. Despite the spectral spread of the pulse-like signals that characterize these recordings when a sinusoidal basis is employed, Bessel filters are commonly used to denoise these signals to acceptable signal-to-noise ratios (SNRs) at the cost of losing many of the faster temporal features. Here, we report improvements to the SNR that can be achieved using wavelet denoising instead of Bessel filtering. When combined with state-of-the-art high-bandwidth CMOS recording instrumentation, we can reduce baseline noise levels by over a factor of 4 compared to a 2.5 MHz Bessel filter while retaining transient properties in the signal comparable to this filter bandwidth. Similarly, for ion-channel recordings, we achieve a temporal response better than a 100 kHz Bessel filter with a noise level comparable to that achievable with a 25 kHz Bessel filter. Improvements in SNR can be used to achieve robust statistical analyses of these recordings, which may provide important insights into nanopore translocation dynamics and mechanisms of ion-channel function.

Nanoscale Fluid Vortices and Nonlinear Electroosmotic Flow Drive Ion Current Rectification in the Presence of Concentration Gradients.

Ion current rectification (ICR) is a transport phenomenon in which an electrolyte conducts unequal currents at equal and opposite voltages. Here, we show that nanoscale fluid vortices and nonlinear electroosmotic flow (EOF) drive ICR in the presence of concentration gradients. The same EOF can yield negative differential resistance (NDR), in which current decreases with increasing voltage. A finite element model quantitatively reproduces experimental ICR and NDR recorded across glass nanopipettes under concentration gradients. The model demonstrates that spatial variations of electrical double layer properties induce the nanoscale vortices and nonlinear EOF. Experiments are performed in conditions directly related to scanning probe imaging and show that quantitative understanding of nanoscale transport under concentration gradients requires accounting for EOF. This characterization of nanopipette transport physics will benefit diverse experimentation, pushing the resolution limits of chemical and biophysical recordings.

A 512-Pixel, 51-kHz-Frame-Rate, Dual-Shank, Lens-less, Filter-less Single Photon Avalanche Diode CMOS Neural Imaging Probe.

We present an implantable single photon shank-based imager, monolithically integrated onto a single CMOS IC. The imager comprises of 512 single photon avalanche diodes distributed along two shanks, with a 6-bit depth in-pixel memory and an on-chip digital-to-time converter. To scale down the system to a minimally invasive form factor, we substitute optical filtering and focusing elements with a time-gated, angle-sensitive detection system. The imager computationally reconstructs the position of fluorescent sources within a three-dimensional volume of 3.4 mm × 600 µm × 400 µm.

Developing Next-generation Brain Sensing Technologies - A Review.

Advances in sensing technology raise the possibility of creating neural interfaces that can more effectively restore or repair neural function and reveal fundamental properties of neural information processing. To realize the potential of these bioelectronic devices, it is necessary to understand the capabilities of emerging technologies and identify the best strategies to translate these technologies into products and therapies that will improve the lives of patients with neurological and other disorders. Here we discuss emerging technologies for sensing brain activity, anticipated challenges for translation, and perspectives for how to best transition these technologies from academic research labs to useful products for neuroscience researchers and human patients.

Ultrafast Graphene Light Emitters.

Ultrafast electrically driven nanoscale light sources are critical components in nanophotonics. Compound semiconductor-based light sources for the nanophotonic platforms have been extensively investigated over the past decades. However, monolithic ultrafast light sources with a small footprint remain a challenge. Here, we demonstrate electrically driven ultrafast graphene light emitters that achieve light pulse generation with up to 10 GHz bandwidth across a broad spectral range from the visible to the near-infrared. The fast response results from ultrafast charge-carrier dynamics in graphene and weak electron-acoustic phonon-mediated coupling between the electronic and lattice degrees of freedom. We also find that encapsulating graphene with hexagonal boron nitride (hBN) layers strongly modifies the emission spectrum by changing the local optical density of states, thus providing up to 460% enhancement compared to the gray-body thermal radiation for a broad peak centered at 720 nm. Furthermore, the hBN encapsulation layers permit stable and bright visible thermal radiation with electronic temperatures up to 2000 K under ambient conditions as well as efficient ultrafast electronic cooling via near-field coupling to hybrid polaritonic modes under electrical excitation. These high-speed graphene light emitters provide a promising path for on-chip light sources for optical communications and other optoelectronic applications.

CMOS-Integrated Low-Noise Junction Field-Effect Transistors for Bioelectronic Applications.

In this work, we present a CMOS-integrated low-noise junction field-effect transistor (JFET) developed in a standard 0.18 pm CMOS process. These JFETs reduce input-referred flicker noise power by more than a factor of 10 when compared to equally sized n-channel MOS devices by eliminating oxide interfaces in contact with the channel. We show that this improvement in device performance translates into a factor-of-10 reduction in the input-referred noise of integrated CMOS operational amplifiers when JFET devices are used at the input, significant for many applications in bioelectronics.

Single-Walled Carbon Nanotubes: Mimics of Biological Ion Channels.

Here we report on the ion conductance through individual, small diameter single-walled carbon nanotubes. We find that they are mimics of ion channels found in natural systems. We explore the factors governing the ion selectivity and permeation through single-walled carbon nanotubes by considering an electrostatic mechanism built around a simplified version of the Gouy-Chapman theory. We find that the single-walled carbon nanotubes preferentially transported cations and that the cation permeability is size-dependent. The ionic conductance increases as the absolute hydration enthalpy decreases for monovalent cations with similar solid-state radii, hydrated radii, and bulk mobility. Charge screening experiments using either the addition of cationic or anionic polymers, divalent metal cations, or changes in pH reveal the enormous impact of the negatively charged carboxylates at the entrance of the single-walled carbon nanotubes. These observations were modeled in the low-to-medium concentration range (0.1-2.0 M) by an electrostatic mechanism that mimics the behavior observed in many biological ion channel-forming proteins. Moreover, multi-ion conduction in the high concentration range (>2.0 M) further reinforces the similarity between single-walled carbon nanotubes and protein ion channels.

Complementary Metal-Oxide-Semiconductor Integrated Carbon Nanotube Arrays: Toward Wide-Bandwidth Single-Molecule Sensing Systems.

There is strong interest in realizing genomic molecular diagnostic platforms that are label-free, electronic, and single-molecule. One attractive transducer for such efforts is the single-molecule field-effect transistor (smFET), capable of detecting a single electronic charge and realized with a point-functionalized exposed-gate one-dimensional carbon nanotube field-effect device. In this work, smFETs are integrated directly onto a custom complementary metal-oxide-semiconductor chip, which results in an array of up to 6000 devices delivering a measurement bandwidth of 1 MHz. In a first exploitation of these high-bandwidth measurement capabilities, point functionalization through electrochemical oxidation of the devices is observed with microsecond temporal resolution, which reveals complex reaction pathways with resolvable scattering signatures. High-rate random telegraph noise is detected in certain oxidized devices, further illustrating the measurement capabilities of the platform.

Measurement of DNA Translocation Dynamics in a Solid-State Nanopore at 100 ns Temporal Resolution.

Despite the potential for nanopores to be a platform for high-bandwidth study of single-molecule systems, ionic current measurements through nanopores have been limited in their temporal resolution by noise arising from poorly optimized measurement electronics and large parasitic capacitances in the nanopore membranes. Here, we present a complementary metal-oxide-semiconductor (CMOS) nanopore (CNP) amplifier capable of low noise recordings at an unprecedented 10 MHz bandwidth. When integrated with state-of-the-art solid-state nanopores in silicon nitride membranes, we achieve an SNR of greater than 10 for ssDNA translocations at a measurement bandwidth of 5 MHz, which represents the fastest ion current recordings through nanopores reported to date. We observe transient features in ssDNA translocation events that are as short as 200 ns, which are hidden even at bandwidths as high as 1 MHz. These features offer further insights into the translocation kinetics of molecules entering and exiting the pore. This platform highlights the advantages of high-bandwidth translocation measurements made possible by integrating nanopores and custom-designed electronics.

Single-Molecule Reaction Chemistry in Patterned Nanowells.

A new approach to synthetic chemistry is performed in ultraminiaturized, nanofabricated reaction chambers. Using lithographically defined nanowells, we achieve single-point covalent chemistry on hundreds of individual carbon nanotube transistors, providing robust statistics and unprecedented spatial resolution in adduct position. Each device acts as a sensor to detect, in real-time and through quantized changes in conductance, single-point functionalization of the nanotube as well as consecutive chemical reactions, molecular interactions, and molecular conformational changes occurring on the resulting single-molecule probe. In particular, we use a set of sequential bioconjugation reactions to tether a single-strand of DNA to the device and record its repeated, reversible folding into a G-quadruplex structure. The stable covalent tether allows us to measure the same molecule in different solutions, revealing the characteristic increased stability of the G-quadruplex structure in the presence of potassium ions (K(+)) versus sodium ions (Na(+)). Nanowell-confined reaction chemistry on carbon nanotube devices offers a versatile method to isolate and monitor individual molecules during successive chemical reactions over an extended period of time.

Integrated nanopore sensing platform with sub-microsecond temporal resolution.

Nanopore sensors have attracted considerable interest for high-throughput sensing of individual nucleic acids and proteins without the need for chemical labels or complex optics. A prevailing problem in nanopore applications is that the transport kinetics of single biomolecules are often faster than the measurement time resolution. Methods to slow down biomolecular transport can be troublesome and are at odds with the natural goal of high-throughput sensing. Here we introduce a low-noise measurement platform that integrates a complementary metal-oxide semiconductor (CMOS) preamplifier with solid-state nanopores in thin silicon nitride membranes. With this platform we achieved a signal-to-noise ratio exceeding five at a bandwidth of 1 MHz, which to our knowledge is the highest bandwidth nanopore recording to date. We demonstrate transient signals as brief as 1 µs from short DNA molecules as well as current signatures during molecular passage events that shed light on submolecular DNA configurations in small nanopores.

Whole-blood immunoassay for γH2AX as a radiation biodosimetry assay with minimal sample preparation.

The current state of the art in high-throughput minimally invasive radiation biodosimetry involves the collection of samples in the field and analysis at a centralized facility. We have developed a simple biological immunoassay for radiation exposure that could extend this analysis out of the laboratory into the field. Such a forward placed assay would facilitate triage of a potentially exposed population. The phosphorylation and localization of the histone H2AX at double-stranded DNA breaks has already been proven to be an adequate surrogate assay for reporting DNA damage proportional to radiation dose. Here, we develop an assay for phosphorylated H2AX directed against minimally processed sample lysates. We conduct preliminary verification of H2AX phosphorylation using irradiated mouse embryo fibroblast cultures. Additional dosimetry is performed using human blood samples irradiated ex vivo. The assay reports H2AX phosphorylation in human blood samples in response to ionizing radiation over a range of 0-5 Gy in a linear fashion, without requiring filtering, enrichment, or purification of the blood sample.

Improving signal-to-noise performance for DNA translocation in solid-state nanopores at MHz bandwidths.

DNA sequencing using solid-state nanopores is, in part, impeded by the relatively high noise and low bandwidth of the current state-of-the-art translocation measurements. In this Letter, we measure the ion current noise through sub 10 nm thick Si3N4 nanopores at bandwidths up to 1 MHz. At these bandwidths, the input-referred current noise is dominated by the amplifier's voltage noise acting across the total capacitance at the amplifier input. By reducing the nanopore chip capacitance to the 1-5 pF range by adding thick insulating layers to the chip surface, we are able to transition to a regime in which input-referred current noise (∼ 117-150 pArms at 1 MHz in 1 M KCl solution) is dominated by the effects of the input capacitance of the amplifier itself. The signal-to-noise ratios (SNRs) reported here range from 15 to 20 at 1 MHz for dsDNA translocations through nanopores with diameters from 4 to 8 nm with applied voltages from 200 to 800 mV. Further advances in bandwidth and SNR will require new amplifier designs that reduce both input capacitance and input-referred amplifier noise.

Graphene field-effect transistors with gigahertz-frequency power gain on flexible substrates.

The development of flexible electronics operating at radio-frequencies (RF) requires materials that combine excellent electronic performance and the ability to withstand high levels of strain. In this work, we fabricate graphene field-effect transistors (GFETs) on flexible substrates from graphene grown by chemical vapor deposition (CVD). Our devices demonstrate unity-current-gain frequencies, f(T), and unity-power-gain frequencies, f(max), up to 10.7 GHz and 3.7 GHz, respectively, with strain limits of 1.75%. These devices represent the only reported technology to achieve gigahertz-frequency power gain at strain levels above 0.5%. As such, they demonstrate the potential of CVD graphene to enable a broad range of flexible electronic technologies which require both high flexibility and RF operation.

Single ion channel recordings with CMOS-anchored lipid membranes.

We present single-ion-channel recordings performed with biomimetic lipid membranes which are directly attached to the surface of a complementary metal-oxide-semiconductor (CMOS) preamplifier chip. With this system we resolve single-channel currents from several types of bacterial ion channels, including fluctuations of a single alamethicin channel at a bandwidth of 1 MHz which represent the fastest single-ion-channel recordings reported to date. The platform is also used for high-resolution α-hemolysin nanopore recordings. These results illustrate the high signal fidelity, fine temporal resolution, small geometry, and multiplexed integration which can be achieved by leveraging integrated semiconductor platforms for advanced ion channel interfaces.