Microsecond-timescale simulations suggest 5-HT-mediated preactivation of the 5-HT3A serotonin receptor.

Aided by efforts to improve their speed and efficiency, molecular dynamics (MD) simulations provide an increasingly powerful tool to study the structure-function relationship of pentameric ligand-gated ion channels (pLGICs). However, accurate reporting of the channel state and observation of allosteric regulation by agonist binding with MD remains difficult due to the timescales necessary to equilibrate pLGICs from their artificial and crystalized conformation to a more native, membrane-bound conformation in silico. Here, we perform multiple all-atom MD simulations of the homomeric 5-hydroxytryptamine 3A (5-HT3A) serotonin receptor for 15 to 20 µs to demonstrate that such timescales are critical to observe the equilibration of a pLGIC from its crystalized conformation to a membrane-bound conformation. These timescales, which are an order of magnitude longer than any previous simulation of 5-HT3A, allow us to observe the dynamic binding and unbinding of 5-hydroxytryptamine (5-HT) (i.e., serotonin) to the binding pocket located on the extracellular domain (ECD) and allosteric regulation of the transmembrane domain (TMD) from synergistic 5-HT binding. While these timescales are not long enough to observe complete activation of 5-HT3A, the allosteric regulation of ion gating elements by 5-HT binding is indicative of a preactive state, which provides insight into molecular mechanisms that regulate channel activation from a resting state. This mechanistic insight, enabled by microsecond-timescale MD simulations, will allow a careful examination of the regulation of pLGICs at a molecular level, expanding our understanding of their function and elucidating key structural motifs that can be targeted for therapeutic regulation.

Rapid, quantitative therapeutic screening for Alzheimer's enzymes enabled by optimal signal transduction with transistors.

We show that commercially sourced n-channel silicon field-effect transistors (nFETs) operating above their threshold voltage with closed loop feedback to maintain a constant channel current allow a pH readout resolution of (7.2 ± 0.3) × 10-3 at a bandwidth of 10 Hz, or ≈3-fold better than the open loop operation commonly employed by integrated ion-sensitive field-effect transistors (ISFETs). We leveraged the improved nFET performance to measure the change in solution pH arising from the activity of a pathological form of the kinase Cdk5, an enzyme implicated in Alzheimer's disease, and showed quantitative agreement with previous measurements. The improved pH resolution was realized while the devices were operated in a remote sensing configuration with the pH sensing element off-chip and connected electrically to the FET gate terminal. We compared these results with those measured by using a custom-built dual-gate 2D field-effect transistor (dg2DFET) fabricated with 2D semi-conducting MoS2 channels and a signal amplification of 8. Under identical solution conditions the nFET performance approached the dg2DFETs pH resolution of (3.9 ± 0.7) × 10-3. Finally, using the nFETs, we demonstrated the effectiveness of a custom polypeptide, p5, as a therapeutic agent in restoring the function of Cdk5. We expect that the straight-forward modifications to commercially sourced nFETs demonstrated here will lower the barrier to widespread adoption of these remote-gate devices and enable sensitive bioanalytical measurements for high throughput screening in drug discovery and precision medicine applications.

The Role of Lipid Interactions in Simulations of the α-Hemolysin Ion-Channel-Forming Toxin.

Molecular dynamics simulations were performed to describe the function of the ion-channel-forming toxin α-hemolysin (αHL) in lipid membranes that were composed of either 1,2-diphytanoyl-sn-glycero-3-phospho-choline or 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-choline. The simulations highlight the importance of lipid type in maintaining αHL structure and function, enabling direct comparison to experiments for biosensing applications. We determined that although the two lipids studied are similar in structure, 1,2-diphytanoyl-sn-glycero-3-phospho-choline membranes better match the hydrophobic thickness of αHL compared to 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-choline membranes. This hydrophobic match is essential to maintaining proper alignment of ß-sheet loops at the trans entrance of αHL, which, when disrupted, creates an additional constriction to ion flow that decreases the channel current below experimental values and creates greater variability in channel conductance. Agreement with experiments was further improved with sufficient lipid membrane equilibration and allowed the discrimination of subtle αHL conduction states with lipid type. Finally, we explore the effects of truncating the extramembrane cap of αHL and its role in maintaining proper alignment of αHL in the membrane and channel conductance. Our results demonstrate the essential role of lipid type and lipid-protein interactions in simulations of αHL and will considerably improve the interpretation of experimental data.

Analytical applications for pore-forming proteins.

Proteinaceous nanometer-scale pores are ubiquitous in biology. The canonical ionic channels (e.g., those that transport Na(+), K(+), Ca(2+), and Cl(-) across cell membranes) play key roles in many cellular processes, including nerve and muscle activity. Another class of channels includes bacterial pore-forming toxins, which disrupt cell function, and can lead to cell death. We describe here the recent development of these toxins for a wide range of biological sensing applications. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale.

Novel nanofluidic chemical cells based on self-assembled solid-state SiO2 nanotubes.

Novel nanofluidic chemical cells based on self-assembled solid-state SiO2 nanotubes on silicon-on-insulator (SOI) substrate have been successfully fabricated and characterized. The vertical SiO2 nanotubes with a smooth cavity are built from Si nanowires which were epitaxially grown on the SOI substrate. The nanotubes have rigid, dry-oxidized SiO2 walls with precisely controlled nanotube inner diameter, which is very attractive for chemical-/bio-sensing applications. No dispersion/aligning procedures were involved in the nanotube fabrication and integration by using this technology, enabling a clean and smooth chemical cell. Such a robust and well-controlled nanotube is an excellent case of developing functional nanomaterials by leveraging the strength of top-down lithography and the unique advantage of bottom-up growth. These solid, smooth, clean SiO2 nanotubes and nanofluidic devices are very encouraging and attractive in future bio-medical applications, such as single molecule sensing and DNA sequencing.

Single Molecule Discrimination of Heteropolytungstates and Their Isomers in Solution with a Nanometer-Scale Pore.

We report a new method to identify metallic nanoclusters (polyoxometalate structures) in solution at the single molecule limit using a nanometer-scale pore. The technique allows the measurement of polyoxometalates with over 2 orders of magnitude lower analyte concentration than conventional analytical chemistry tools. Furthermore, pH-dependent structural changes in phosphotungstic acid are measured with protein nanopores and validated with NMR. We further demonstrate that the method can also discriminate [PW9O34](9-) structural isomers. The results suggest this technique can serve as a complementary approach to traditional methods.

MOSAIC: A Modular Single-Molecule Analysis Interface for Decoding Multistate Nanopore Data.

Biological and solid-state nanometer-scale pores are the basis for numerous emerging analytical technologies for use in precision medicine. We developed Modular Single-Molecule Analysis Interface (MOSAIC), an open source analysis software that improves the accuracy and throughput of nanopore-based measurements. Two key algorithms are implemented: ADEPT, which uses a physical model of the nanopore system to characterize short-lived events that do not reach their steady-state current, and CUSUM+, a version of the cumulative sum statistical method optimized for longer events that do. We show that ADEPT detects previously unreported conductance states that occur as double-stranded DNA translocates through a 2.4 nm solid-state nanopore and reveals new interactions between short single-stranded DNA and the vestibule of a biological pore. These findings demonstrate the utility of MOSAIC and the ADEPT algorithm, and offer a new tool that can improve the analysis of nanopore-based measurements.

Theory of polymer-nanopore interactions refined using molecular dynamics simulations.

Molecular dynamics simulations were used to refine a theoretical model that describes the interaction of single polyethylene glycol (PEG) molecules with α-hemolysin (αHL) nanopores. The simulations support the underlying assumptions of the model, that PEG decreases the pore conductance by binding cations (which reduces the number of mobile ions in the pore) and by volume exclusion, and provide bounds for fits to new experimental data. Estimation of cation binding indicates that four monomers coordinate a single K(+) in a crown-ether-like structure, with, on average, 1.5 cations bound to a PEG 29-mer at a bulk electrolyte concentration of 4 M KCl. Additionally, PEG is more cylindrical and has a larger cross-section area in the pore than in solution, although its volume is similar. Two key experimental quantities of PEG are described by the model: the ratio of single channel current in the presence of PEG to that in the polymer's absence (blockade depth) and the mean residence time of PEG in the pore. The refined theoretical model is simultaneously fit to the experimentally determined current blockade depth and the mean residence times for PEGs with 15 to 45 monomers, at applied transmembrane potentials of -40 to -80 mV and for three electrolyte concentrations. The model estimates the free energy of the PEG-cation complexes to be -5.3 kBT. Finally the entropic penalty of confining PEG to the pore is found to be inversely proportional to the electrolyte concentration.

Temperature sculpting in yoctoliter volumes.

The ability to perturb large ensembles of molecules from equilibrium led to major advances in understanding reaction mechanisms in chemistry and biology. Here, we demonstrate the ability to control, measure, and make use of rapid temperature changes in fluid volumes that are commensurate with the size of single molecules. The method is based on attaching gold nanoparticles to a single nanometer-scale pore formed by a protein ion channel. Visible laser light incident on the nanoparticles causes a rapid and large increase of the adjacent solution temperature, which is estimated from the change in the nanopore ionic conductance. The temperature shift also affects the ability of individual molecules to enter into and interact with the nanopore. This technique could significantly improve sensor systems and force measurements based on single nanopores, thereby enabling a method for single molecule thermodynamics and kinetics.

Anthrax toxin-induced rupture of artificial lipid bilayer membranes.

We demonstrate experimentally that anthrax toxin complexes rupture artificial lipid bilayer membranes when isolated from the blood of infected animals. When the solution pH is temporally acidified to mimic that process in endosomes, recombinant anthrax toxin forms an irreversibly bound complex, which also destabilizes membranes. The results suggest an alternative mechanism for the translocation of anthrax toxin into the cytoplasm.

Significantly improved trapping lifetime of nanoparticles in an optical trap using feedback control.

We demonstrate an increase in trapping lifetime for optically trapped nanoparticles by more than an order of magnitude using feedback control, with no corresponding increase in beam power. Langevin dynamics simulations were used to design the control law, and this technique was then demonstrated experimentally using 100 nm gold particles and 350 nm silica particles. No particle escapes were detected with the controller on, leading to lower limits on the increase in lifetime for 100 nm gold particles of 26 times (at constant average beam power) and 22 times for 350 nm silica particles (with average beam power reduced by one-third). The approach described here can be combined with other techniques, such as counter propagating beams or higher-order optical modes, to trap the smallest nanoparticles and can be used to reduce optical heating of particles that are susceptible to photodamage, such as biological systems.

The effects of diffusion on an exonuclease/nanopore-based DNA sequencing engine.

Over 15 years ago, the ability to electrically detect and characterize individual polynucleotides as they are driven through a single protein ion channel was suggested as a potential method for rapidly sequencing DNA, base-by-base, in a ticker tape-like fashion. More recently, a variation of this method was proposed in which a nanopore would instead detect single nucleotides cleaved sequentially by an exonuclease enzyme in close proximity to one pore entrance. We analyze the exonuclease/nanopore-based DNA sequencing engine using analytical theory and computer simulations that describe nucleotide transport. The available data and analytical results suggest that the proposed method will be limited to reading <80 bases, imposed, in part, by the short lifetime each nucleotide spends in the vicinity of the detection element within the pore and the ability to accurately discriminate between the four mononucleotides.

High resolution voltammetric and field-effect transistor readout of carbon fiber microelectrode biosensors.

Rapid and sensitive pH measurements with increased spatiotemporal resolution are imperative to probe neurochemical signals and illuminate brain function. We interfaced carbon fiber microelectrode (CFME) sensors with both fast scan cyclic voltammetry (FSCV) and field-effect transistor (FET) transducers for dynamic pH measurements. The electrochemical oxidation and reduction of functional groups on the surface of CFMEs affect their response over a physiologically relevant pH range. When measured with FET transducers, the sensitivity of the measurements over the measured pH range was found to be (101 ± 18) mV, which exceeded the Nernstian value of 59 mV by approximately 70%. Finally, we validated the functionality of CFMEs as pH sensors with FSCV ex vivo in rat brain coronal slices with exogenously applied solutions of varying pH values indicating that potential in vivo study is feasible.

Optimal field-effect transistor operation for high-resolution biochemical measurements.

Field-effect transistors (FETs) are powerful tools for sensitive measurements of numerous biomarkers (e.g., proteins, nucleic acids, and antigen) and gaseous species. Most research studies in this field focused on building discrete devices with high performance. We show that instrumentation that is commonly used in multiple areas of physics and engineering can greatly improve the performance of measurement systems that embed FET-based transducers for biological applications. We review the state-of-the-art instrumentation in the field as applied to sensing with FETs. We show how high-performance dual-gate 2D FETs that we recently developed, when operated using closed-loop proportional-integral-derivative control, can drastically improve both the sensitivity and resolution. We further show that this closed-loop control approach can be extended to commonly used single-gate silicon FETs. The generalizability of the results will allow their application to virtually any previously developed FET-based sensor. Finally, we provide insight into further optimization and performance benefits that can be extracted by using the closed-loop feedback approach for applications in biosensing.

Reproducible Performance Improvements to Monolayer MoS2 Transistors through Exposed Material Forming Gas Annealing.

Metal-mediated exfoliation has been demonstrated as a promising approach for obtaining large-area flakes of two-dimensional (2D) materials to fabricate prototypical nanoelectronics. However, several processing challenges related to organic contamination at the interface of a 2D material and gate oxide must be overcome to realize robust devices with high yields. Here, we demonstrate an optimized process to realize high-performance field-effect transistor (FET) arrays from large-area (∼5000 µm2), monolayer MoS2 with a yield of 85%. A central element of this process is an exposed material forming gas anneal (EM-FGA) that results in uniform FET performance metrics (i.e., field-effect mobilities, threshold voltages, and contact performance). Complementary analytical measurements show that the EM-FGA process reduces deleterious channel doping effects by decreasing organic contamination while also reducing the prevalence of insulating molybdenum oxide, effectively improving the MoS2-gate oxide interface. The uniform FET performance metrics and high device yield achieved by applying the EM-FGA technique on large-area 2D material flakes will help advance the fabrication of complex 2D nanoelectronic devices and demonstrate the need for improved engineering of the 2D material-gate oxide interface.

Quantum capacitance-limited MoS2 biosensors enable remote label-free enzyme measurements.

We have demonstrated atomically thin, quantum capacitance-limited, field-effect transistors (FETs) that enable the detection of pH changes with 75-fold higher sensitivity (≈4.4 V per pH) over the Nernst value of 59 mV per pH at room temperature when used as a biosensor. The transistors, which are fabricated from monolayer films of MoS2, use a room temperature ionic liquid (RTIL) in place of a conventional oxide gate dielectric and exhibit very low intrinsic noise resulting in a pH resolution of 92 × 10-6 at 10 Hz. This high device performance, which is a function of the structure of our device, is achieved by remotely connecting the gate to a pH sensing element allowing the FETs to be reused. Because pH measurements are fundamentally important in biotechnology, the increased resolution demonstrated here will benefit numerous applications ranging from pharmaceutical manufacturing to clinical diagnostics. As an example, we experimentally quantified the function of the kinase Cdk5, an enzyme implicated in Alzheimer's disease, at concentrations that are 5-fold lower than physiological values, and with sufficient time-resolution to allow the estimation of both steady-state and kinetic parameters in a single experiment. The high sensitivity, increased resolution, and fast turnaround time of the measurements will allow the development of early diagnostic tools and novel therapeutics to detect and treat neurological conditions years before currently possible.

Quantifying short-lived events in multistate ionic current measurements.

We developed a generalized technique to characterize polymer-nanopore interactions via single channel ionic current measurements. Physical interactions between analytes, such as DNA, proteins, or synthetic polymers, and a nanopore cause multiple discrete states in the current. We modeled the transitions of the current to individual states with an equivalent electrical circuit, which allowed us to describe the system response. This enabled the estimation of short-lived states that are presently not characterized by existing analysis techniques. Our approach considerably improves the range and resolution of single-molecule characterization with nanopores. For example, we characterized the residence times of synthetic polymers that are three times shorter than those estimated with existing algorithms. Because the molecule's residence time follows an exponential distribution, we recover nearly 20-fold more events per unit time that can be used for analysis. Furthermore, the measurement range was extended from 11 monomers to as few as 8. Finally, we applied this technique to recover a known sequence of single-stranded DNA from previously published ion channel recordings, identifying discrete current states with subpicoampere resolution.

PEG-labeled nucleotides and nanopore detection for single molecule DNA sequencing by synthesis.

We describe a novel single molecule nanopore-based sequencing by synthesis (Nano-SBS) strategy that can accurately distinguish four bases by detecting 4 different sized tags released from 5'-phosphate-modified nucleotides. The basic principle is as follows. As each nucleotide is incorporated into the growing DNA strand during the polymerase reaction, its tag is released and enters a nanopore in release order. This produces a unique ionic current blockade signature due to the tag's distinct chemical structure, thereby determining DNA sequence electronically at single molecule level with single base resolution. As proof of principle, we attached four different length PEG-coumarin tags to the terminal phosphate of 2'-deoxyguanosine-5'-tetraphosphate. We demonstrate efficient, accurate incorporation of the nucleotide analogs during the polymerase reaction, and excellent discrimination among the four tags based on nanopore ionic currents. This approach coupled with polymerase attached to the nanopores in an array format should yield a single-molecule electronic Nano-SBS platform.