Narrowing Signal Distribution by Adamantane Derivatization for Amino Acid Identification Using an α-Hemolysin Nanopore.

The rapid progress in nanopore sensing has sparked interest in protein sequencing. Despite recent notable advancements in amino acid recognition using nanopores, chemical modifications usually employed in this process still need further refinements. One of the challenges is to enhance the chemical specificity to avoid downstream misidentification of amino acids. By employing adamantane to label proteinogenic amino acids, we developed an approach to fingerprint individual amino acids using the wild-type α-hemolysin nanopore. The unique structure of adamantane-labeled amino acids (ALAAs) improved the spatial resolution, resulting in distinctive current signals. Various nanopore parameters were explored using a machine-learning algorithm and achieved a validation accuracy of 81.3% for distinguishing nine selected amino acids. Our results not only advance the effort in single-molecule protein characterization using nanopores but also offer a potential platform for studying intrinsic and variant structures of individual molecules.

Engineering Biological Nanopore Approaches toward Protein Sequencing.

Biotechnological innovations have vastly improved the capacity to perform large-scale protein studies, while the methods we have for identifying and quantifying individual proteins are still inadequate to perform protein sequencing at the single-molecule level. Nanopore-inspired systems devoted to understanding how single molecules behave have been extensively developed for applications in genome sequencing. These nanopore systems are emerging as prominent tools for protein identification, detection, and analysis, suggesting realistic prospects for novel protein sequencing. This review summarizes recent advances in biological nanopore sensors toward protein sequencing, from the identification of individual amino acids to the controlled translocation of peptides and proteins, with attention focused on device and algorithm development and the delineation of molecular mechanisms with the aid of simulations. Specifically, the review aims to offer recommendations for the advancement of nanopore-based protein sequencing from an engineering perspective, highlighting the need for collaborative efforts across multiple disciplines. These efforts should include chemical conjugation, protein engineering, molecular simulation, machine-learning-assisted identification, and electronic device fabrication to enable practical implementation in real-world scenarios.

Nanopore sensing: A physical-chemical approach.

Protein nanopores have emerged as an important class of sensors for the understanding of biophysical processes, such as molecular transport across membranes, and for the detection and characterization of biopolymers. Here, we trace the development of these sensors from the Coulter counter and squid axon studies to the modern applications including exquisite detection of small volume changes and molecular reactions at the single molecule (or reactant) scale. This review focuses on the chemistry of biological pores, and how that influences the physical chemistry of molecular detection.

Laser-based temperature control to study the roles of entropy and enthalpy in polymer-nanopore interactions.

Single-molecule approaches for probing the free energy of confinement for polymers in a nanopore environment are critical for the development of nanopore biosensors. We developed a laser-based nanopore heating approach to monitor the free energy profiles of such a single-molecule sensor. Using this approach, we measure the free energy profiles of two distinct polymers, polyethylene glycol and water-soluble peptides, as they interact with the nanopore sensor. Polyethylene glycol demonstrates a retention mechanism dominated by entropy with little sign of interaction with the pore, while peptides show an enthalpic mechanism, which can be attributed to physisorption to the nanopore (e.g., hydrogen bonding). To manipulate the energetics, we introduced thiolate-capped gold clusters [Au25(SG)18] into the pore, which increases the charge and leads to additional electrostatic interactions that help dissect the contribution that enthalpy and entropy make in this modified environment. These observations provide a benchmark for optimization of single-molecule nanopore sensors.

A comparison of ion channel current blockades caused by individual poly(ethylene glycol) molecules and polyoxometalate nanoclusters.

Proteinaceous nanometer-scale pores have been used to detect and physically characterize many different types of analytes at the single-molecule limit. The method is based on the ability to measure the transient reduction in the ionic channel conductance caused by molecules that partition into the pore. The distribution of blockade depth amplitudes and residence times of the analytes in the pore are used to physically and chemically characterize them. Here we compare the current blockade events caused by flexible linear polymers of ethylene glycol (PEGs) and structurally well-defined tungsten polyoxymetallate nanoparticles in the nanopores formed by Staphylococcus aureusα-hemolysin and Aeromonas hydrophila aerolysin. Surprisingly, the variance in the ionic current blockade depth values for the relatively rigid metallic nanoparticles is much greater than that for the flexible PEGs, possibly because of multiple charged states of the polyoxymetallate clusters.

The Utility of Nanopore Technology for Protein and Peptide Sensing.

Resistive pulse nanopore sensing enables label-free single-molecule analysis of a wide range of analytes. An increasing number of studies have demonstrated the feasibility and usefulness of nanopore sensing for protein and peptide characterization. Nanopores offer the potential to study a variety of protein-related phenomena that includes unfolding kinetics, differences in unfolding pathways, protein structure stability, and free-energy profiles of DNA-protein and RNA-protein binding. In addition to providing a tool for fundamental protein characterization, nanopores have also been used as highly selective protein detectors in various solution mixtures and conditions. This review highlights these and other developments in the area of nanopore-based protein and peptide detection.

Determining the Physical Properties of Molecules with Nanometer-Scale Pores.

Nanometer-scale pores have been developed for the detection, characterization, and quantification of a wide range of analytes (e.g., ions, polymers, proteins, anthrax toxins, neurotransmitters, and synthetic nanoparticles) and for DNA sequencing. We describe the key requirements that made this method possible and how the technique evolved. Finally, we show that, despite sound theoretical work, which advanced both the conceptual framework and quantitative capability of the method, there are still unresolved questions that need to be addressed to further improve the technique.

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 Nanopore Spectrometry for Peptide Detection.

Sensing and characterization of water-soluble peptides is of critical importance in a wide variety of bioapplications. Single molecule nanopore spectrometry (SMNS) is based on the idea that one can use biological protein nanopores to resolve different sized molecules down to limits set by the blockade duration and noise. Previous work has shown that this enables discrimination between polyethylene glycol (PEG) molecules that differ by a single monomer unit. This paper describes efforts to extend SMNS to a variety of biologically relevant, water-soluble peptides. We describe the use of Au25(SG)18 clusters, previously shown to improve PEG detection, to increase the on- and off-rate of peptides to the pore. In addition, we study the role that fluctuations play in the single molecule nanopore spectrometry (SMNS) methodology and show that modifying solution conditions to increase peptide flexibility (via pH or chaotropic salt) leads to a nearly 2-fold reduction in the current blockade fluctuations and a corresponding narrowing of the peaks in the blockade distributions. Finally, a model is presented that connects the current blockade depths to the mass of the peptides, which shows that our enhanced SMNS detection improves the mass resolution of the nanopore sensor more than 2-fold for the largest cationic peptides studied.

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.

Biochip for the Detection of Bacillus anthracis Lethal Factor and Therapeutic Agents against Anthrax Toxins.

Tethered lipid bilayer membranes (tBLMs) have been used in many applications, including biosensing and membrane protein structure studies. This report describes a biosensor for anthrax toxins that was fabricated through the self-assembly of a tBLM with B. anthracis protective antigen ion channels that are both the recognition element and electrochemical transducer. We characterize the sensor and its properties with electrochemical impedance spectroscopy and surface plasmon resonance. The sensor shows a sensitivity similar to ELISA and can also be used to rapidly screen for molecules that bind to the toxins and potentially inhibit their lethal effects.

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.

Attachment of a diruthenium compound to Au and SiO2/Si surfaces by "click" chemistry.

Fabrication of electrodes with functionalized properties is of interest in many electronic applications with the surface impacting the electrical and electronic properties of devices. We report the formation of molecular monolayers containing a redox-active diruthenium(II,III) compound to gold and silicon surfaces via "click" chemistry. The use of Cu-catalyzed azide-alkyne cycloaddition enables modular design of molecular surfaces and interfaces and allows for a variety of substrates to be functionalized. Attachment of the diruthenium compound is monitored by using infrared and photoelectron spectroscopies. The highest occupied molecular (or system) orbital of the "clicked-on" diruthenium is clearly seen in the photoemission measurements and is mainly attributed to the presence of the Ru atoms. The "click" attachment is robust and provides a route to investigate the evolution of the electronic structure and properties of novel molecules attached to a variety of electrodes. The ability to attach this redox-active Ru molecule onto SiO2 and Au surfaces is important for the development of functional molecular devices such as charge-based memory devices.

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.

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.

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.

Electrical and physical characterization of bilayer carboxylic acid-functionalized molecular layers.

We have used flip chip lamination (FCL) to form monolayer and bilayer molecular junctions of carboxylic acid-containing molecules with Cu atom incorporation. Carboxylic acid-terminated monolayers are self-assembled onto ultrasmooth Au by using thiol chemistry and grafted onto n-type Si. Prior to junction formation, monolayers are physically characterized by using polarized infrared absorption spectroscopy, X-ray photoelectron spectroscopy, and near-edge X-ray absorption fine structure spectroscopy, confirming the molecular quality and functional group termination. FCL was used to form monolayer junctions onto H-terminated Si or bilayer junctions of carboxylic acid monolayers on Au and Si. From the electrical measurements, we find that the current through the junction is attenuated as the effective molecular length within the junction increases, indicating that molecules are electrically active within the junction. We find that the electronic transport through the bilayer junction saturates at very thick effective distances possibly because of another electron-transport mechanism that is not nonresonant tunneling as a result of trapped defects or sequential tunneling. In addition, bilayer junctions are fabricated with and without Cu atoms, and we find that the electron transport is not distinguishably different when Cu atoms are within the bilayer.

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.