Quantitative Microscopic Observation of Base-Ligand Interactions via Hydrogen Bonds by Single-Molecule Counting.

Chemical properties have been based on statistical averages since the introduction of Avogadro's number. The lack of suitable methods for counting identified single molecules has posed challenges to counting statistics. The selectivity, affinity, and mode of hydrogen bonding between base and small molecules that make up DNA, which is vital for living organisms, have not yet been revealed at the single molecule level. Here, we show the quantitation of the above-mentioned parameters via single-molecule counting based on the combination of single-molecule electrical measurements and AI. The binding selectivity values of five ligands to four different base molecules were evaluated quantitatively by determining the ratio of the number of aggregates in a solution mixture of base molecules and a ligand. In addition, we show the ligand dependence of the mode and number of microscopic hydrogen bonds via single-molecule counting and quantum chemical calculations.

Self-Assembled Monolayers of Gemini-Type Amphiphilic Hexabenzocoronenes on Gold: Contribution of Their Triethylene Glycol Side Chains to Self-Assembly Formation.

We report on a two-dimensional self-assembled structure of a supramolecule with hydrophilic oligoethylene glycol (EG) units, which are capable of stronger electrostatic interactions than van der Waals (vdW) interactions between alkyl chains. For this purpose, hexabenzocoronene (HBC) with two hydrophobic dodecyl chains on one side of the HBC core and two hydrophilic triethylene glycol (TEG) chains on the other side of the HBC core (HBCGemini) and HBCGemini with a trinitrofluorenone (TNF) added to the end of one TEG chain (HBCTNFGemini) were employed. Scanning tunneling microscopy (STM) revealed the presence of multiple two-dimensional self-assembled structures in each of HBCGemini and HBCTNFGemini deposited on the gold substrate in vacuum. The role of polar functional groups in these observations is discussed based on semiempirical molecular orbital simulations. Two types of 2D organized structures of HBC-TEG were observed: one with rectangular and relatively dense unit cells and the other with nearly square and relatively sparse unit cells. In both organized structures, the phenyl group TEG units and alkyl chains were considered to be the main molecular interactions with each other. On the other hand, in HBCTNFGemini, three types of organized structures were observed, which could be explained by the mechanism of interdigitation of the TEG-containing side-chain moieties to form a dimeric core. The EG units are more flexible than the alkyl chains and thus can interact flexibly with the hydrophobic HBC core, and the glycol side chains facilitate the intermolecular interactions as well as the alkyl chains.

Review of the use of nanodevices to detect single molecules.

The development of methodologies to identify single molecules and/or to detect/monitor molecular behavior at the single-molecule level is one of the important research topics in chemistry and biology. In this review, we summarized the state-of-the-art of single molecule measurement methods and its latest applications using nanodevices integrated with molecular-size functional nanostructures, nanopores, nanogaps, and nanofluidic channels, which detect differences in chemical species, presence or absence of translational modifications, changes in steric structure, and changes in interactions between molecules. Besides these fundamental analytical achievements of molecular identification abilities, the latest applications include the single-molecule electrical sequencing, disease diagnosis, viral testing, single-molecule drug screening, and environmental monitoring. Finally, we added some discussion on the current status of single-molecule measurement as a method and technology to solve the problems to expand the future application needs of single-molecule measurement.

Salt Gradient Control of Translocation Dynamics in a Solid-State Nanopore.

Tuning capture rates and translocation time of analytes in solid-state nanopores are one of the major challenges for their use in detecting and analyzing individual nanoscale objects via ionic current measurements. Here, we report on the use of salt gradient for the fine control of capture-to-translocation dynamics in 300 nm sized SiNx nanopores. We demonstrated a decrease up to a factor of 3 in the electrophoretic speed of nanoparticles at the pore exit along with an over 3-fold increase in particle detection efficiency by subjecting a 5-fold ion concentration difference across the dielectric membrane. The improvement in the sensor performance was elucidated to be a result of the salt-gradient-mediated electric field and electroosmotic flow asymmetry at nanochannel orifices. The present findings can be used to enhance nanopore sensing capability for detecting biomolecules such as amyloids and proteins.

Rapid Discrimination of Extracellular Vesicles by Shape Distribution Analysis.

A rapid and simple cancer detection method independent of cancer type is an important technology for cancer diagnosis. Although the expression profiles of biological molecules contained in cancer cell-derived extracellular vesicles (EVs) are considered candidates for discrimination indexes to identify any cancerous cells in the body, it takes a certain amount of time to examine these expression profiles. Here, we report the shape distributions of EVs suspended in a solution and the potential of these distributions as a discrimination index to discriminate cancer cells. Distribution analysis is achieved by low-aspect-ratio nanopore devices that enable us to rapidly analyze EV shapes individually in solution, and the present results reveal a dependence of EV shape distribution on the type of cells (cultured liver, breast, and colorectal cancer cells and cultured normal breast cells) secreting EVs. The findings in this study provide realizability and experimental basis for a simple method to discriminate several types of cancerous cells based on rapid analyses of EV shape distributions.

Direct Observation of Distinctive Electronic States of Ferrocene Moieties in Ferrocene-Bridged Trisporphyrin on Au(111) Using Scanning Tunneling Microscopy/Spectroscopy.

The mechanical and electronic properties of ferrocene-bridged trisporphyrin (Fc2P3), which consists of three porphyrin units bridged via a ferrocene (Fc) moiety and adsorbed onto Au(111), were investigated by a pulse injection method using low-temperature scanning tunneling microscopy (STM) and scanning tunneling spectroscopy (STS). STM images revealed that Fc2P3 adsorbed onto Au(111) adopts a linear arrangement with a herringbone reconstruction structure comprising both Fc and porphyrin moieties. Sequential submolecular-resolution STM images and conformational analyses using a simple molecular model revealed that the ferrocene moiety acts as a molecular ball bearing. The electronic states originating from the ferrocene group were clearly observed by bias voltage-dependent STM images, the dI/dV map, and STS. Distinct electronic states are found approximately 1 eV below Ef in the STS spectrum acquired of Fc, whereas these are absent in the porphyrin spectrum.

Chemical-Labeling-Assisted Detection of Nucleobase Modifications by Quantum-Tunneling-Based Single-Molecule Sensing.

Quantum-tunneling-based DNA sensing is a single-molecule technique that promises direct mapping of nucleobase modifications. However, its applicability is seriously limited because of the small difference in conductivity between modified and unmodified nucleobases. Herein, a chemical labeling strategy is presented that facilitates the detection of modified nucleotides by quantum tunneling. We used 5-Formyl-2'-deoxyuridine as a model compound and demonstrated that chemical labeling dramatically alters its molecular conductance compared with that of canonical nucleotides; thus, facilitating statistical discrimination, which is impeded in the unlabeled state. This work introduces a chemical strategy that overcomes the intrinsic difficulty in quantum-tunneling-based modification analysis-the similarity of the molecular conductance of the nucleobases of interest.

COVID-19 Drug Discovery Using Intensive Approaches.

Since the infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was reported in China during December 2019, the coronavirus disease 2019 (COVID-19) has spread on a global scale, causing the World Health Organization (WHO) to issue a warning. While novel vaccines and drugs that target SARS-CoV-2 are under development, this review provides information on therapeutics which are under clinical trials or are proposed to antagonize SARS-CoV-2. Based on the information gained from the responses to other RNA coronaviruses, including the strains that cause severe acute respiratory syndrome (SARS)-coronaviruses and Middle East respiratory syndrome (MERS), drug repurposing might be a viable strategy. Since several antiviral therapies can inhibit viral replication cycles or relieve symptoms, mechanisms unique to RNA viruses will be important for the clinical development of antivirals against SARS-CoV-2. Given that several currently marketed drugs may be efficient therapeutic agents for severe COVID-19 cases, they may be beneficial for future viral pandemics and other infections caused by RNA viruses when standard treatments are unavailable.

Significant epitranscriptomes in heterogeneous cancer.

Precision medicine places significant emphasis on techniques for the identification of DNA mutations and gene expression by deep sequencing of gene panels to obtain medical data. However, other diverse information that is not easily readable using bioinformatics, including RNA modifications, has emerged as a novel diagnostic and innovative therapy owing to its multifunctional aspects. It is suggested that this breakthrough innovation might open new avenues for the elucidation of uncharacterized cancer cellular functions to develop more precise medical applications. The functional characteristics and regulatory mechanisms of RNA modifications, ie, the epitranscriptome (ETR), which reflects RNA metabolism, remains unclear, mainly due to detection methods being limited. Recent studies have revealed that N6-methyl adenosine, the most common modification in mRNA in eukaryotes, is affected in various types of cancer and, in some cases, cancer stem cells, but also affects cellular responses to viral infections. The ETR can control cancer cell fate through mRNA splicing, stability, nuclear export, and translation. Here we report on the recent progress of ETR detection methods, and biological findings regarding the significance of ETR in cancer precision medicine.

Paving the way to single-molecule chemistry through molecular electronics.

Since our understanding of single-molecule junctions, in which single molecules are connected between nanoelectrodes, has deepened, we have paved the way to single-molecule chemistry. Herein, we review fundamental properties, including the number of molecules connected to the electrode, their structure and type, the bonding force between the single molecule and electrode and the thermopower and quantum interference in single-molecule junctions. Additionally, we review the application of single-molecule junctions to biomolecules. Finally, we explore single-molecule chemical reaction analysis, which is one direction of single-molecule junction research.

Volume discrimination of nanoparticles via electrical trapping using nanopores.

Electrophoretic capture of an oversized object on a solid-state nanopore is a useful approach for single-particle analyses via post electrical and optical measurements. Here we report on nanoparticle discriminations by the volume through combining this nanopore trap method with the cross-membrane ionic current measurements. We investigated ion transport through a pore channel being partially occluded by an electrophoretically-drawn nanoparticle at the orifice. We found distinct difference in the amount of current blockage by particles of different sizes. Multiphysics simulations revealed dominant contribution of particle volume over the other properties. We also demonstrated single-particle discriminations of two different sizes in a mixture solution. The present results demonstrate that this electrical capturing is a promising technique to immobilize a target at a single particle level that concomitantly offer wealth of information concerning their volume.

Identifying Single Viruses Using Biorecognition Solid-State Nanopores.

Immunosensing is a bioanalytical technique capable of selective detections of pathogens by utilizing highly specific and strong intermolecular interactions between recognition probes and antigens. Here, we exploited the molecular mechanism in artificial nanopores for selective single-virus identifications. We designed hemagglutinin antibody mimicking oligopeptides with a weak affinity to influenza A virus. By functionalizing the pore wall surface with the synthetic peptides, we rendered specificity to virion-nanopore interactions. The ligand binding thereof was found to perturb translocation dynamics of specific viruses in the nanochannel, which facilitated digital typing of influenza by the resistive pulse bluntness. As amino acid sequence degrees of freedom can potentially offer variety of recognition ability to the molecular probes, this peptide nanopore approach can be used as a versatile immunosensor with single-particle sensitivity that promises wide applications in bioanalysis including bacterial and viral screening to infectious disease diagnosis.

Identification of Individual Bacterial Cells through the Intermolecular Interactions with Peptide-Functionalized Solid-State Pores.

Bioinspired pore sensing for selective detection of flagellated bacteria was investigated. The Au micropore wall surface was modified with a synthetic peptide designed from toll-like receptor 5 (TLR5) to mimic the pathogen-recognition capability. We found that intermolecular interactions between the TLR5-derived recognition peptides and flagella induce ligand-specific perturbations in the translocation dynamics of Escherichia coli, which facilitated the discrimination between the wild-type and flagellin-deletion mutant (ΔfliC) by the resistive pulse patterns thereby demonstrating the sensing of bacteria at a single-cell level. These results provide a novel concept of utilizing weak intermolecular interactions as a recognition probes for single-cell microbial identification.

Rapid structural analysis of nanomaterials in aqueous solutions.

Rapid structural analysis of nanoscale matter in a liquid environment represents innovative technologies that reveal the identities and functions of biologically important molecules. However, there is currently no method with high spatio-temporal resolution that can scan individual particles in solutions to gain structural information. Here we report the development of a nanopore platform realizing quantitative structural analysis for suspended nanomaterials in solutions with a high z-axis and xy-plane spatial resolution of 35.8 ± 1.1 and 12 nm, respectively. We used a low thickness-to-diameter aspect ratio pore architecture for achieving cross sectional areas of analyte (i.e. tomograms). Combining this with multiphysics simulation methods to translate ionic current data into tomograms, we demonstrated rapid structural analysis of single polystyrene (Pst) beads and single dumbbell-like Pst beads in aqueous solutions.

Detecting Single-Nucleotides by Tunneling Current Measurements at Sub-MHz Temporal Resolution.

Label-free detection of single-nucleotides was performed by fast tunneling current measurements in a polar solvent at 1 MHz sampling rate using SiO2-protected Au nanoprobes. Short current spikes were observed, suggestive of trapping/detrapping of individual nucleotides between the nanoelectrodes. The fall and rise features of the electrical signatures indicated signal retardation by capacitance effects with a time constant of about 10 microseconds. The high temporal resolution revealed current fluctuations, reflecting the molecular conformation degrees of freedom in the electrode gap. The method presented in this work may enable direct characterizations of dynamic changes in single-molecule conformations in an electrode gap in liquid.

Impact of Water-Depletion Layer on Transport in Hydrophobic Nanochannels.

Recent experiments showed that by fabricating nanofluidic channels with hydrophobic materials, the measured amplitudes of both electroosmotic flow (EOF) and ionic current deviated significantly from the conventional electrokinetic modeling indication. Among these unexpected observations, the complicated dependence of EOF on the surface charge concentration of the channel wall remains most confusing. In this work we give a complete and unified picture for the phenomena by outlining the competing two mechanisms in the water-depletion layer around the channel wall: the decreasing trend of fluidic flow due to the redistribution of net charges, and the increasing trend because of the reduced solution viscosity there. Our quantitative evaluation illustrates that the alternate dominating by the two mechanisms leads to the observed transport behaviors. Furthermore, by considering the decreasing of ionic mobility in the depletion layer, our calculations show quantitative agreement with the latest experiments using BN nanotube channels.

Total variation denoising-based method of identifying the states of single molecules in break junction data.

Break junction (BJ) measurements provide insights into the electrical properties of diverse molecules, enabling the direct assessment of single-molecule conductances. The BJ method displays potential for use in determining the dynamics of individual molecules, single-molecule chemical reactions, and biomolecules, such as deoxyribonucleic acid and ribonucleic acid. However, conductance data obtained via single-molecule measurements may be susceptible to fluctuations due to minute structural changes within the junctions. Consequently, clearly identifying the conduction states of these molecules is challenging. This study aims to develop a method of precisely identifying conduction state traces. We propose a novel single-molecule analysis approach that employs total variation denoising (TVD) in signal processing, focusing on the integration of information technology with measured single-molecule data. We successfully applied this method to simulated conductance traces, effectively denoise the data, and elucidate multiple conduction states. The proposed method facilitates the identification of well-defined plateau lengths and supervised machine learning with enhanced accuracies. The introduced TVD-based analytical method is effective in elucidating the states within the measured single-molecule data. This approach exhibits the potential to offer novel perspectives regarding the formation of molecular junctions, conformational changes, and cleavage.

Single-molecule detection of modified amino acid regulating transcriptional activity.

Acetylation of lysine, a component of histones, regulates transcriptional activity. Simple detection methods for acetyl lysine are essential for early diagnosis of diseases and understanding of the physiological effects. We have detected and recognized acetyl lysine at the single-molecule level by combining MCBJ measurement and machine learning.

Enhanced Nanoparticle Sensing in a Highly Viscous Nanopore.

Slowing down translocation dynamics is a crucial challenge in nanopore sensing of small molecules and particles. Here, it is reported on nanoparticle motion-mediated local viscosity enhancement of water-organic mixtures in a nanofluidic channel that enables slow translocation speed, enhanced capture efficiency, and improved signal-to-noise ratio by transmembrane voltage control. It is found that higher detection rates of nanoparticles under larger electrophoretic voltage in the highly viscous solvents. Meanwhile, the strongly pulled particles distort the liquid in the pore at high shear rates over 103 s-1 which leads to a counterintuitive phenomenon of slower translocation speed under higher voltage via the induced dilatant viscosity behavior. This mechanism is demonstrated as feasible with a variety of organic molecules, including glycerol, xanthan gum, and polyethylene glycol. The present findings can be useful in resistive pulse analyses of nanoscale objects such as viruses and proteins by allowing a simple and effective way for translocation slowdown, improved detection throughput, and enhanced signal-to-noise ratio.

Immunological assay using a solid-state pore with a low limit of detection.

Emerging infectious diseases, cancer, and other diseases are quickly tested mainly via immune reactions based on specific molecular recognition between antigens and antibodies. By changing the diameter of solid-state pores, biomolecules of various sizes can be rapidly detected at the single-molecule level. The combination of immunoreactions and solid-state pores paves the way for an efficient testing method with high specificity and sensitivity. The challenge in developing this method is achieving quantitative analysis using solid-state pores. Here, we demonstrate a method with a low limit of detection for testing tumor markers using a combination of immunoreactions and solid-state pore technology. Quantitative analysis of the mixing ratio of two and three beads with different diameters was achieved with an error rate of up to 4.7%. The hybrid solid-state pore and immunoreaction methods with prostate-specific antigen (PSA) and anti-PSA antibody-modified beads achieved a detection limit of 24.9 fM PSA in 30 min. The hybrid solid-state pore and immunoreaction enabled the rapid development of easy-to-use tests with lower limit of detection and greater throughput than commercially available immunoassay for point-of-care testing.