Identifying Viral Vector Characteristics by Nanopore Sensing.

Using viral vectors as gene delivery vehicles for gene therapy necessitates their quality control. Here, we report on nanopore sensing for nondestructively inspecting genomes inside the nanoscale cargoes at the single-molecule level. Using ionic current measurements, we motion-tracked the adeno-associated virus (AAV) vectors as they translocated through a solid-state nanopore. Considering the varying contributions of the electrophoretic forces from the negatively charged internal polynucleotides of different lengths, the nanocargoes carrying longer DNA moved more slowly in the nanochannel. Moreover, ion blockage characteristics revealed their larger volume by up to approximately 3600 nm3 in proportion to the length of single-stranded DNA packaged inside, thereby allowing electrical discriminations of AAV vectors by the gene-derived physical features. The present findings can be a promising tool for the enhanced quality control of AAV products by enabling the screening of empty and intermediate vectors at the single-particle level.

Oil Inclusions Found in Skeleton Crystals of Quartz Indicated the Existence of Organic Matter Surrounding Ancient Growth Environments.

In nature, minerals record various origins and information for geology and geobiochemistry. Here, we investigated the origin of organic matter and growth mechanism of quartz with oil inclusion revealing fluorescence under short ultraviolet (UV) light, obtained from the clay vein at Shimanto-cho, Kochi, Shikoku Island, Japan. Geological investigation indicated that the oil-quartz was formed in hydrothermal metamorphic veins found in the late Cretaceous interbedded sandstone and mudstone. The obtained oil-quartz crystals are mostly double-terminated. Micro-X-ray computed tomography (microCT) indicated that oil-quartz crystals have various veins originating as skeleton structures along the quartz crystal {111} and {1-11} faces. Spectroscopic and chromatographic studies indicated that aromatic ester and tetraterpene (lycopene) molecules, which revealed fluorescence, were detected. Large molecular weight sterol molecules, such as C40, were also detected in the vein of oil-quartz. This investigation indicated that organic inclusions in mineral crystals would form with ancient microorganism culture environments.

Effect of Electrolyte Concentration on Cell Sensing by Measuring Ionic Current Waveform through Micropores.

Immunostaining has been widely used in cancer prognosis for the quantitative detection of cancer cells present in the bloodstream. However, conventional detection methods based on the target membrane protein expression exhibit the risk of missing cancer cells owing to variable protein expressions. In this study, the resistive pulse method (RPM) was employed to discriminate between cultured cancer cells (NCI-H1650) and T lymphoblastoid leukemia cells (CCRF-CEM) by measuring the ionic current response of cells flowing through a micro-space. The height and shape of a pulse signal were used for the simultaneous measurement of size, deformability, and surface charge of individual cells. An accurate discrimination of cancer cells could not be obtained using 1.0 × phosphate-buffered saline (PBS) as an electrolyte solution to compare the size measurements by a microscopic observation. However, an accurate discrimination of cancer cells with a discrimination error rate of 4.5 ± 0.5% was achieved using 0.5 × PBS containing 2.77% glucose as the electrolyte solution. The potential application of RPM for the accurate discrimination of cancer cells from leukocytes was demonstrated through the measurement of the individual cell size, deformability, and surface charge in a solution with a low electrolyte concentration.

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.

Quantitative analysis of DNA with single-molecule sequencing.

Cancer can be diagnosed by identifying DNA and microRNA base sequences that have the same base length yet differ in a few base sequences, if the abundance ratios of these slightly deviant base sequences can be determined. However, such quantitative analyses cannot be performed using the current DNA sequencers. Here we determine entire base sequences of four types of DNA corresponding to the let-7 microRNA, which is a 22-base cancer marker. We record the single-molecule conductances of the base molecules using current-tunneling measurements. In addition, we count the numbers of molecules in a solution to determine the abundance ratios of two DNA strands that differ by a single base sequence.

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.

Detection of post-translational modifications in single peptides using electron tunnelling currents.

Post-translational modifications alter the properties of proteins through the cleavage of peptide bonds or the addition of a modifying group to one or more amino acids. These modifications allow proteins to perform their primary biological functions, but single-protein studies of post-translational modifications have been hindered by a lack of suitable analysis methods. Here, we show that single amino acids can be identified using electron tunnelling currents measured as the individual molecules pass through a nanoscale gap between electrodes. We identify 12 different amino acids and the post-translational modification phosphotyrosine, which is involved in the process that switches enzymes on and off. Furthermore, we show that the conductance measurements can be used to partially sequence peptides of an epidermal growth factor receptor substrate, and can discriminate a peptide from its phosphorylated variant.