Selective miR-21 detection technology based on photocrosslinkable artificial nucleic acid-modified magnetic particles and hybridization chain reaction.

Recently, microRNA (miRNA) detection in blood has attracted attention as a new early detection technology for cancer. The extraction of target miRNA is a necessary preliminary step for detection; however, currently, most extraction methods extract all RNA from the blood, which limits the detection selectivity. Therefore, a method for the selective extraction and detection of target miRNA from blood is very important. In this study, we utilized photocrosslinkable artificial nucleic acids and the hybridization chain reaction (HCR) in an attempt to improve upon the current standard method RT-qPCR, which is hampered by problems with primer design and enzymatic amplification. By introducing photocrosslinkable artificial nucleic acids to oligonucleotide probes modified with magnetic particles with a sequence complementary to that of the target miRNA and irradiating them with light, covalent bonds were formed between the target miRNA and the oligonucleotide probes. These tight covalent bonds enabled the capture of miRNA in blood, and intensive washing ensured that only the target miRNA were extracted. After extraction, two types of DNA (H1 and H2) modified with fluorescent dyes were added and the fluorescence signals were amplified by the HCR in the presence of the target miRNA bound to the photocrosslinkable artificial nucleic acids, allowing for isothermal and enzyme-free miRNA detection. The novel method is suitable for selective miRNA detection in real blood samples. Because the reaction proceeds isothermally and no specialized equipment is used for washing, this detection technology is simple and selective and suitable for application to point-of-care technology using microfluidic devices.

Self-assembly of Aeropyrum pernix bacilliform virus 1 (APBV1) major capsid protein and its application as building blocks for nanomaterials.

Virus capsid proteins have various applications in diverse fields such as biotechnology, electronics, and medicine. In this study, the major capsid protein of bacilliform clavavitus APBV1, which infects the hyperthermophilic archaeon Aeropyrum pernix, was successfully expressed in Escherichia coli. The gene product was expressed as a histidine-tagged protein in E. coli and purified to homogeneity using single-step nickel affinity chromatography. The purified recombinant protein self-assembled to form bacilliform virus-like particles at room temperature. The particles exhibited tolerance against high concentrations of organic solvents and protein denaturants. In addition, we succeeded in fabricating functional nanoparticles with amine functional groups on the surface of ORF6-81 nanoparticles. These robust protein nanoparticles can potentially be used as a scaffold in nanotechnological applications.

Time-Series Clustering of Single-Cell Trajectories in Collective Cell Migration.

Collective invasion drives multicellular cancer cells to spread to surrounding normal tissues. To fully comprehend metastasis, the methodology of analysis of individual cell migration in tissue should be well developed. Extracting and classifying cells with similar migratory characteristics in a colony would facilitate an understanding of complex cell migration patterns. Here, we used electrospun fibers as the extracellular matrix for the in vitro modeling of collective cell migration, clustering of mesenchymal and epithelial cells based on trajectories, and analysis of collective migration patterns based on trajectory similarity. We normalized the trajectories to eliminate the effect of cell location on clustering and used uniform manifold approximation and projection to perform dimensionality reduction on the time-series data before clustering. When the clustering results were superimposed on the trajectories before normalization, the results still exhibited positional similarity, thereby demonstrating that this method can identify cells with similar migration patterns. The same cluster contained both mesenchymal and epithelial cells, and this result was related to cell location and cell division. These data highlight the reliability of this method in identifying consistent migration patterns during collective cell migration. This provides new insights into the epithelial-mesenchymal interactions that affect migration patterns.

Quantitative Analysis of Collective Migration by Single-Cell Tracking Aimed at Understanding Cancer Metastasis.

Metastasis is a major complication of cancer treatments. Studies of the migratory behavior of cells are needed to investigate and control metastasis. Metastasis is based on the epithelial-mesenchymal transition, in which epithelial cells acquire mesenchymal properties and the ability to leave the population to invade other regions of the body. In collective migration, highly migratory "leader" cells are found at the front of the cell population, as well as cells that "follow" these leader cells. However, the interactions between these cells are not well understood. We examined the migration properties of leader-follower cells during collective migration at the single-cell level. Different mixed ratios of "leader" and "follower" cell populations were compared. Collective migration was quantitatively analyzed from two perspectives: cell migration within the colony and migration of the entire colony. Analysis of the effect of the cell mixing ratio on migration behavior showed that a small number of highly migratory cells enhanced some of the migratory properties of other cells. The results provide useful insights into the cellular interactions in collective cell migration of cancer cell invasion.

Electrospun Porous Nanofibers with Imprinted Patterns Induced by Phase Separation of Immiscible Polymer Blends.

Nanofibrous nonwoven fabrics have attracted attention as porous adsorbents with high specific surface areas for the safe and efficient treatment of spilled organic dyes and petroleum. For this purpose, a method of fabricating porous nanofibers with high specific surface areas would be highly beneficial. In this study, the phase separation in nanofibers electrospun from blended solutions of immiscible polymers [poly(styrene) (PS) and poly(vinylpyrrolidone) (PVP)] was investigated. The removal of PVP as a sacrificial polymer afforded the imprinting of mesopores (40-70 nm) in the PS nanofibers. The effects of solution composition (PS/PVP in N,N-dimethylformamide) on the structure formation in the fibers were investigated. The nanofibers thus obtained could selectively adsorb low-molecular-weight hydrophobic dyes, such as Nile Red and Oil Red O. Thus, it is expected that the combined approach of electrospinning of immiscible polymer blends and phase separation-induced patterning can be applied to the fabrication of functional nanofibers for diverse applications.

Photo-Cross-Linked Probe-Modified Magnetic Particles for the Selective and Reliable Recovery of Nucleic Acids.

Polymerase chain reaction (PCR) assays are used to diagnose various infectious diseases such as Coronavirus disease 2019 by detecting the nucleic acids of the pathogen. However, in practice, the yield of the extraction process and the inhibition of the reverse transcription reaction and PCR by foreign substances reduce the sensitivity and may yield false negative results. The sensitivity of the PCR test can be improved by using technologies that can reliably capture the target nucleic acid and remove foreign substances. In this study, we developed photo-cross-linkable probe-modified magnetic particles (PPMPs) for the sequence-specific recovery of target nucleic acids using photo-cross-linkable artificial nucleic acid probes and magnetic particles. Nucleic acid probes modified with photo-cross-linkable artificial nucleic acids can hybridize with the target nucleic acids in a sequence-specific manner and then securely capture the target nucleic acids by UV irradiation-mediated covalent bonding. Then the target nucleic acid is detected by trapping the target-bound probe on the surface of the magnetic particles and subjecting these collected magnetic particles to PCR. Recovery of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) N gene pseudo-DNA (120 bp) was performed using PPMPs. We confirmed that the PPMPs captured the target consistently even after washes were done with denaturing agents and surfactants. Even in the presence of foreign DNA fragments, PPMPs were able to specifically recover the target DNA. This method allows for a more accurate detection by recovering only the target DNA for PCR. Hence, PPMPs can be successfully used for PCR-mediated detection of SARS-CoV-2 and other pathogens whose nucleic acid sequences are known.

Characterization and preliminary in vivo evaluation of a self-expandable hydrogel stent with anisotropic swelling behavior and endoscopic deliverability for use in biliary drainage.

We demonstrate the potential of a novel self-expandable biliary stent comprised of poly(vinyl alcohol) (PVA) hydrogel with anisotropic swelling behavior and endoscopic deliverability in vivo, using a porcine stent model. The mechanism underlying the anisotropic swelling behavior and endoscopic deliverability (i.e., flexibility) was investigated by scanning electron microscopy (SEM), small-angle X-ray scattering (SAXS), evaluation of the water content and swelling ratio, and three-point bending tests. The in vivo experiment using a porcine stent model indicated that the tube-shaped PVA hydrogel could effectively expand the biliary tract, without disturbing bile flow. SEM and SAXS showed that PVA hydrogels prepared by drying under extension showed structural orientation along the extension axis, leading to anisotropic swelling. The water content of the PVA hydrogel was found to be crucial for maintaining flexibility as well as endoscopic deliverability. In conclusion, this study demonstrated the novel concept of using a hydrogel stent as a self-expandable biliary stent.

Characterization of a Novel Thermostable Dye-Linked l-Lactate Dehydrogenase Complex and Its Application in Electrochemical Detection.

Flavoenzyme dye-linked l-lactate dehydrogenase (Dye-LDH) is primarily involved in energy generation through electron transfer and exhibits potential utility in electrochemical devices. In this study, a gene encoding a Dye-LDH homolog was identified in a hyperthermophilic archaeon, Sulfurisphaera tokodaii. This gene was part of an operon that consisted of four genes that were tandemly arranged in the Sf. tokodaii genome in the following order: stk_16540, stk_16550 (dye-ldh homolog), stk_16560, and stk_16570. This gene cluster was expressed in an archaeal host, Sulfolobus acidocaldarius, and the produced enzyme was purified to homogeneity and characterized. The purified recombinant enzyme exhibited Dye-LDH activity and consisted of two different subunits (products of stk_16540 (α) and stk_16550 (ß)), forming a heterohexameric structure (α3ß3) with a molecular mass of approximately 253 kDa. Dye-LDH also exhibited excellent stability, retaining full activity upon incubation at 70 °C for 10 min and up to 80% activity after 30 min at 50 °C and pH 6.5-8.0. A quasi-direct electron transfer (DET)-type Dye-LDH was successfully developed by modification of the recombinant enzyme with an artificial redox mediator, phenazine ethosulfate, through amine groups on the enzyme's surface. This study is the first report describing the development of a quasi-DET-type enzyme by using thermostable Dye-LDH.

Highly Efficient Multi-Step Oxidation Bioanode Using Microfluidic Channels.

With the rapid decline of fossil fuels, various types of biofuel cells (BFCs) are being developed as an alternative energy source. BFCs based on multi-enzyme cascade reactions are utilized to extract more electrons from substrates. Thus, more power density is obtained from a single molucule of substrate. In the present study, a bioanode that could extract six electrons from a single molecule of L-proline via a three-enzyme cascade reaction was developed and investigated for its possible use in BFCs. These enzymes were immobilized on the electrode to ensure highly efficient electron transfer. Then, oriented immobilization of enzymes was achieved using two types of self-assembled monolayers (SAMs). In addition, a microfluidic system was incorporated to achieve efficient electron transfer. The microfluidic system, in which the electrodes were arranged in a tooth-shaped comb, allowed for substrates to be supplied continuously to the cascade, which resulted in smooth electron transfer. Finally, we developed a high-performance bioanode which resulted in the accumulation of higher current density compared to that of a gold disc electrode (205.8 µA cm-2: approximately 187 times higher). This presents an opportunity for using the bioanode to develop high-performance BFCs in the future.

One-Step Surface Immobilization of Protein A on Hydrogel Nanofibers by Core-Shell Electrospinning for Capturing Antibodies.

Nanofibers (NFs) are potential candidates as filter materials for affinity separation owing to their high liquid permeability based on their high porosity. Multiple and complex processes were conventionally performed to immobilize proteins for modifying NF surfaces. A simple method must be developed to immobilize proteins without impairing their biological activity. Herein, we succeeded in fabricating NFs with a core of cellulose acetate and a shell of hydrophilic polyvinyl alcohol immobilized with staphylococcal recombinant protein A by a one-step process based on core-shell electrospinning. A total of 12.9 mg/cm3 of antibody was captured in the fiber shell through high affinity with protein A immobilized in an aqueous environment of the hydrogel. The maximum adsorption site and dissociation constant evaluated by the Langmuir model were 87.8 µg and 1.37 µmol/L, respectively. The fiber sheet withstood triplicate use. Thus, our NF exhibited high potential as a material for membrane chromatography.

A high performance nanocomposite based bioanode for biofuel cell and biosensor application.

Herein, to improve the current density and sensitivity for biofuel cell and glucose sensing application, a bioanode based on redox polymer (PEI-Fc) binding polydopamine (PDA) coated MWCNTs (PEI-Fc/PDA/MWCNTs) nanocomposite and glucose oxidase (GOD) was fabricated. PDA/MWCNTs nanocomposite was prepared by spontaneous self-polymerization of dopamine on MWCNTs surface and the PEI-Fc/PDA/MWCNTs nanocomposite was prepared by a simple self-assembly method. The PEI-Fc/PDA/MWCNTs nanocomposite and the resulting bioanode were fully characterized. A maximum current density of 0.73 mA cm-2 at the resulting bioanode was obtained by linear sweep voltammetry (LSV) at the scan rate of 50 mV s-1 with 20 mM glucose concentration. Moreover, a linear range up to 4 mM, a high sensitivity of 57.2 µA mM-1 cm-2, a fast response time reaching 95% of the steady current (2 s) and a low limit of detection (0.024 mM) were achieved. The amperometric method demonstrated both the sensitivity and the stability of the bioanode for glucose-sensing was improved by the employed PDA layer. Finally, the biosensor was used for glucose detection in human serum samples showing good recoveries. This study proposed an excellent functional material prepared by a facile self-assembled method for applying in biofuel cells and second-generation biosensors.

Nanofiber-Mâché Hollow Ball Mimicking the Three-Dimensional Structure of a Cyst.

The occasional malignant transformation of intracranial epidermoid cysts into squamous cell carcinomas remains poorly understood; the development of an in vitro cyst model is urgently needed. For this purpose, we designed a hollow nanofiber sphere, the "nanofiber-mâché ball." This hollow structure was fabricated by electrospinning nanofiber onto alginate hydrogel beads followed by dissolving the beads. A ball with approximately 230 mm3 inner volume provided a fibrous geometry mimicking the topography of the extracellular matrix. Two ducts located on opposite sides provided a route to exchange nutrients and waste. This resulted in a concentration gradient that induced oriented migration, in which seeded cells adhered randomly to the inner surface, formed a highly oriented structure, and then secreted a dense web of collagen fibrils. Circumferentially aligned fibers on the internal interface between the duct and hollow ball inhibited cells from migrating out of the interior, similar to a fish bottle trap. This structure helped to form an adepithelial layer on the inner surface. The novel nanofiber-mâché technique, using a millimeter-sized hollow fibrous scaffold, is excellently suited to investigating cyst physiology.

Electrochemical characteristics of a gold nanoparticle-modified controlled enzyme-electrode contact junction electrode.

Biodevices in which biomolecules such as enzymes and antibodies are immobilized on the surface of electrode materials are capable of converting chemical energy into electrical energy, and are expected to contribute to solving energy problems and developing medical measurements especially as biobatteries and biosensors. Device performance depends on the interface formed between the biomolecule layer and electrode material, and the interface is required to simultaneously achieve a highly efficient enzymatic reaction and electron transfer. However, when enzymes were immobilized on a material surface, the enzymes undergoes a structural change due to the interaction between the enzyme and the electrode surface, making it difficult to maximize the function of the enzyme molecule on the material surface. In this study, we postulate that the structural change of the enzyme would be reduced and the electrochemical performance improved by making the contact area between the enzyme and the electrode extremely small and adsorbing it as a point. Therefore, we aimed to develop a high-power biodevice that retains enzyme structure and activity by interposing gold nanoparticles (AuNPs) between the enzyme and the electrode. The enzymatic and electrochemical properties of pyrroloquinoline quinone-dependent glucose dehydrogenase adsorbed on AuNPs of 5-40 nm diameter were investigated. We found that the characteristics differed among the particles, and the enzyme adsorbed on 20 nm AuNPs showed the best electrochemical characteristics.

Cell Trapping via Migratory Inhibition within Density-Tuned Electrospun Nanofibers.

Cell migration is an essential bioprocess that occurs during wound healing and tissue regeneration. Abnormal cell migration is observed in various pathologies, including cancer metastasis. Glioblastoma multiforme (GBM) is an aggressive and highly infiltrative brain tumor. The white matter tracts are considered the preferred routes for GBM invasion and the subsequent spread throughout the brain tissue. In the present study, a platform based on electrospun nanofibers with a consistent alignment and controlled density was designed to inhibit cell migration. The observation of the cells cultured on the nanofibers with different fiber densities revealed an inverse correlation between the cell migration velocity and nanofiber density. This was attributed to the formation of focal adhesions (FAs). The FAs in the sparse fiber matrix were small, whereas those in the dense fiber matrix were large, aligned with the nanofibers, and distributed throughout the cells. A nanofiber-based platform with stepwise different fiber densities was designed based on the aforementioned observation. A time-lapse observation of the GBM cells cultured on the platform revealed a directional one-way migration that induced the entrapment of cells in the dense-fiber zone. The designed platform mimicked the structure of the white matter tracts and enabled the entrapment of migrating cells. The demonstrated approach is suitable for inhibiting metastasis and understanding the biology of invasion, thereby functioning as a promising therapeutic strategy for GBM.

Electrospun collagen core/poly-l-lactic acid shell nanofibers for prolonged release of hydrophilic drug.

The development of sustained control drug release for delivering hydrophilic drugs has been challenging due to a burst release. Nanofibers are used as materials that enable efficient drug delivery systems. In this study, we designed drug-encapsulated core-shell nanofibers comprising a hydrophilic core of collagen (Col) incorporated with berberine chloride (BC), an anti-inflammatory and anti-cancer agent used as a model drug, and a hydrophobic shell of poly-l-lactic acid (PLLA). Long-term drug release profiles under both the physiological and hydrolysis-accelerated conditions were measured and analyzed using a Korsmeyer-Peppas kinetics model. We found that the Col/PLLA core-shell fiber achieved a controllable long-term release of the hydrophilic drug incorporated inside the core by the slow degradation of the PLLA shell to prevent the burst release while PLLA monolithic fibers showed early release due to the dissolution of drug and the following rapid hydrolysis of fibers. As shown by the results of Col/PLLA core-shell fiber under a hydrolysis-accelerated condition to promote the release of drugs test, it would provide sustained release over 16 days under physiological conditions. Here, the development of the nanomaterial for the long-term drug release of hydrophilic drugs was achieved, leading to its potential medical application including cancer treatment.

Activity enhancement of multicopper oxidase from a hyperthermophile via directed evolution, and its application as the element of a high performance biocathode.

Although multicopper oxidase from the hyperthermophilic archaeon Pyrobaculum aerophilum (McoP) can be particularly useful in biotechnological applications, e.g., as a specific catalyst at the biocathode of biofuel cells (BFCs), owing to its high stability against extremely high temperatures and across a wide range of pH values, this application potential remains limited due to the enzyme's low catalytic activity. A directed evolution strategy was conducted to improve McoP catalytic activity, and the No. 571 mutant containing four amino acid substitutions was identified, with specific activity approximately 9-fold higher than that of the wild type enzyme. Among the substitutions, the single amino acid mutant F290I was essential in enhancing catalytic activity, with a specific activity approximately 12-fold higher than that of the wild type enzyme. F290I thermostability and pH stability were notably comparable with values obtained for the wild type. Crystal structure analysis suggested that the F290I mutant increased loop flexibility near the T1 Cu center, and affected electron transfer between the enzyme and substrate. Additionally, electric current density of the F290I mutant-immobilized electrode was 7-fold higher than that of the wild type-immobilized one. These results indicated that F290I mutant was a superior catalyst with potential in practical biotechnological applications.

Biocathode design with highly-oriented immobilization of multi-copper oxidase from Pyrobaculum aerophilum onto a single-walled carbon nanotube surface via a carbon nanotube-binding peptide.

Biofuel cells generate electric energy using an enzyme as a catalyst for an electrode but their stability and low battery output pose problems for practical use. To solve these problems, this study aimed to build a long-lasting and high-output biocathode as a catalyst using a highly stable hyperthermophilic archaeal enzyme, multi-copper oxidase, from Pyrobaculum aerophilum (McoP). To increase output, McoP was oriented and immobilized on single-walled carbon nanotubes (SWCNT) with a high specific surface area, and the electrode interface was designed to achieve highly efficient electron transfer between the enzyme and electrode. Type 1 copper (T1Cu), an electron-accepting site in the McoP molecule, is located near the C-terminus. Therefore, McoP was prepared by genetically engineering a CNT-binding peptide with the sequence LLADTTHHRPWT, at the C-terminus of McoP (McoP-CBP). We then constructed an electrode using a complex in which McoP-CBP was aligned and immobilized on SWCNT, and then clarified the effect of CBP. The amounts of immobilized enzymes on McoP-SWCNT and (McoP-CBP)-SWCNT complexes were almost equal. CV measurement of the electrode modified with both complexes showed 5.4 times greater current density in the catalytic reaction of the (McoP-CBP)-SWCNT/GC electrode than in the McoP-SWCNT/GC electrode. This is probably because CBP fusion immobilize the enzyme on SWCNTs in an orientational manner, and T1Cu, the oxidation-reduction site in McoP, is close to the electrode, which improves electron transfer efficiency.

Site-Directed Mutagenesis of Multicopper Oxidase from Hyperthermophilic Archaea for High-Voltage Biofuel Cells.

Enzymes from hyperthermophilic archaea are potential candidates for industrial use because of their superior pH, thermal, and long-term stability, and are expected to improve the long-term stability of biofuel cells (BFCs). However, the reported multicopper oxidase (MCO) from hyperthermophilic archaea has lower redox potential than MCOs from other organisms, which leads to a decrease in the cell voltage of BFCs. In this study, we attempted to positively shift the redox potential of the MCO from hyperthermophilic archaeon Pyrobaculum aerophilum (McoP). Mutations (M470L and M470F) were introduced into the axial ligand of the T1 copper atom of McoP, and the enzymatic chemistry and redox potentials were compared with that of the parent (M470). The redox potentials of M470L and M470F shifted positively by about 0.07 V compared with that of M470. In addition, the catalytic activity of the mutants towards 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) increased 1.2-1.3-fold. The thermal stability of the mutants and the electrocatalytic performance for O2 reduction of M470F was slightly reduced compared with that of M470. This research provides useful enzymes for application as biocathode catalysts for high-voltage BFCs.

Enhanced and Prolonged Activity of Enzymes Adsorbed on TEMPO-Oxidized Cellulose Nanofibers.

2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO)-oxidized cellulose nanofibers (TOCNs) have a width of about 4 nm and a very large specific surface area. TOCN is a negatively charged bionanomaterial having carboxy groups on the surface and promising physical properties. In particular, TOCN can be used as an adsorbent for biomolecules for biotechnological applications, but the adsorption behavior of biomolecules on the TOCN surface requires investigation. Thus, in this study, we investigated the adsorption behavior of pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH) on TOCN and evaluated the activity, structure, and long-term stability of the adsorbed enzyme. Transmission electron microscopy observation revealed that the enzyme was aligned and adsorbed on the TOCNs, and circular dichroism measurements were used to determine the structure of the enzyme adsorbed on TOCN. Interestingly, the adsorbed enzyme showed higher activity after adsorption, resulting in long-term retention of enzyme activity, probably because the stability of PQQ-GDH was improved by adsorption. These results suggest that TOCN is an excellent biomolecule immobilization material. Our results can be used for the development of biomaterials using TOCN as a scaffold for the adsorption of enzymes with increased stability and activity.

Formation of one-dimensional arrays of nanoparticles using segmented polyurethane nanofibers prepared by electrospinning.

The number density and the arrangement of metal nanoparticles in composite materials have a significant effect on their performance and hence their suitability for use in sensors and devices. Forming one-dimensional arrays of metal nanoparticles is one way of controlling their number density and arrangement in the devices. In this study, we fabricated one-dimensional arrays of gold nanoparticles by adsorbing them on linearly distributed hard segments present on the surfaces of segmented polyurethane nanofibers, which were produced by electrospinning under a stretching force. The length of the one-dimensional array was approximately 500 nm. Furthermore, the interparticle distance was almost constant at approximately 14 nm. Thus, the proposed method is suitable for fabricating one-dimensional arrays of metal nanoparticles with high precision.