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.

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.

Development of Biofuel Cell Using a Complex of Highly Oriented Immobilized His-Tagged Enzyme and Carbon Nanotube Surface Through a Pyrene Derivative.

For increasing the output of biofuel cells, increasing the cooperation between enzyme reaction and electron transfer on the electrode surface is essential. Highly oriented immobilization of enzymes onto a carbon nanotube (CNT) with a large specific surface area and excellent conductivity would increase the potential for their application as biosensors and biofuel cells, by utilizing the electron transfer between the electrode-molecular layer. In this study, we prepared a CNT-enzyme complex with highly oriented immobilization of enzyme onto the CNT surface. The complex showed excellent electrical characteristics, and could be used to develop biodevices that enable efficient electron transfer. Multi-walled carbon nanotubes (MWCNT) were dispersed by pyrene butyric acid N-hydroxysuccinimide ester, and then N-(5-amino-1-carboxypentyl) iminodiacetic acid (AB-NTA) and NiCl2 were added to modify the NTA-Ni2+ complex on the CNT surface. Pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase (GDH) was immobilized on the CNT surface through a genetically introduced His-tag. Formation of the MWCNT-enzyme complex was confirmed by monitoring the catalytic current electrochemically to indicate the enzymatic activity. PQQ-GDH was also immobilized onto a highly ordered pyrolytic graphite surface using a similar process, and the enzyme monolayer was visualized by atomic force microscopy to confirm its structural properties. A biofuel cell was constructed using the prepared CNT-enzyme complex and output evaluation was carried out. As a result, an output of 32 µW/cm² could be obtained without mediators.

DNA photo-cross-linking using a pyranocarbazole-modified oligodeoxynucleotide with a d-threoninol linker.

An alternative photo-cross-linker having a d-threoninol skeleton instead of the 2'-deoxyribose backbone in 3-cyanovinylcarbazole (CNVK) was investigated to improve the photoreactivity of photo-cross-linkers; the photo-cross-linking rate of 3-cyanovinylcarbazole with d-threoninol (CNVD) was found to be greater than that of CNVK. Therefore, in this study, a novel photo-cross-linker having pyranocarbazole (PCX) and d-threoninol instead of the 2'-deoxyribose backbone in PCX (PCXD) was developed. The PCXD in double-stranded DNA photo-cross-linked to a pyrimidine base at the -1 position of a complementary strand similar to PCX. Furthermore, the photoreactivity of PCXD was significantly higher than that of PCX. The introduction of d-threoninol improved the reactivity of pyranocarbazole to cytosine, the use of PCXD may extend the applicability of the photo-cross-linking reaction for DNA manipulation. In particular, this novel photo-cross-linker can contribute to the photochemical regulation of gene expression or biological events in a living cell.

Construction of a biocathode using the multicopper oxidase from the hyperthermophilic archaeon, Pyrobaculum aerophilum: towards a long-life biobattery.

OBJECTIVES: The life of biobatteries remains an issue due to loss of enzyme activity over time. In this study, we sought to develop a biobattery with a long life using a hyperthermophilic enzyme. RESULTS: We hypothesized that use of such hyperthermophilic enzymes would allow for the biofuel cells to have a long battery life. Using pyrroloquinoline quinone-glucose dehydrogenase and the multicopper oxidase from Pyrobaculum aerophilum, we constructed an anode and cathode. The maximum output was 11 µW at 0.2 V, and the stability of the both electrode was maintained at 70 % after 14 days. CONCLUSION: The biofuel cells that use hyperthermophilic enzymes may prolong their life.