Papers - USUI Kenji
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Microwave-induced modulation of intracellular distribution of peptides based on mitochondrial targeting sequences Reviewed International journal
Fumihiro Kayamori, Takuto Kariya, Momo Hirata, Yonejiro Arimoto, Tamaki Endoh, Nobuhiro Nakanishi & Kenji Usui
Scientific Reports 2026.6
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Development of a Microwave-Assisted Method for Enhancing the Cellular Uptake of Arginine-Rich Peptides Reviewed International journal
Fumihiro Kayamori, Momo Hirata, Takuto Kariya, Yonejiro Arimoto, Ryuji Osawa, Mami Ishikawa, Tamaki Endoh, Nobuhiro Nakanishi, Kenji Usui
Processes 14 ( 3 ) 497 2026.1
Joint Work
Authorship：Last author, Corresponding author
Cell-penetrating peptides (CPPs) have been extensively explored as tools for the intracellular delivery of diverse molecular cargoes. Although substantial progress has been made in elucidating their uptake mechanisms and sequence-dependent functions, limitations in cellular internalization efficiency remain a major challenge, hindering their broader biomedical application. To address this issue, the present study investigated whether microwave (MW) irradiation at 2.45 GHz can enhance CPP-mediated delivery. Using confocal laser scanning microscopy and fluorescence spectroscopy, we examined the effects of MW irradiation on the cellular uptake of arginine-rich peptides. Our results suggested that MW irradiation enhanced the cellular uptake of the peptides. These findings imply that CPP-mediated delivery assisted by MW irradiation is an effective method for improving intracellular transport and may open new avenues for the development of advanced drug delivery systems.
DOI： 10.3390/pr14030497
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Synthesis of Biocompatible Gold Nanoparticles for Photothermal Therapy by Mineralization Using Peptides Reviewed International journal
Shuhei Yoshida, Koki Yoshida, Yoshiki Shitamukai, Makoto Ozaki, Takaaki Tsuruoka, Kenji Usui
RSC Advances 15 48521 - 48528 2025.12
Joint Work
Authorship：Corresponding author
DOI： https://doi.org/10.1039/d5ra08717a
Other Link： https://pubs.rsc.org/en/content/articlepdf/2025/ra/d5ra08717a
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One-Pot Synthesis and Immobilization of Gold Nanoparticles Using Peptidyl Microbeads Reviewed International journal
Shuhei Yoshida, Koki Yoshida, Taichi Isozaki, Maho Oura, Makoto Ozaki, Takaaki Tsuruoka, Kenji Usui
Molecules 30 1689 2025.4
Joint Work
Authorship：Last author, Corresponding author
Gold nanoparticles (AuNPs) have surface plasmon resonance (SPR) and catalytic activity that are not found in bulk gold and have been studied in various fields. Among these, immobilization of AuNPs on various solid-phase substrates is known to produce stable catalytic activity and specific SPRs and research on the immobilization of AuNPs has been conducted actively. However, the conventional method requires the preparation and immobilization of AuNPs in separate processes, making it difficult to prepare immobilized AuNPs in a one-pot process. In this study, we attempted to synthesize and immobilize AuNPs using peptidyl beads, which are microbeads having immobilized a peptide capable of reducing gold ions. We successfully reduced Au ions from 0.5 to 1000 µM of HAuCl4 and immobilized them on peptidyl beads in the form of AuNPs. The immobilized AuNPs have a constant particle size independent of the HAuCl4 concentration. Furthermore, the peptidyl beads with AuNPs have catalytic activity. The quantity of the AuNPs on the peptidyl beads and, subsequently, the catalytic reaction rate of the sample, could be controlled. This study would also be expected to be applied to the immobilization of metallic nanomaterials other than AuNPs by modifying the peptide sequence.
DOI： 10.3390/molecules30081689
Other Link： https://www.mdpi.com/1420-3049/30/8/1689
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Development of detection system for lead ions in mixture solutions using UV-Vis measurements with peptide immobilized microbeads Reviewed International journal
Shuhei Yoshida, Koki Yoshida, Yoshio Hamada, Takaaki Tsuruoka, Kenji Usui
Scientific Reports 15 3249 2025.1
Joint Work
Authorship：Last author, Corresponding author
Environmental pollution caused by heavy metals are problems worldwide. In particular, pollution and poisoning by lead ions (Pb2+) continue to be common and serious problems. Hence, there is a need for a widely usable method to easily detect Pb2+ from solutions containing organic materials from environmental water such as seas, ponds, etc. Here, we established a system to easily detect Pb2+ from such mixture solutions using Pb2+ binding peptide immobilized beads (peptidyl beads) and ultraviolet (UV) absorption measurements. This method could detect Pb2+ at low concentrations equivalent to inductively coupled plasmon-atomic emission spectroscopy (ICP-AES). Using the detected values to create a calibration curve, it was found that there was a positive correlation between the concentration of Pb2+ and absorbance, which also made it possible to quantify sub-µM Pb2+ in the solutions. Furthermore, Pb2+ was detected and quantified under mixed conditions of environmental water such as seas, rivers, and ponds. This method is expected to become a versatile and easy-to-use Pb2+ detection method for end-users worldwide.
DOI： 10.1038/s41598-025-86082-z
Other Link： https://www.nature.com/articles/s41598-025-86082-z
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Complex and Non-sequential Current Signatures of a β-Hairpin Peptide Confined in a Nanopore Reviewed International coauthorship International journal
Misa Yamaji, Mauro Chinappi, Blasco Morozzo della Rocca, Kenji Usui, Ryuji Kawano
Analytical Chemistry 97 2044 - 2051 2025.1
Joint Work
Publisher：American Chemical Society (ACS)
Nanopore sensing is widely used for single-molecule detection, originally applied to nucleic acids and now extended to protein sensing. Our study focuses on the complex conformational changes of peptides in nanopores, which may have implications for peptide fingerprinting and protein identification. Specifically, we investigated the interaction of a β-hairpin peptide (SV28) within an α-hemolysin (αHL) nanopore. Our experiments revealed that SV28 is captured via dielectrophoresis and exhibits long dwell times within the nanopore, leading to multiple current blockade levels. Unlike DNA hairpins, the peptide showed non-sequential transitions among four distinct blockade levels. This complex behavior indicates that the peptide dynamics in nanopores cannot be simply modeled along a single reaction coordinate. Our findings provide insights into peptide-nanopore interactions, which are potentially useful for developing nanopore-based peptide identification technologies.
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Development of a CaCO3 Precipitation Method Using a Peptide and Microwaves Generated by a Magnetron Reviewed International journal
Fumihiro Kayamori, Hiroyuki Togashi, Natsumi Endo, Makoto Ozaki, Kan Hirao, Yonejiro Arimoto, Ryuji Osawa, Takaaki Tsuruoka, Takahito Imai, Kin-ya Tomizaki, Tomohiro Umetani, Nobuhiro Nakanishi, Kenji Usui
Processes 12 1327 - 1327 2024.6
Joint Work
Authorship：Last author, Corresponding author Publisher：MDPI AG
Microwave applications, such as microwave ovens and mobile phones, are ubiquitous and indispensable in modern society. As the utilization of microwave technology is becoming more widespread, the effects of microwaves on living organisms and physiological processes have received increased attention. This study aimed to investigate the effects of microwaves on calcium carbonate biomineralization as a model biochemical process. A magnetron oscillator was used to generate 2450 MHz microwaves because magnetrons are relatively inexpensive and widespread. We conducted transmission electron microscopy (TEM), atomic force microscopy (AFM), TEM-electron energy-loss spectroscopy (EELS), dynamic light scattering (DLS), and high-performance liquid chromatography (HPLC) measurements to analyze the calcium carbonate precipitates. Our findings showed the formation of string-like precipitates of calcium carbonate upon microwave irradiation from one direction, similar to those obtained using a semiconductor oscillator, as reported previously. This implied that the distribution of the frequency had little effect on the morphology. Furthermore, spherical precipitates were obtained upon microwave irradiation from two directions, indicating that the morphology could be controlled by varying the direction of microwave irradiation. Magnetrons are versatile and also used in large-scale production; thus, this method has potential in medical and industrial applications.
DOI： 10.3390/pr12071327
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Effect of linearly polarized microwaves on nanomorphology of calcium carbonate mineralization using peptides Reviewed International journal
Kenji Usui, Makoto Ozaki, Kan Hirao, Tsubasa Kosaka, Natsumi Endo, Shuhei Yoshida, Shin-ichiro Yokota, Yonejiro Arimoto, Ryuji Osawa, Nobuhiro Nakanishi, Kin-ya Tomizaki, Tomohiro Umetani, Fumihiro Kayamori
Scientific Reports 13 12027 2023.7
Joint Work
Authorship：Lead author, Corresponding author
Microwaves are used for diverse applications such as mobile phones, ovens, and therapy devices. However, there are few reports on the effects of microwaves on diseases other than cancer, and on physiological processes. Here, we focused on CaCO3 mineralization as a model of biomineralization and attempted to elucidate the effect of microwaves on CaCO3 mineralization using peptides. We conducted AFM, ζ potential, HPLC, ICP-AES, and relative permittivity measurements. Our findings show that microwaves alter the nanomorphology of the CaCO3 precipitate, from sphere-like particles to string-like structures. Furthermore, microwaves have little effect on the mineralization when the mineralization ability of a peptide is high, but a large effect when the precipitation ability is low. Our findings may be applicable to not only the treatment of teeth and bones but also the development of organic–inorganic nanobiomaterials. This methodology can be expanded to other molecular/atomic reactions under various microwave conditions to alter reaction activity parameters.
DOI： 10.1038/s41598-023-37473-7
Other Link： https://www.nature.com/articles/s41598-023-37473-7
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Construction of a Method to Design Fibril-Forming Peptides Applied to Biomaterials from 20 Beta-Sheet Peptides by Statistical Analysis Reviewed International journal
Kazuya Iwata, Taisei Terao, Akira Takekawa, Tomohiro Umetani, Kenji Usui
Peptide Science 2022 141 - 142 2023.3
Joint Work
Authorship：Last author, Corresponding author
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De novo design of a nanopore for single-molecule detection that incorporates a β-hairpin peptide. Reviewed International coauthorship International journal
Keisuke Shimizu, Batsaikhan Mijiddorj, Masataka Usami, Ikuro Mizoguchi, Shuhei Yoshida, Shiori Akayama, Yoshio Hamada, Akifumi Ohyama, Kenji Usui, Izuru Kawamura, Ryuji Kawano
Nature nanotechnology 17 67 - 75 2022.1
Joint Work
The amino-acid sequence of a protein encodes information on its three-dimensional structure and specific functionality. De novo design has emerged as a method to manipulate the primary structure for the development of artificial proteins and peptides with desired functionality. This paper describes the de novo design of a pore-forming peptide, named SV28, that has a β-hairpin structure and assembles to form a stable nanopore in a bilayer lipid membrane. This large synthetic nanopore is an entirely artificial device for practical applications. The peptide forms multidispersely sized nanopore structures ranging from 1.7 to 6.3 nm in diameter and can detect DNAs. To form a monodispersely sized nanopore, we redesigned the SV28 by introducing a glycine-kink mutation. The resulting redesigned peptide forms a monodisperse pore with a diameter of 1.7 nm leading to detection of a single polypeptide chain. Such de novo design of a β-hairpin peptide has the potential to create artificial nanopores, which can be size adjusted to a target molecule.
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ウォッシャーディスインフェクターのすすぎ排水中に含まれる洗浄剤残留量の測定によるすすぎ性能評価 Reviewed
三軒隼人、藤田敏、川田原瑠勇、武川公、臼井健二、原田陽滋
医療機器学 91 315 - 332 2021.8
Joint Work
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Intracellular mineralization of gold nanoparticles using gold ion-binding peptides with cell-penetrating ability Reviewed International journal
Ozaki, M., Yoshida, S., Tsuruoka, T., Usui, K.
Chemical Communications 57 2021
Joint Work
Authorship：Last author, Corresponding author
We developed a system to directly produce gold nanoparticles in cells by intracellular mineralization in lower concentration than conventional methods using a peptide consisting of a cell-penetrating sequence and a gold ion-binding sequence. Furthermore, we could control the uniquely shaped gold nanostructures that were produced by changing peptide structures.
DOI： 10.1039/d0cc06117d
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Elemental composition control of gold-titania nanocomposites by site-specific mineralization using artificial peptides and DNA Reviewed International journal
Ozaki, M., Imai, T., Tsuruoka, T., Sakashita, S., Tomizaki, K.-Y., Usui, K.
Communications Chemistry 4 2021
Joint Work
Authorship：Last author, Corresponding author
Biomineralization, the precipitation of various inorganic compounds in biological systems, can be regulated in terms of the size, morphology, and crystal structure of these compounds by biomolecules such as proteins and peptides. However, it is difficult to construct complex inorganic nanostructures because they precipitate randomly in solution. Here, we report that the elemental composition of inorganic nanocomposites can be controlled by site-specific mineralization by changing the number of two inorganic-precipitating peptides bound to DNA. With a focus on gold and titania, we constructed a gold-titania photocatalyst that responds to visible light excitation. Both microscale and macroscale observations revealed that the elemental composition of this gold-titania nanocomposite can be controlled in several ten nm by changing the DNA length and the number of peptide binding sites on the DNA. Furthermore, photocatalytic activity and cell death induction effect under visible light (>450 nm) irradiation of the manufactured gold-titania nanocomposite was higher than that of commercial gold-titania and titania. Thus, we have succeeded in forming titania precipitates on a DNA terminus and gold precipitates site-specifically on double-stranded DNA as intended. Such nanometer-scale control of biomineralization represent a powerful and efficient tool for use in nanotechnology, electronics, ecology, medical science, and biotechnology.
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Development of a chromophore-solid phase peptide reaction assay (C-SPRA) for assessing skin sensitization: In vitro Reviewed International journal
Miyazaki, H., Hamada, Y., Takaishi, H., Minamino, Y., Ikeda, H., Mekata, H., Takaishi, M., Yamashita, K., Usui, K.
Analyst 145 3211 - 3216 2020.5
Joint Work
Authorship：Last author, Corresponding author
We developed an in vitro chromophore-solid phase peptide reaction assay (C-SPRA) using microbead-immobilized peptides and chromophores. Peptide-resins (microbeads) reacted with 14 representative chemicals to demonstrate the test's capacity to predict skin sensitization. C-SPRA enables accurate and high-throughput assessments of various chemicals, including poorly water-soluble sensitizers that are regarded as weakly potent by other methods.
DOI： 10.1039/c9an02514f
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Novel purification process for amyloid beta peptide(1-40) Reviewed International journal
Usui, K., Yokota, S.-I., Iwata, K., Hamada, Y.
Processes 8 2020
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Mass spectrometry-based solid phase peptide reaction assay for detecting allergenicity using an immobilized peptide-conjugating photo-cleavable linker Reviewed International journal
Miyazaki, H., Samejima, Y., Iwata, K., Minamino, Y., Hikida, S., Ariumi, H., Ikeda, H., Hamada, Y., Yamashita, K., Usui, K.
International Journal of Molecular Sciences 21 2020
Joint Work
Authorship：Last author, Corresponding author
The biological process of skin sensitization depends on the ability of a sensitizer to modify endogenous proteins. A direct peptide reactivity assay (DPRA), based on the biological process of skin sensitization, was developed as an alternative to controversial animal experiments. Although DPRA has been endorsed by industries and is internationally accepted as promising, it has several drawbacks, such as incompatibility with hydrophobic chemicals, inability to perform detailed reaction analysis, and ability to evaluate only single components. Here, we demonstrated that sensitizers and peptide adducts can be easily identified using a mass spectrometry-based solid-phase peptide reaction assay (M-SPRA). We synthesized peptides with a photo-cleavable linker immobilized on resins. We showed the potential of M-SPRA in predicting skin sensitization by measuring the peptide adducts that were selectively eluted from the resin after cleaving the linker post-reaction. M-SPRA provides more detailed information regarding chemical reactivity and accurate assessment of test samples, including mixtures. M-SPRA may be helpful for understanding the binding mechanism of sensitizers (toxicology), which may assist in further refining reactivity assays and aiding in the interpretation of reactivity data.
DOI： 10.3390/ijms21218332
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Synthesis of peptide-immobilized magnetic beads, and peptide reactivity assay for assessing skin sensitization utilizing chromophore Reviewed International journal
Miyazaki, H., Takaishi, H., Ikeda, H., Ariumi, H., Hamada, Y., Yamashita, K., Usui, K.
Processes 8 2020
Joint Work
Authorship：Last author, Corresponding author
DPRA (direct peptide reactivity assay) and ADRA (amino acid derivative reactivity assay), which are based on the biological events of skin sensitization, were developed as alternatives to the controversial animal experiments. These assays are described in the OECD (Organization for Economic Co-operation and Development) guideline, Test No. 442C. Although these assays have been endorsed by the industries and internationally accepted as promising and effective tests for in vitro skin sensitization, they suffer from several drawbacks, such as incompatibility with hydrophobic chemicals and complicated sample processing. Here, we demonstrated a chromophore-based solid phase peptide reaction assay in vitro using peptides immobilized on magnetic beads (C-SPRA-MB). We successfully synthesized lysine (Lys) and cysteine (Cys) immobilized on magnetic microbeads. However, Cys immobilized magnetic microbeads showed gradual decomposition of the magnetic beads due to SH oxidation. Using Lys immobilized magnetic microbeads, we demonstrated the capacity of C-SPRA-MB to predict skin sensitization by measuring free amino groups of the Lys after reaction with test chemicals. First, the free amines on the microbeads were reacted with bromophenol blue (BB). Then, by treatment with a saturated solution of Lys, the bound BBs were released and quantified. C-SPRA-MB provides high-throughput and accurate assays for assessments of chemicals, including with low-potency as skin sensitizers and poor water solubility. C-SPRA-MB may be useful for effective prediction of their skin sensitization potential in the process of compound screening, especially in the case of misclassified by DPRA and ADRA. Thus, C-SPRA-MB can be applied to assessing the sensitization potential of medical, pharmaceutical, cosmetics, and industrial compounds.
DOI： 10.3390/pr8101257
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Effect of tryptophan residues on gold mineralization by a gold reducing peptide Reviewed International journal
Ozaki, M., Yoshida, S., Oura, M., Tsuruoka, T., Usui, K.
RSC Advances 10 2020
Joint Work
Authorship：Last author, Corresponding author
AuBP1, obtained by phage display selection, was previously shown to produce gold nanoparticles without reducing agents. The tryptophan (Trp) residue located at the N-terminus of this peptide contributes to the reduction of Au3+ to Au0 and is involved in the nucleation and crystal growth of gold nanoparticles. However, clear guidelines for relationships between the number of Trp residues in the peptide and its gold reducing ability have not been established. We focused on gold mineralization and attempted to elucidate aspects of the underlying mechanism. We performed a detailed evaluation of the effects of modifying the N-terminus of the core sequence on gold mineralization without reducing agents. Besides, advantages of utilizing peptides in manufacturing gold nanoparticles are shown. UV-Vis measurements, TEM observations, and kinetic analyses were used to show that increasing the number of Trp residues in the peptide increases the reducing ability, causing predominance of the nucleation reaction and the production of small gold nanoparticles. In addition, these peptides also had the ability as a dispersant to protect the surface of gold nanoparticles. Furthermore, the catalytic activity of mineralized gold nanoparticles with peptides was higher than that of a commercial gold nanoparticle. This study should help to elucidate the relationship between peptide sequence and mineralization ability for use in materials chemistry.
DOI： 10.1039/d0ra07098j
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Modification of the N-terminus of a calcium carbonate precipitating peptide affects calcium carbonate mineralization Reviewed International journal
Kenji Usui, Shin-Ichiro Yokota, Makoto Ozaki, Shungo Sakashita, Takahito Imai, Kin-Ya Tomizaki
Protein and Peptide Letters 25 42 - 47 2018.4
Joint Work
Authorship：Lead author, Corresponding author Publisher：Bentham Science Publishers B.V.
Background: A core sequence (the 9 C-terminal residues) of calcification-associated peptide (CAP-1) isolated from the exoskeleton of the red swamp crayfish was previously shown to control calcium carbonate precipitation with chitin. In addition, a modified core sequence in which the phosphorylated serine at the N terminus is replaced with serine exhibits was also previously shown to alter precipitation characteristics with chitin. Objectives: We focused on calcium carbonate precipitation and attempted to elucidate aspects of the mechanism underlying mineralization. We attempted to evaluate in detail the effects of modifying the N-terminus in the core sequence on calcium carbonate mineralization without chitin. Methods: The peptide modifications included phosphorylation, dephosphorylation, and a free or acetylated Nterminus. The peptides were synthesized manually on Wang resin using the DIPCI-DMAP method for the first residue, and Fmoc solid phase peptide synthesis with HBTU-HOBt for the subsequent residues. Prior to calcium carbonate precipitation, calcium carbonate was suspended in MilliQ water. Carbon dioxide gas was bubbled into the stirred suspension, then the remaining solid CaCO3 was removed by filtration. The concentration of calcium ions in the solution was determined by standard titration with ethylenediaminetetraacetate. Calcium carbonate precipitation was conducted in a micro tube for 3 h at 37°C. We used the micro-scale techniques AFM (atomic force microscopy) and TEM (transmission electron microscopy), and the macro-scale techniques chelate titration, HPLC, gel filtration, CD (circular dichroism) and DLS (dynamic light scattering). Results: We determined the morphologies of the calcium carbonate deposits using AFM and TEM. The pS peptide provided the best control of the shape and size of the calcium carbonate round particles. The acetylated peptides (Ac-S and Ac-pS) provided bigger particles with various shapes. S peptide provided a mixture of bigger particles and amorphous particles. We verified these findings using DLS. All the peptide samples produced nanostructures of the expected size in agreement with the AFM and TEM results. We estimated the abilities of these peptides to precipitate calcium carbonate by determining the residual calcium hydrogen carbonate concentration by standard titration with ethylenediaminetetraacetate after calcium carbonate precipitation. The Ac-pS peptide showed the lowest residual calcium hydrogen carbonate concentration whereas the S peptide showed the highest, suggesting that the precipitating activities of these peptides towards calcium carbonate correlated with peptide net charge. Then the gel filtration results showed a large oligomer peak and a small oligomer/monomer peak for all peptide samples in agreement with the AFM, TEM and DLS results. CD measurements showed that all the peptides formed random-coil-like structures. Thus, we used both macro-and micro-observation techniques such as chelate titration, DLS, AFM and TEM to show that the calcium carbonate precipitating activities of four derivatives of the core sequence of CAP-1 may correlate with the peptide net charge. Conclusion: These peptides mainly act as a catalyst rather than as a binder or component of the calcium carbonate deposits (as a template). On the other hand, the morphologies of the calcium carbonate deposits appeared to be dependent on the ability of the peptide to assemble and act as a template. Consequently, elucidating the relationship between peptide sequence and the ability of the peptide to assemble would be indispensable for controlling precipitate morphologies in the near future. This knowledge would provide important clues for elucidating the relationship between peptide sequence and mineralization ability, including deposit morphology and precipitating activity, for use in nanobiochemistry and materials chemistry research.
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Yuki Tominaga, Kenji Usui, Akiyoshi Hirata, Hiro-O Ito, Kiyoshi Nokihara
Bioorganic and Medicinal Chemistry 26 3210 - 3216 2018.4
Joint Work
Authorship：Lead author Publisher：Elsevier Ltd
A fundamental method has been developed focusing on a facile and rapid examination of periodontal disease. Periodontal disease is an oral disease thought to affect 80% of adults, and early detection with treatment is desirable for the improvement of the quality of life. Unfortunately conventional methods are not consistent as the disease is caused by a number of undefined bacteria and detection relies on the skills of the dentist. Thus an objective detection system is required. We have performed an experiment on saliva using a novel biodetection system, designated PepTenChip®. A disease model for saliva was prepared using a specimen from a healthy subject and a mixture of hemoglobin (f-Hb) and lactate dehydrogenase (LDH), which is used as a periodontal disease marker protein with healthy saliva. PepTenChip® is a peptide microarray in which fluorescent labelled structured peptides are immobilized on a novel amorphous carbon substrate. Since the peptides used as capture molecules are fluorescently labelled, labeling of analytes is not necessary. The fluorescence intensity change before and after application of analytes are detected rather than the ON/OFF detection common to conventional microarrays using a set of antigen–antibody. The fluorescence intensity value changes according to the concentration of captured protein allowing the generation of protein fingerprint (PFP) and dendrograms. The present method does not rely on a “one to one” interaction, unlike conventional biodetection, and advantages can be envisaged in the case of an undefined or unknown cause of disease. The statistical analyses, such as multivariate analyses, allow classification of the type of proteins added in saliva as mimetics of disease. PepTenChip® system is useful and convenient for examination of periodontal disease in health care.