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Simple and effective detection methods for circulating tumor cells are essential for early detection and progression monitoring of tumors. The use of DNA aptamer and bioluminescence is expected to be a key tool for the simple, effective, and sensitive detection of tumor cells. Herein, we designed multifunctional protein nanoparticles for the detection of tumor cells using DNA aptamer and bioluminescence. Fusion proteins (ELP-poly(d)-POIs), composed of elastin-like polypeptide (ELP) fused with protein of interests (POIs) via poly(aspartic acid) (poly(d)), formed the protein nanoparticles based on the temperature responsivity of ELP sequences, leading to multiply displayed POIs on the protein nanoparticles. In the present study, we focused on porcine circovirus type 2 replication initiation protein (Rep), which covalently conjugated with DNA aptamers, and NanoLuc luciferase (Nluc), which emitted a strong bioluminescence, as POIs. ELP-poly(d)-Rep and ELP-poly(d)-Nluc were constructed and formed the protein nanoparticles with multiply displayed Nluc and Rep (DNA aptamer) that amplified the bioluminescence signal and tumor recognition ability. Mucin-1 (MUC1)-overexpressing human breast tumor MCF7 cells and MUC1-recognizing aptamer (MUC1 aptamer) were selected as models. The MUC1 aptamer-conjugated protein nanoparticles exhibited a 13.7-fold higher bioluminescence signal to MCF-7 cells than to human embryonic kidney 293 (HEK293) cells, which express low levels of MUC1. Furthermore, the protein nanoparticles could detect up to 70.7 cells/mL of MCF-7 cells from a cell suspension containing HEK-293. The protein nanoparticles with multiple Rep and Nluc show a great potential as a material for detecting CTCs.

DOI： 10.1021/acsbiomaterials.3c00712

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BACKGROUND: Rapid increase in COVID-19 suspected cases has rendered disease diagnosis challenging, mainly depending upon RT-qPCR. Reliable, rapid, and cost-effective diagnostic assays that complement RT-qPCR should be introduced after thoroughly evaluating their performance upon various disease phases, viral load, and sample storage conditions. OBJECTIVE: We investigated the correlation of cycle threshold (Ct) value, which implies the viral load and infection phase, and the storage condition of the clinical specimen with the diagnosis of SARS-CoV-2 through our newly developed in-house rapid enzyme-linked immunosorbent assay (ELISA) system. METHOD: Naso-oropharyngeal samples of 339 COVID-19 suspected cases were collected and evaluated through RT-qPCR that were stored up to 30 days in different conditions (i.e. -80°C, -20°C and initially at 4°C followed by -80°C). The clinical specimens were evaluated with our in-house ELISA system after finalizing the assay method through checkerboard assay and minimizing the signal/noise ratio. RESULT: The ELISA system showed the highest sensitivity (92.9%) for samples with Ct ≤30 and preserving at -80°C temperature. The sensitivity reduced proportionally with increasing Ct value and preserving temperature. However, the specificity ranged between 98.3% and 100%. CONCLUSION: The results indicate the necessity of early infection phase diagnosis and lower temperature preservation of samples to perform rapid antigen ELISA tests.

DOI： 10.1080/14787210.2021.1976144

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Detection of small amounts of target molecules with high sensitivity is important for the diagnosis of many diseases, including cancers, and is particularly important to detect early stages of disease. Here, we report the development of a temperature-responsive fusion protein (ELP-DCN) comprised of an elastin-like polypeptide (ELP), poly-aspartic acid (D), antibody-binding domain C (C), and NanoLuc luciferase (N). ELP-DCN proteins form nanoparticles above a certain threshold temperature that display an antibody-binding domain and NanoLuc luciferase on their surface. ELP-DCN nanoparticles can be applied for enhancement of immunoassay systems because they provide more antibody-binding sites and an increased number of luciferase molecules, resulting in an increase in assay signal. Here, we report the detection of human serum albumin (HSA) as a model protein using anti-HSA and ELP-DCN proteins. Upon formation of ELP-DCN nanoparticles, the detection limit improved tenfold compared to the monomeric form of ELP-DCN.

DOI： 10.1007/s00216-021-03842-2

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BACKGROUND: Vaccination with the Oxford-AstraZeneca COVID-19 vaccine (AZD1222) initially started in the UK and quickly implemented around the Globe, including Bangladesh. Up to date, more than nine million doses administrated to the Bangladeshi public. METHOD: Herein, we studied the antibody response to the first dose of AZD1222 in 86 Bangladeshi individuals using in-house ELISA kits. Study subjects were categorized into two groups, convalescent and uninfected, based on prior infection history and SARS-CoV-2 nucleocapsid-IgG profiles. RESULTS: All the convalescent individuals presented elevated spike-1-IgG compared to 90% of uninfected ones after the first dose. Day >28 post-vaccination, the convalescent group showed six times higher antibody titer than the uninfected ones. The most elevated antibody titers for the former and later group were found at Day 14 and Days >28 post-vaccination, respectively. The spike-1-IgA titer showed a similar pattern as spike-1-IgG, although in a low-titer. In contrast, the IgM titer did not show any significant change in either group. CONCLUSION: High antibody titer in the convalescent group, signify the importance of the first dose among the uninfected group. This study advocates the integration of antibody tests in vaccination programs in the healthcare system for maximizing benefit.

DOI： 10.1080/14760584.2021.1977630

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Artificial materials have no biological functions, but they are important for medical devices such as artificial organs and matrices for regenerative medicine. In this study, mitogenic and differentiation-inducible materials are devised via the simple coating of polypeptides, which contain the sequence of epidermal growth factor or insulin-like growth factor with a key amino acid (3,4-dihydroxyphenylalanine) of underwater adhesive proteins. The adhesive polypeptides prepared via solid-phase synthesis form layers on various substrates involving organic and inorganic materials to provide biological surfaces. Through the direct activation of cognate receptors on interactive surfaces, the materials enable increased cell growth and differentiation compared to that achieved by soluble growth factors. This superior growth and differentiation are attributed to the long-lasting signal transduction (triggered by the bound growth factors), which do not cause receptor internalization and subsequent downregulation.

DOI： 10.1002/advs.202100961

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BACKGROUND: Dynamics and persistence of neutralizing and non-neutralizing antibodies can give us the knowledge required for serodiagnosis, disease management, and successful vaccine design and development. The disappearance of antibodies, absence of humoral immunity activation, and sporadic reinfection cases emphasize the importance of longitudinal antibody dynamics against variable structural antigens. METHODS: In this study, twenty-five healthy subjects working in a SARS-COV-2 serodiagnostic assay development project were enrolled, and their sign and symptoms were followed up to six months. Three subjects showed COVID-19-like symptoms, and three subjects' antibody dynamics were followed over 120 days by analyzing 516 samples. We have developed 12 different types of in-house ELISAs to observe the kinetics of IgG, IgM, and IgA against four SARS-CoV-2 proteins, namely nucleocapsid, RBD, S1, and whole spike (S1+S2). For the development of these assays, 30-104 pre-pandemic samples were taken as negative controls and 83 RT-qPCR positive samples as positive ones. RESULTS: All three subjects presented COVID-19-like symptoms twice, with mild symptoms in the first episode were severe in the second, and RT-qPCR confirmed the latter. The initial episode did not culminate with any significant antibody development, while a multifold increase in IgG antibodies characterized the second episode. Interestingly, IgG antibody development concurrent with IgM and IgA and persisted, whereas the latter two weans off rather quickly if appeared. CONCLUSION: Antibody kinetics observed in this study can provide a pathway to the successful development of sero-diagnostics and epidemiologists to predict the fate of vaccination currently in place.

DOI： 10.2147/JIR.S313188

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BACKGROUND: Serological tests detecting severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are widely used in seroprevalence studies and evaluating the efficacy of the vaccination program. Some of the widely used serological testing techniques are enzyme-linked immune-sorbent assay (ELISA), chemiluminescence immunoassay (CLIA), and lateral flow immunoassay (LFIA). However, these tests are plagued with low sensitivity or specificity, time-consuming, labor-intensive, and expensive. We developed a serological test implementing flow-through dot-blot assay (FT-DBA) for SARS-CoV-2 specific IgG detection, which provides enhanced sensitivity and specificity while being quick to perform and easy to use. METHODS: SARS-CoV-2 antigens were immobilized on nitrocellulose membrane to capture human IgG, which was then detected with anti-human IgG conjugated gold nanoparticle (hIgG-AuNP). A total of 181 samples were analyzed in-house. Within which 35 were further evaluated in US FDA-approved CLIA Elecsys SARS-CoV-2 assay. The positive panel consisted of RT-qPCR positive samples from patients with both <14 days and >14 days from the onset of clinical symptoms. The negative panel contained samples collected from the pre-pandemic era dengue patients and healthy donors during the pandemic. Moreover, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of FT-DBA were evaluated against RT-qPCR positive sera. However, the overall efficacies were assessed with sera that seroconverted against either nucleocapsid (NCP) or receptor-binding domain (RBD). RESULTS: In-house ELISA selected a total of 81 true seropositive and 100 seronegative samples. The sensitivity of samples with <14 days using FT-DBA was 94.7%, increasing to 100% for samples >14 days. The overall detection sensitivity and specificity were 98.8% and 98%, respectively, whereas the overall PPV and NPV were 99.6% and 99%. Moreover, comparative analysis between in-house ELISA assays and FT-DBA revealed clinical agreement of Cohen's Kappa value of 0.944. The FT-DBA showed sensitivity and specificity of 100% when compared with commercial CLIA kits. CONCLUSION: The assay can confirm past SARS-CoV-2 infection with high accuracy within 2 minutes compared to commercial CLIA or in-house ELISA. It can help track SARS-CoV-2 disease progression, population screening, and vaccination response. The ease of use of the assay without requiring any instruments while being semi-quantitative provides the avenue of its implementation in remote areas around the globe, where conventional serodiagnosis is not feasible.

DOI： 10.2147/IJN.S313140

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BACKGROUND: In the ongoing pandemic situation of COVID-19, serological tests can complement the molecular diagnostic methods, and can be one of the important tools of sero-surveillance and vaccine evaluation. AIM: To develop and evaluate a rapid SARS-CoV-2 specific ELISA for detection of anti-SARS-CoV2 IgG from patients' biological samples. METHODS: In order to develop this ELISA, three panels of samples (n = 184) have been used: panel 1 (n = 19) and panel 2 (n = 60) were collected from RT-PCR positive patients within 14 and after 14 days of onset of clinical symptoms, respectively; whereas panel 3 consisted of negative samples (n = 105) collected either from healthy donors or pre-pandemic dengue patients. As a capturing agent full-length SARS-CoV2 specific recombinant nucleocapsid was immobilized. Commercial SARS-CoV2 IgG kit based on chemiluminescent assay was used for the selection of samples and optimization of the assay. The threshold cut-off point, inter-assay and intra-assay variations were determined. RESULTS: The incubation/reaction time was set at a total of 30 minutes with the sensitivity of 84% (95% confidence interval, CI, 60.4%, 96.6%) and 98% (95% CI, 91.1%, 100.0%), for panel 1 and 2, respectively; with overall 94.9% sensitivity (95% CI 87.5%, 98.6%). Moreover, the clinical specificity was 97.1% (95% CI, 91.9%, 99.4%) with no cross reaction with dengue samples. The overall positive and negative predictive values are 96.2% (95% CI 89.2%, 99.2%) and 96.2% (95% CI, 90.6% 99.0%), respectively. In-house ELISA demonstrated 100% positive and negative percent agreement with Elecsys Anti-SARS-CoV-2, with Cohen's kappa value of 1.00 (very strong agreement), while comparing 13 positive and 17 negative confirmed cases. CONCLUSION: The assay is rapid and can be applied as one of the early and retrospective sero-monitoring tools in all over the affected areas.

DOI： 10.1371/journal.pone.0246346

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Monoclonal antibodies have been developed as anticancer agents to block immune checkpoint pathways associated with programmed cell death 1 (PD-1) and its ligand PD-L1. However, the high cost of antibodies has encouraged researchers to develop other inhibitor types. Here, biphenyl compounds were conjugated with poly(ethylene glycol) (PEG) to enhance the activity of small molecular inhibitors. Immunoassay results revealed the decrease in the inhibition activity following conjugation with linear PEG, suggesting that the PEG moiety reduced the interaction between the biphenyl structure and PD-L1. However, the inhibitory effect on PD-1/PD-L1 interaction was further enhanced by using branched PEG conjugates. The increase in the number of conjugated biphenyl compounds resulted in increased inhibitory activity. The highest IC50 value was 0.33 μM, which was about 5 times higher than that observed for a non-conjugated monovalent compound. The inhibitory activity was more than 20 times the activity reported for the starting compound. Considering the increase in the inhibition activity, this multivalent strategy can be useful in the design of new immune checkpoint inhibitors.

DOI： 10.1039/d0tb01729a

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Control of the cross-linking reaction is imperative when developing a sophisticated in situ forming hydrogel in the body. In this study, a heteroarmed thermoresponsive (TR) nanoparticle was designed to investigate the mechanism of controlling reactivity of the functional groups introduced into the nanoparticles. The coupling reaction was suppressed/proceeded by utilizing temperature-induced morphological changes of the TR polymer. The heteroarmed TR nanoparticle was prepared by the coassembly of amphiphilic block copolymers possessing both a TR segment and hydrophilic segment with reactive functional groups of succinimide. The longer TR chain on the nanoparticle covered the succinimide group and suppressed the reaction with the primary amine on the external nanoparticle. In contrast, the coupling reaction was promoted at a high temperature to create the chemical cross-linking structure between the nanoparticles because of the exposure of the succinimide group on the surface of the particle as a consequence of the morphological change of the TR polymer. In addition, the thermally controlled chemical reaction modulated initiation of the gelation using a highly concentrated nanoparticle solution. The heteroarmed TR nanoparticle offers great practical advantages for clinical uses, such as embolization agents, through precise control of the reaction.

DOI： 10.1021/acsami.0c12875

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E-cadherin is a key Ca-dependent cell adhesion molecule, which is expressed on many cell surfaces and involved in cell morphogenesis, embryonic development, EMT, etc. The fusion protein E-cad-Fc consists of the extracellular domain of E-cadherin and the IgG Fc domain. On plates coated with this chimeric protein, ES/iPS cells are cultivated particularly well and induced to differentiate. The cells adhere to the plate via E-cad-Fc in the presence of Ca2+ and detach by a chelating agent. For the purpose of clarifying the structures of E-cad-Fc in the presence and absence of Ca2+, we analyzed the molecular structure of E-cad-Fc by AFM in liquid. Our AFM observations revealed a rod-like structure of the entire extracellular domain of E-cad-Fc in the presence of Ca2+ as well as trans-binding of E-cad-Fc with adjacent molecules, which may be the first, direct confirmation of trans-dimerization of E-cadherin. The observed structures were in good agreement with an X-ray crystallographic model. Furthermore, we succeeded in visualizing the changes in the rod-like structure of the EC domains with and without calcium. The biomatrix surface plays an important role in cell culture, so the analysis of its structure and function may help promote cell engineering based on cell recognition.

DOI： 10.1038/s41598-020-72517-2

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Photoreactive polymers that generate active species upon irradiation with light are very useful for modifying the surfaces of substrates. However, water solubility decreases as the number of photoreactive functional groups on the polymer increases because most photoreactive functional groups are hydrophobic. In order to improve the hydrophilicity of the photoreactive polymer, we synthesized polyethylene glycol-based photoreactive polymers bearing hydrophobic azidophenyl groups on their side chains. Because of the hydrophilicity of the ethylene glycol main chain, polymers with large numbers of azidophenyl groups were solubilized in protic solvents compared to hydrophobic alkylene chain-based polymers prepared by radical polymerization of methacrylate monomers. Polymers were immobilized on various substrates by irradiation with ultraviolet light and were shown to suppress nonspecific interactions between proteins and cells on the substrate. We conclude that such polymers are useful, highly water soluble antifouling agents.

DOI： 10.1021/acsabm.0c00628

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There is growing interest in the functional roles of the extracellular matrix (ECM) in regulating the fate of pluripotent stem cells (PSCs). An artificially bioengineered ECM provides an excellent model for studying the molecular mechanisms underlying self-renewal and differentiation of PSCs, without multiple unknown and variable factors associated with natural substrates. Here, we have engineered multifunctional fusion proteins that are based on peptides from laminin, including p20, RGD, and elastin-like polypeptide (ELP), where laminin peptides work as cell adhesion molecules (CAMs) and ELP to promote anchorage. The functionality of these chimeric proteins, referred to as ERE-p20 and E-p20, was assessed by determining their ability to immobilize cells on a hydrophobic polystyrene surface, improve mouse induced pluripotent stem cells (miPSCs) attachment, and promote miPSC differentiation to neural progenitors. ERE-p20 and E-p20 proteins showed hydrophobic binding saturation to the polystyrene plates around 500 nM (2.39 μg/cm2 ) and 750 nM (2.27 μg/cm2 ) protein concentrations, respectively. The apparent maximum cell binding to ERE-p20 and E-p20 was approximately 81% and 73%, respectively, relative to gelatin. For neural precursors, neurite outgrowth was enhanced by the presence of RGD and p20 peptides. The expression levels of neuronal marker protein MAP2 were upregulated approximately 2.5-fold and threefold by ERE-p20 and E-p20, respectively, relative to laminin. Overall, we have shown that elastin-mimetic fusion proteins consisting of p20 with and without RGD peptides are able to induce neuronal differentiation. In conclusion, our newly designed bioengineered fusion proteins allow preparation of specific bioactive matrices or coating/scaffold for miPSCs differentiation.

DOI： 10.1002/jbm.b.34600

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Cancer immunotherapy has been revolutionized by the development of monoclonal antibodies (mAbs) that inhibit interactions between immune checkpoint molecules, such as programmed cell-death 1 (PD-1), and its ligand PD-L1. However, mAb-based drugs have some drawbacks, including poor tumor penetration and high production costs, which could potentially be overcome by small molecule drugs. BMS-8, one of the potent small molecule drugs, induces homodimerization of PD-L1, thereby inhibiting its binding to PD-1. Our assay system revealed that BMS-8 inhibited the PD-1/PD-L1 interaction with IC50 of 7.2 μM. To improve the IC50 value, we designed and synthesized a small molecule based on the molecular structure of BMS-8 by in silico simulation. As a result, we successfully prepared a biphenyl-conjugated bromotyrosine (X) with IC50 of 1.5 μM, which was about five times improved from BMS-8. We further prepared amino acid conjugates of X (amino-X), to elucidate a correlation between the docking modes of the amino-Xs and IC50 values. The results suggested that the displacement of amino-Xs from the BMS-8 in the pocket of PD-L1 homodimer correlated with IC50 values. This observation provides us a further insight how to derivatize X for better inhibitory effect.

DOI： 10.3390/ijms21103639

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DNA-displaying nanoparticles comprised of conjugates of single-stranded DNA (ssDNA) and elastin-like polypeptide (ELP) were developed. ssDNA was enzymatically conjugated to ELPs via a catalytic domain of Porcine Circovirus type 2 replication initiation protein (pRep) fused to ELPs. Nanoparticles were formed upon heating to temperatures above the phase transition temperature due to the hydrophobicity of ELPs and the hydrophilicity of conjugated ssDNA. We demonstrated the applicability of the resultant nanoparticles as drug carriers with tumor-targeting properties by conjugating a DNA aptamer, which is known to bind to Mucin 1 (MUC1), to ELPs. DNA aptamer-displaying nanoparticles encapsulating the anti-cancer drug paclitaxel were able to bind to cells overexpressing MUC1 and induce cell death.

DOI： 10.1088/1361-6528/ab8042

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A fusion protein, designated ELP-D-C, comprised of a hydrophobic elastin-like polypeptide unit, a hydrophilic aspartic acid-rich peptide unit, and an antibody-binding domain as a functional unit, was constructed. Upon heat induction, ELP-D-C forms micellar nanoparticles displaying antibody-binding domains on their surfaces. The protein nanoparticles were able to incorporate hydrophobic fluorescent compounds and subsequently detect target molecules via antibody binding by the resulting fluorescence intensity, which was proportional to the log of the concentration of the target molecule.

DOI： 10.2116/analsci.19N024

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Supramolecular protein hydrogels with tunable properties represent promising candidates for advanced designer extracellular matrices (ECMs). To control cellular functions, ECMs should be able to spatiotemporally regulate synergistic signaling between transmembrane receptors and growth factor (GF) receptors. In this study, we developed genetically engineered temperature-responsive multifunctional protein hydrogels. The designed hydrogel was fabricated by combining the following four peptide blocks: thermosensitive elastin-like polypeptides (ELPs), a polyaspartic acid (polyD) chain to control aggregation and delivery of GFs, a de novo-designed helix peptide that forms antiparallel homotetrameric coiled-coils, and a biofunctional peptide. The resultant coiled-coil unit bound ELPs (CUBEs) exhibit a controllable sol-gel transition with tunable mechanical properties. CUBEs were functionalized with bone sialoprotein-derived RGD (bRGD), and human umbilical vein endothelial cells (HUVECs) were three-dimensionally cultured in bRGD-modified CUBE (bRGD-CUBE) hydrogels. Proangiogenic activity of HUVECs was promoted by bRGD. Moreover, heparin-binding angiogenic GFs were immobilized to bRGD-CUBEs via electrostatic interactions. HUVECs cultured in GF-tethered bRGD-CUBE hydrogels formed three-dimensional (3-D) tubulelike structures. The designed CUBE hydrogels may demonstrate utility as advanced smart biomaterials for biomedical applications. Further, the protein hydrogel design strategy may provide a novel platform for constructing designer 3-D microenvironments for specific cell types.

DOI： 10.1021/acs.biomac.9b01496

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OBJECTIVE: We developed a DNA-NanoLuc luciferase (NnaoLuc) conjugates for DNA aptamer-based sandwich assay using the catalytic domain of the replication initiator protein derived from porcine circovirus type 2 (pRep). RESULTS: For construction of DNA aptamer and NanoLuc conjugate using the catalytic domain of Rep from PCV2. pRep fused to NanoLuc was genetically constructed and expressed in E. coli. After purification, the activities of fused pRep and NanoLuc were evaluated, and DNA-NanoLuc conjugates were constructed via the fused pRep. Finally, constructed DNA-NanoLuc conjugates were applied for use in a DNA aptamer-based sandwich assay. Here, pRep was used not only for conjugation of the NanoLuc to the detection aptamer, but also for immobilization of the capture aptamer on the plate surface. CONCLUSION: We have demonstrated that DNA-NanoLuc conjugates via the catalytic domain of PCV2 Rep could be applied for DNA aptamer-based sandwich assay system.

DOI： 10.1007/s10529-018-02641-7

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Modification of protein-based drug carriers with tumor-targeting properties is an important area of research in the field of anticancer drug delivery. To this end, we developed nanoparticles comprised of elastin-like polypeptides (ELPs) with fused poly-aspartic acid chains (ELP-D) displaying DNA aptamers. DNA aptamers were enzymatically conjugated to the surface of the nanoparticles via genetic incorporation of Gene A* protein into the sequence of the ELP-D fusion protein. Gene A* protein, derived from bacteriophage ϕX174, can form covalent complexes with single-stranded DNA via the latter's recognition sequence. Gene A* protein-displaying nanoparticles exhibited the ability to deliver the anticancer drug paclitaxel (PTX), whilst retaining activity of the conjugated Gene A* protein. PTX-loaded protein nanoparticles displaying DNA aptamers known to bind to the MUC1 tumor marker resulted in increased cytotoxicity with MCF-7 breast cancer cells compared to PTX-loaded protein nanoparticles without the DNA aptamer modification.

DOI： 10.1007/s11033-018-4467-2

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Publishing type：Research paper (scientific journal)   Publisher：MyJove Corporation  

DOI： 10.3791/57377

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OBJECTIVE: We explored the co-localization of multiple enzymes on a DNA backbone via a DNA-binding protein, Gene-A* (A*-tag) to increase the efficiency of cascade enzymatic reactions. RESULTS: Firefly luciferase (FLuc) and pyruvate orthophosphate dikinase (PPDK) were genetically fused with A*-tag and modified with single-stranded (ss) DNA via A*-tag. The components were assembled on ssDNA by hybridization, thereby enhancing the efficiency of the cascading bioluminescent reaction producing light emission from pyrophosphate. The activity of A*-tag in each enzyme was investigated with dye-labeled DNA. Co-localization of the enzymes via hybridization was examined using a gel shift assay. The multi-enzyme complex showed significant improvement in the overall efficiency of the cascading reaction in comparison to a mixture of free enzymes. CONCLUSION: A*-tag is highly convenient for ssDNA modification of versatile enzymes, and it can be used for construction of functional DNA-enzyme complexes.

DOI： 10.1007/s10529-018-2517-4

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Nanoparticles are small size-controlled particles from 1 to 100 nm diameters and characterized by their structure, base material and functional units displayed on their surfaces. In this study, protein-based nanoparticles composed of a hydrophobic elastin-like peptide unit, a hydrophilic aspartic acid-rich peptide unit and displaying antibody binding domains on their surfaces, were designed and genetically synthesized. The constituent fusion proteins, termed ELP-D-C, were found to exist in monomeric form (ELP-D-C/monomer) at low temperature. Above the phase transition temperature, however, ELP-D-C was found to rapidly self-assemble to form spherical micelles (ELP-D-C/micelle) with a hydrophobic core and diameters of ∼40 nm. Furthermore, ELP-D-C/micelle were shown to display antibody binding domains on their surfaces, which allowed for immobilization of antibodies and subsequent formation of large, visually detectable complexes in the presence of target molecule (antigen), whose sizes increased in proportion to the target molecule concentration. The observed target molecule concentration-dependent complex formation suggests that ELP-D-C/micelle may be useful as base particles in applications such as homogeneous turbidity immunoassays.

DOI： 10.1016/j.ab.2017.12.023

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Self-assembling peptides are attractive materials for tissue engineering applications because of their functionality including high biocompatibility and biodegradability. Modification of self-assembling peptides with functional motifs, such as the cell-adhesive tripeptide sequence RGD leads to functional artificial extracellular matrices (ECMs). In this study, we developed an artificial self-assembling ECM protein tethered with a growth factor via heterotrimer triple-helix (helix A/B/C) formation. The helix A and helix C peptides, which are capable of forming a heterodimer coiled-coil structure, were fused to both ends of a matrix protein composed of the elastin-derived structural unit (APGVGV)12 with an RGD motif. The helix B peptide, which constituents the third helix of the triple-helix structure, was fused with basic fibroblast growth factor (bFGF) for tethering to the artificial ECM proteins. Each recombinant protein exhibited cell adhesion and cell proliferation activities similar to the original, while the designed bFGF-tethered ECM protein exhibited superior cell proliferation activity. These results demonstrate that the approach of creating growth factor-tethered self-assembling proteins via triple-helix formation can be applied to develop functional ECMs for tissue engineering applications.

DOI： 10.1088/1748-605X/aa7616

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The amyloid beta (Aβ) peptide is regarded as a causative agent of Alzheimer's disease. In this study, fluorescent and luminescent fusion proteins were constructed to analyze Aβ aggregation. A system was developed to monitor changes in luminescence that provides information about Aβ aggregation. In the presence of monomeric Aβ, the fusion protein exhibits higher luminescence intensity, and the luminescence intensity is diminished after aggregation of the fusion protein and Aβ. In contrast, the fluorescence is sustained in the presence of Aβ. In the absence of Aβ, the fusion protein self-aggregates, and its luminescence and fluorescence are quenched, thus decreasing the background fluorescence and enhancing the detection of Aβ inside and outside the cells. The ratio of the luminescence intensity to the fluorescence intensity would allow the aggregation degrees of Aβ to be distinguished. This study would be a promising method for analyzing the aggregation state of a particular amyloid protein/peptide (monomer, oligomer, or fibril), as well as the distribution of the amyloid protein/peptide within and at the cell surface, by using a single fusion protein. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

DOI： 10.1002/psc.3003

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DOI： 10.1002/smll.201603104

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:The feasibility of assembling enzymes, catalyzing consecutive reactions, on to a double-stranded DNA (dsDNA) scaffold utilizing zinc finger motifs is described. The catalytic activities of two zinc finger motif-fused enzymes catalyzing a bioluminescence reaction with energy recycling, namely pyruvate phosphate dikinase and firefly luciferase, have been evaluated. Bioluminescence measurements with dsDNA scaffolds coding a different distance between the binding sites for each zinc finger motif-fused enzyme confirmed the effect of the distance, proving the proximity effect of ATP recycling presumed to be the result of efficient intermediate diffusion. Thus, fusion to zinc finger motifs offers a promising option for the assembly of bi-enzymes, catalyzing a consecutive reaction, onto a dsDNA scaffold with a proximity effect.

DOI： 10.1007/s10529-014-1644-9

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DOI： 10.1371/journal.pone.0049235

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We applied Systematic Evolution of Ligands by EXponential enrichment using Small Cell Lung Cancer (SCLC) cells. A DNA aptamer was identified and evaluated by fluorescent confocal microscopy and flow cytometry. Our results showed that the DNA aptamer binds to molecules that exist predominantly on target SCLC cell surfaces compared with other types of SCLC cells.

DOI： 10.1039/c0an00962h

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:A method for the evaluation of interactions between protein and ligand using DNA-modified ligands, including signal enhancement of the DNA ligation reactions, is described. For proof of principle, a DNA probe modified by biotin was used. Two DNA probes were prepared with complementary sticky-ends. While one DNA probe was modified at the 5&#039;-end of the sticky-end, the other was not modified. The probes could be ligated together by T4 DNA ligase along the strand without biotin modification. However, in the presence of streptavidin or anti-biotin Fab, the ligation reaction joining the two probes could not occur on either strand.

DOI： 10.1007/s10529-009-0109-z

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DOI： 10.1016/j.jbiotec.2008.09.011

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DOI： 10.1016/j.biomaterials.2008.04.006

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DOI： 10.1021/bc060351o

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:It is widely recognized that stimuli-responsive nanostructures play a promising role in nanodevices for medical treatments and experimental tools. We have designed and constructed a basic structure which controls the distance between two termini domains through temperature reversibility. Our structure, shaped like a bouquet, is composed of two proteins, alpha-helix and elastin-like protein (ELP). Alpha-helices align and bundle the ELP while ELP twists and forms a fiber-like structure at warm temperatures. This ELP conformational change alters the distance between the structure termini at the site opposite the alpha-helix. We connected enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP) at the structure&#039;s two termini to evaluate the distance using fluorescence resonance energy transfer (FRET) efficiency. These proteins spontaneously formed a complex which decreased the distance between the two fluorescent proteins located at its termini, at physiologically relevant temperatures. This change was repeated with complete reversibility (n = 5).

DOI： 10.1021/bc070120x

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Publishing type：Research paper (scientific journal)  

:The interaction between a small ligand and a protein were assessed by electrophoretic mobility shift assay. A sensing probe was created by modifying the model ligand, biotin, with DNA. The complex of DNA-modified ligand and anti-biotin antibody or streptavidin as a target protein was analyzed by agarose gel electrophoresis. The band corresponding to the DNA-modified ligand was shifted in the presence of the target protein, and the intensities of the shifted bands were decreased by adding increasing concentrations of free ligand ranging from 0.1 microM to 100 microM. From this calibration the concentration of ligand in the samples could be determined, allowing for evaluation of the interaction between a small ligand and its target.

DOI： 10.1007/s10529-006-9301-6

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DOI： 10.1016/j.bbrc.2006.05.029

Scopus

PubMed

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Publishing type：Research paper (scientific journal)  

:A novel protein for controlling cellular functions was constructed by combining functional units of various proteins. The Arg-Gly-Asp (RGD) sequence functioning as a cell adhesive function, an epidermal growth factor (EGF) as a cell growth function, and a hydrophobic sequence (E12) as an efficient assembling function, were combined and incorporated into one molecule. The fusion protein, designated ERE-EGF, was produced in Escherichia coli and purified with affinity chromatography using a His-tag. The ERE-EGF coated on an unmodified hydrophobic surface of a cell-culture plate (through the hydrophobic E12 moiety) retained both cell adhesive activity (through the RGD sequence) and cell growth activity (through the EGF moiety).

DOI： 10.1016/j.biomaterials.2006.02.003

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Publishing type：Research paper (scientific journal)  

:Protein transduction domain (PTD)-mediated protein delivery into animal cells is a useful technique for regulating cellular functions. Proteins captured by antibodies were delivered into living cells using an antibody/PTD-fused protein A complex. As a model protein, fluorescent-modified antibodies, captured by their respective primary antibody, were analyzed by fluorescence-activated cell sorting (FACS) which showed that the fluorescent-modified antibodies were directly delivered into cells. Peroxidase, captured by its specific antibody, was also delivered into cells and retained its activity.

DOI： 10.1007/s10529-006-9076-9

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Publishing type：Research paper (scientific journal)  

:NeuroD2, one of the neurospecific basic helix-loop-helix transcription factors, has the ability to induce neural differentiation in undifferentiated cells. In this paper, we show that transduction of NeuroD2 protein induced mouse neuroblastoma cell line N1E-115 into neural differentiation. NeuroD2 has two basic-rich domains, one is nuclear localization signal (NLS) and the other is basic region of basic helix-loop-helix (basic). We constructed some mutants of NeuroD2, ND2(Delta100-115) (lack of NLS), ND2(Delta123-134) (lack of basic) and ND2(Delta100-134) (lack of both NLS and basic) for transduction experiments. Using these proteins, we have shown that NLS region of NeuroD2 plays a role of protein transduction. Continuous addition of NeuroD2 protein resulted in N1E-115 cells adopting neural morphology after 4 days and Tau mRNA expression was increased. These results suggest that neural differentiation can be induced by direct addition of NeuroD2 protein.

DOI： 10.1016/j.jbiotec.2006.04.021

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Publishing type：Research paper (scientific journal)  

:One of the promising methods of preparing antibody arrays is immobilizing antibodies with protein A or protein G, each of which binds specifically to the heavy chain constant (Fc) region of immunoglobulin G (IgG). In this system, antibody immobilization efficiency depends on the number of active Fc binding proteins that need to be immobilized on the surface. Here we have designed and constructed an Fc binding protein with a self-adhering ability that can be immobilized on the hydrophobic surface by simple adsorption. It consists of an Fc binding domain of protein G (G3) and hydrophobic domain of elastin (E72). Direct observation revealed its self-adhering ability on the hydrophobic surface. The enzyme-linked immunosorbent assay (ELISA) showed that it retained antibody binding ability on the surface. The antibody array model was prepared on a hydrophobic microwell glass slide with E72G3, which specifically detect the antigen with a sevenfold greater sensitivity than the G3-treated slide. These results suggest that the E72G3 is useful for simple and effective immobilization of antibodies and can be used to fabricate any immuno devices.

DOI： 10.1016/j.ab.2005.12.034

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Publishing type：Research paper (scientific journal)  

:Although the idea of homogeneous electrochemical immunoassay using antibody and an electroactive modified antigen as a probe looks to be very useful for high-throughput drug screening, there have been few reports. One reason for this is the difficulty experienced making an electroactive probe, because the introduction of electroactive compounds to antigens often interferes with the antigen-antibody interaction. To apply a homogeneous electrochemical assay to drug screening, we have designed new probes referring to the information of immobilization on beads which could identify the drug receptor. FK506 (also called Tacrolimus), immunosuppressive agent is modified with ferrocene derivatives as an electron mediator between glucose oxidase and an electrode, at a non-obstructing part. One of the probes still indicated the electrochemical activity as a mediator and had the specific binding capability for FKBP12 (FK506 binding protein). The current decrease in response to the additional FKBP12, detected with constant voltage amperometry using the probe, was observed within 5 min. Then, free FK506 as a leader drug, rapamycin and cyclosporine A as unknown drugs were used as a model for dru

DOI： 10.1016/j.bios.2005.08.002

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DOI： 10.1016/j.bbrc.2005.08.200

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PubMed

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Publishing type：Research paper (scientific journal)  

:We developed an electrochemical detection method for evaluating cellular physiological status based on the stringent response as a means to monitor cell viability. A reporter plasmid was constructed by inserting the beta-galactosidase gene (lacZ) under the control of the rpoS promoter, and then used to transform E. coli cells. Electrochemical responses from the products catalyzed by beta-galactosidase expressed by these E. coli cells were detected using the chronoamperometric technique in a nondestructive manner. Comparisons of response currents between the relA-positive strain and relA-negative strain revealed that increases in these currents were caused by the stringent response due to the stressful alcoholic environment, and thus as a model of stressful cultivating conditions. The current was proportional to the beta-galactosidase activity assayed by a conventional method that required the destruction of cells. The cellular physiological status, which depends on the stringent response as a viability marker, therefore, could then be evaluated online with a current using the rpoS-lacZ reporter gene in the relA-positive strain without pretreatment.

DOI： 10.1002/bit.20459

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Publishing type：Research paper (scientific journal)  

:Detection of specific nucleic acids is important to understand cellular mechanisms and functions of gene regulation. Here, we demonstrated a novel method to detect specific nucleic acids using recombinant protein and oligonucleotides. A recombinant protein YRGnC-11ad, which has a Rev-peptide between enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP) was constructed and expressed in HeLa cells. Rev-peptide, which corresponds to amino acids 34-50 of the HIV-1 Rev protein, indicates disordered structure in solution but forms alpha-helical and elongated conformation upon binding to Rev response element RNA (RRE-RNA) and Rev-aptamer, respectively. We confirmed that YRGnC-11ad could specifically bind to RRE-RNA and Rev-aptamer in cell lysate, and fluorescent resonance energy transfer (FRET) signal was changed upon binding following the conformational change of Rev-peptide. To utilize this FRET signal change toward the detection of specific nucleic acids, we split the RRE-RNA sequence and connected to the complementary oligonucleotide for target nucleic acids. When each two oligonucleotides hybridized to an adjacent region of target nucleic acids corr

DOI： 10.1021/ac048491j

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Publishing type：Research paper (scientific journal)  

:A novel method for detecting interaction between DNA and DNA-binding protein at single molecular level has been proposed. In this study, estrogen receptor-alpha (ER-alpha) was used for biosensing as the proof-example. A 518 bp-long (ca. 176 nm) DNA probe labeled with streptavidin at its 5&#039;-terminus was prepared by inserting a consensus oligonucleotide sequence that binds to ER-alpha. A solution containing ER-alpha was dropped onto the Ni-treated mica substrate on which the DNA prove was previously immobilized, and it was observed by AFM. Specific binding of ER-alpha could be observed by measuring the distance between the site where binding occur, to the streptavidin label.

DOI： 10.1016/j.bios.2003.12.028

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Publishing type：Research paper (scientific journal)  

:A fusion protein consisting of streptavidin and firefly luciferase was constructed to establish an accurate measuring technique of local ATP concentration. The fusion protein retained the binding ability of streptavidin and enzymatic activity of luciferase. Also, it could detect the concentration of antigens and could determine nanomolar concentrations of ATP in its fixed form via interactions with biotin-conjugated antibodies.

DOI： 10.1023/B:BILE.0000032966.17759.08

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Language：English   Publisher：The Society of Materials Engineering for Resources of Japan  

Living cells contain a whole set of intracellular information networks, including signal transduction pathways that control gene expression, intracellular ion influxes and cellular excitability. Stimulations by extracellular molecular messenger or physical stimuli activates these intracellular information networks, modulate physical functions. According to the special characteristics cells could be used as a part of a biological information network within biodevices. Recently, the studies about bioelectronic access to such biological informations as cellular and biomolecular signals were accelerated. This paper describes new concepts of electrochemical methods using cell-based biodevices.

DOI： 10.5188/ijsmer.12.11

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Language：English  

DOI： 10.1023/A:1022339929026

PubMed

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Publishing type：Research paper (scientific journal)  

:Internalization of antibodies into mammalian cells is a useful method for analyzing and regulating cellular function. In this study, we developed a novel method for the delivery of antibodies into cells using the TAT-fused protein. This fusion protein consists of two functional domains, the protein transduction domain of HIV-1 TAT and the B domain of staphylococcal protein A (SpA), which has an ability to bind to the IgG. The TAT-SpA fusion protein was mixed with fluorescence-labeled rabbit IgG and added to cells. The internalization of antibody was analyzed using confocal microscopy and flow cytometry in living cells. As a result, fluorescence-labeled IgG with the TAT-SpA fusion protein was observed intracellularly. Flow cytometry results demonstrated time course and dose dependence relationships of antibody internalization. These results suggest that the TAT-SpA fusion protein can be a useful reagent for the delivery of antibody into cells.

DOI： 10.1016/j.bbrc.2003.09.071

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Publishing type：Research paper (scientific journal)  

:To monitor the extent of cellular physiological stress, the activity of the rpoS promoter was evaluated as a marker of the stress pathway. A reporter plasmid was constructed by inserting the GFPuv gene under the rpoS promoter and used to transform Escherichia coli cells. The fluorescence of the GFPuv protein was measured in intact cells in a non-destructive manner. The physiological status of the cells could be conveniently monitored using the rpoS-GFPuv reporter gene with respect to the cellular growth phase and to elevated ethanol and NaCl concentrations as two examples of environmental stress factors. Comparison of the response of different E. coli strains demonstrated an essential role of the relA gene in the induction of the rpoS-GFPuv reporter gene.

DOI： 10.1016/S0168-1656(01)00446-1

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Language：English   Publishing type：Research paper (international conference proceedings)  

Web of Science

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DOI： 10.1016/S0168-1656(99)00134-0

PubMed

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Language：English   Publishing type：Research paper (international conference proceedings)  

Web of Science

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Language：English  

DOI： 10.5796/electrochemistry.76.529

Web of Science

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Language：Japanese   Publisher：The Society of Polymer Science, Japan  

DOI： 10.1295/kobunshi.56.842

CiNii Books

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Language：Japanese   Publisher：The Society for Biotechnology, Japan  

CiNii Books

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Language：English  

CiNii Books

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Language：Japanese   Publisher：The Society for Biotechnology, Japan  

CiNii Books

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Language：Japanese   Publisher：羊土社  

CiNii Books

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Language：Japanese   Publisher：シーエムシー出版  

CiNii Books

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Language：Japanese   Publisher：The Society for Biotechnology, Japan  

CiNii Books

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Language：Japanese  

CiNii Books

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Publisher：The Japan Society for Precision Engineering  

Recently, the devices of neural network fabrication for basic and applied research of brain are needed. In this study, it is aimed to fabricate device for controlling neurite extension using hole for separate individual cell, conduit for extend neurite, and electrode for stimulate on conduit floor. We fabricated holes and conduits using SU?8 on ITO patterned electrode. We succeeded in controlling neurite extension by culturing cells and electric stimulating on this device.

DOI： 10.11522/pscjspe.2005A.0.951.0

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Language：Japanese   Publisher：The Society for Biotechnology, Japan  

CiNii Books

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Language：Japanese   Publisher：The Society for Biotechnology, Japan  

CiNii Books

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Language：Japanese   Publisher：The Society for Biotechnology, Japan  

CiNii Books

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Language：Japanese   Publisher：共立出版  

CiNii Books

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Language：English  

DOI： 10.1016/j.bios.2003.06.001

Web of Science

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Language：English  

DOI： 10.1016/j.snb.2003.11.003

Web of Science

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Language：English  

Web of Science

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Language：English   Publisher：Akita University  

CiNii Books

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Language：Japanese   Publisher：エヌ・ティー・エス  

CiNii Books

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Language：English  

DOI： 10.1016/S0003-2697(03)00360-9

Web of Science

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Language：Japanese   Publisher：工業調査会  

CiNii Books

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Language：Japanese   Publisher：一般社団法人日本機械学会  

CiNii Books

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Language：Japanese  

CiNii Books

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J-GLOBAL

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Language：Japanese   Publisher：酵素工学研究会  

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Language：Japanese   Publisher：The Society of Polymer Science, Japan  

DOI： 10.1295/kobunshi.50.458

CiNii Books

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J-GLOBAL

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J-GLOBAL

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J-GLOBAL

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Language：Japanese   Publisher：The Society for Biotechnology, Japan  

CiNii Books

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Language：Japanese   Publisher：電気化学会化学センサ研究会  

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Language：Japanese   Publisher：シ-エムシ-  

CiNii Books

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Language：Japanese  

CiNii Books

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Language：Japanese   Publisher：The Society for Biotechnology, Japan  

CiNii Books

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Language：English  

CiNii Books

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Language：Japanese  

CiNii Books

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Language：Japanese   Publisher：The Society for Biotechnology, Japan  

CiNii Books

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Language：Japanese   Publisher：The Society for Biotechnology, Japan  

CiNii Books

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Language：Japanese  

CiNii Books

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J-GLOBAL

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Language：Japanese   Publisher：The Society for Biotechnology, Japan  

CiNii Books

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Language：Japanese  

CiNii Books

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Language：Japanese  

CiNii Books

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Language：Japanese  

CiNii Books

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Language：Japanese   Publisher：The Society for Biotechnology, Japan  

CiNii Books

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Language：Japanese   Publisher：The Society for Biotechnology, Japan  

CiNii Books

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Language：Japanese   Publisher：The Society for Biotechnology, Japan  

CiNii Books

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Language：Japanese   Publisher：社団法人日本農芸化学会  

CiNii Books

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Language：Japanese   Publisher：The Society for Biotechnology, Japan  

CiNii Books

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.32.98

CiNii Books

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