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In this study, the influence of the content of Al and Co in the diffusion source on the magnetic performance and microstructure of the diffused magnet was discussed by grain boundary diffusion treatment with Pr70Al30–xCox (x=0 at%, 10 at%, 15 at%, 20 at%, 30 at%) alloys. When the Co content in the diffusion source increases from 0 at% to 10 at%, the coercivity enhancement in the Pr70Al20Co10 diffused magnet is the highest, increased from 1.62 to 2.24 T, higher than 2.01 T of the Pr70Al30 diffused magnet. With the further increase of Co content in the diffused source, the coercivity of the diffused magnet decreases gradually, the coercivity of Pr70Al15Co15, Pr70Al20Co10 and Pr70Co30 diffused magnet is 2.15, 1.99 and 1.81 T, respectively. Microstructural analysis shows that plenty of continuous grain boundary phases (CGBPs) can be formed in the Pr70Al20Co10 diffused magnet under the synergistic effect of Al and Co, which leads to the enhancement of magnetic isolation between more adjacent grains. However, the amount of CGBP in the diffused magnets gradually decreases with the further increase of Co content in the diffusion source.

Supervisor:前之園 信也, 先端科学技術研究科, 博士

Exosomes are cell-derived membrane-enclosed biological nanoparticles that carry lots of parental molecular information, and are recognized as an ideal biomarker for non-invasive diagnosis. However, due to the low abundance of exosomes in plasma samples and the interferences from complex biological matrices, the sensitive and direct detection of exosomes still remains a challenge. Here, by combining the direct magnetic isolation with in situ fluorescence imaging, we developed a Single Microbead-based Fluorescent Aptasensor (SMFA) for specific enrichment and sensitive quantification of exosomes from plasma. In the SMFA, a single aptamer-modified microbead (MB) served as the reaction carrier so that the specific exosomes inserted with a fluorescent anchor will be highly enriched on the single MB. By in situ fluorescence imaging to monitor the fluorescence signals on the single MB, sensitive detection of exosomes can be realized without the requirement of any signal amplification routes, and as low as 4.9 × 104 particles per μL of exosomes could be simply detected. More importantly, the SMFA could be applied for direct detection of the exosomes from small amounts of clinical plasma samples without prior purification procedures, indicating its great potential applications in clinical diagnostics.

Tumor-derived exosomes carrying unique surface proteins have shown great promise as novel biomarkers for liquid biopsies. However, point-of-care analysis for tumor-derived exosomes in the blood with low-cost and easy processing is still challenging. Herein, we develop an integrated approach, homogenous magneto-fluorescent exosome (hMFEX) nanosensor, for rapid and on-site tumor-derived exosomes analysis. Tumor-derived exosomes are captured immunomagnetically, which further initiates the aptamer-triggered assembly of DNA three-way junctions in homogenous solution containing aggregation-induced emission luminogens and graphene oxide, resulting in an amplified fluorescence signal. By integrating magnetic isolation and enhanced fluorescence measurement, the hMFEX nanosensor detects tumor-derived exosomes in the dynamic range spanning 5 orders of magnitude with high specificity, and the limit of detection is 6.56 × 104 particles/μL. Analyzing tumor-derived exosomes in limited volume plasma from breast cancer patients demonstrates the excellent clinical diagnostic efficacy of the hMFEX nanosensor. This study provides new insights into the point-of-care testing of tumor-derived exosomes for cancer diagnostics.

Isolating tumor exosomes (TEX) secreted by cancer cells can provide valuable information about the state of a tumor. Here, we present a method to rapidly isolate TEX using magnetic nanowires (MNWs)...

The detection of a low concentration of biomarkers required for an early diagnosis remains a tricky task to pursue. In this context, magnetic nanoparticles (MNPs) have been widely used to isolate/concentrate diluted samples. However, by applying the magnetic field, all MNPs are isolated, both those conjugated with the analyte and free MNPs. This excess of MNP can interfere with the analyte quantification. Herein, we propose a novel and versatile approach that ensures the selective isolation of the analyte-conjugated MNP by exploiting the electrophoretic and magnetophoretic behavior of respectively charged silica nanoparticles (SiNPs) and MNPs. In this work, the robustness and sensitivity of the developed isolation and fluorescence-based detection procedure were confirmed by comparing them with the standard method based on simple magnetic isolation of the analyte. The limit of detection (LOD) is improved by two orders of magnitude, namely, from 1 nM to 10 pM. This finding confirms the potentiality of the method and its possible application in the development of rapid and easy-to-use point-of-care devices.

Methicillin-resistant Staphylococcus aureus (MRSA) is considered to be one of the important hospital-acquired pathogens. MRSA is also commonly associated with hospital-acquired infections and mortality. Quantitative and precise detection of MRSA is essential for rapid diagnosis and subsequent effective disease management strategies. We herein developed a highly specific method for rapid MRSA detection that combines surface-enhanced Raman spectroscopy (SERS) nanotags and polymerase chain reaction (PCR). SERS provided the sensitivity and spectral multiplexing capability while PCR provided the specificity required for the assay. The method was tested by the simultaneous detection of two MRSA specific genes (mecA and femA) amplified from genomic DNA isolated from clinical specimens. Magnetic isolation and rapid duplex detection were performed to obtain a detectable signal down to 104 input copies within 80 min. This demonstrated the potential of the SERS-PCR based approach for the accurate identification of MRSA at an early-diagnosis stage. This study also provides an alternative approach to the existing methods for detecting clinical targets without compromising sensitivity and selectivity, and with minimal handling steps. We thus believe that this approach will find a broad application in clinical applications.

International audience; Macropinocytosis refers to the nonselective uptake of extracellular molecules into many different types of eukaryotic cells within large fluid-filled vesicles named macropinosomes. Macropinosomes are relevant for a wide variety of cellular processes, such as antigen sampling in immune cells, homeostasis in the kidney, cell migration or pathogen uptake. Understanding the molecular composition of the different macropinosomes formed during these processes has helped to differentiate their regulations from other endocytic events. Here, we present a magnetic purification protocol that segregates scarce macropinosomes from other endocytic vesicles at a high purity and in a low-cost and unbiased manner. Our protocol takes advantage of moderate-sized magnetic beads of 100 nm in diameter coupled to mass-spectrometry-based proteomic analysis. Passing the cell lysate through a table-top magnet allows the quick retention of the bead-containing macropinosomes. Unlike other cell-fractionation-based methodologies, our protocol minimizes sample loss and production cost without prerequisite knowledge of the macropinosomes and with minimal laboratory experience. We describe a detailed procedure for the isolation of infection-associated macropinosomes during bacterial invasion and the optimization steps to readily adapt it to various studies. The protocol can be performed in 3 d to provide highly purified and enriched macropinosomes for qualitative proteomic composition analysis.

A Surface Plasmon Resonance (SPR) aptasensor was developed for the detection of Brucella melitensis (B. melitensis) in milk samples. Brucellosis is a bacterial zoonotic disease with global distribution caused mostly by contaminated milk or their products. Aptamers recognizing B. melitensis were selected following a whole bacteria-SELEX procedure. Two aptamers were chosen for high affinity and high specificity. The high affinity aptamer (B70 aptamer) was immobilized on the surface of magnetic silica core-shell nanoparticles for initial purification of the target bacteria cells from milk matrix. Another aptamer, highly specific for B. melitensis cells (B46 aptamer), was used to prepare SPR sensor chips for sensitive determination of Brucella in eluted samples from magnetic purification since direct injection of milk samples to SPR sensor chips is known for a high background unspecific signal. Thus, we integrated a quick and efficient magnetic isolation step for subsequent instant detection of B. melitensis contamination in one ml of milk sample by SPR with a LOD value as low as 27 ± 11 cells.

Enumeration and morphological characterization of circulating tumor cells (CTCs) can be useful in diagnosis and prognosis of metastatic cancer patients. The bio-ferrograph (BF) with its five flow channels, which was developed in the late 1990s for magnetic isolation of biological cells and tissue fragments from fluids, is a modification of the analytical ferrograph. Its use for isolation of rare CTCs from human whole blood (HWB) is a novel approach for the detection of cancer at a cellular level. The isolation process is facilitated by the interaction of specifically magnetized cells with a strong external magnetic field, yielding high recovery rates with no morphological alternation of cells that are isolated on a coverslip glass slide, thus allowing complementary microscopic, chemical, biological, and mechanical analyses. Here, a full mechanical and magnetostatic design of a novel high-throughput BF is presented. The system design is based on an optimized procedure for bio-ferrographic isolation of CTCs from HWB. It incorporates a semi-automated CTC separation system consisting of sample preparation, labeling, and staining; magnetic isolation; and system recovery. The design process was optimized based on experimental feasibility tests and finite element analyses. The novel bench-top system consists of 100 flow channels, allowing simultaneous analysis of multiple samples from 20 patients in each run, with the potential to become a decision-making tool for medical doctors when monitoring patients in a hospital setting. It opens a new route for early diagnosis, prognosis, and treatment of cancers, as well as other diseases, such as osteoarthritis.

Grain boundary diffusion process (GBDP) with low melting-point alloy ribbons, namely Pr70Cu15Al15 (PCA), Pr65Tb5Cu15Al15 (PTCA-5) and Pr50Tb20Cu15Al15 (PTCA-20, at%), was applied to Nd-Fe-B sintered magnet (52 M) as thick as 6 mm. The coercivities of diffused magnets by PCA, PTCA-5 and PTCA-20 increase significantly to 19.6 kOe, 24.1 kOe and 24.3 kOe, respectively, from original 14.7 kOe. Microstructural investigations indicate that the coercivity enhancement of PCA magnet is mainly attributed to magnetic isolation by continuous Nd-rich grain boundary phases (GBP). However, the coercivity enhancement of PTCA-20 magnet is mainly owing to magnetic hardening by Tb-rich shells (TRS), and that of PTCA-5 magnet results from a combined strengthening of magnetic isolation by GBP and magnetic hardening by TRS. Meanwhile, the coercivity of PTCA-5 magnet is as high as that of PTCA-20 magnet, but the former consumes Tb only 0.60 wt%, lower than 1.23 wt% for the latter.

Background Highly efficient capture and detection of circulating tumor cells (CTCs) remain elusive mainly because of their extremely low concentration in patients’ peripheral blood. Methods We present an approach for the simultaneous capturing, isolation, and detection of CTCs using an immuno-fluorescent magnetic nanobead system (iFMNS) coated with a monoclonal anti-EpCAM antibody. Results The developed antibody nanobead system allows magnetic isolation and fluorescent-based quantification of CTCs. The expression of EpCAM on the surface of captured CTCs could be directly visualized without additional immune-fluorescent labeling. Our approach is shown to result in a 70–95% capture efficiency of CTCs, and 95% of the captured cells remain viable. Using our approach, the isolated cells could be directly used for culture, reverse transcription-polymerase chain reaction (RT-PCR), and immunocytochemistry (ICC) identification. We applied iFMNS for testing CTCs in peripheral blood samples from a lung cancer patient. Conclusions It is suggested that our iFMNS approach would be a promising tool for CTCs enrichment and detection in one step. Graphic abstract

The presence of iron in circulating monocytes is well known as they play an essential role in iron recycling. It has been demonstrated that the iron content of blood cells can be measured through their magnetic behavior; however, the magnetic properties of different monocyte subtypes remain unknown. In this study, we report for the first time, the magnetic behavior of classical, intermediate and non-classical monocytes, which is related to their iron storage capacity. The magnetic properties of monocytes were compared to other blood cells, such as lymphocytes and red blood cells in the oxyhemoglobin and methemoglobin states, and a cancer cell type. For this analysis, we used an instrument referred to as Cell Tracking Velocimetry (CTV), which quantitatively characterizes the magnetic behavior of biological entities. Our results demonstrate that significant fractions of the intermediate and non-classical monocytes have high magnetophoretic mobilities, equivalent to methemoglobin red blood cells and higher than the classical subset, suggesting their higher iron storage capacities. Moreover, our findings have implications for the immunomagnetic separation industry; we demonstrate that negative magnetic isolation techniques for recovering monocytes from blood should be used with caution, as it is possible to lose magnetic monocytes when using this technique.

Highly efficient capture and detection of circulating tumor cells (CTCs) remain elusive mainly because of their extremely low concentration in the peripheral blood of patients. Herein, we present an approach for the simultaneous capturing, isolation, and detection of CTCs using an immuno-fluorescent magnetic nanobead system (iFMNS) coated with a monoclonal anti-EpCAM antibody. The developed antibody nanobead system allows magnetic isolation and fluorescent-based quantification of CTCs. The expression of EpCAM on the surface of captured CTCs could be directly visualized without additional immune-fluorescent labeling. Our approach is shown to result in a 70 - 95% capture efficiency of CTCs, and 95% of the captured cells remain viable. Using our approach, the isolated cells could be directly used for culture, reverse transcription-polymerase chain reaction (RT-PCR), and immunocytochemistry (ICC) identification. We applied iFMNS for testing CTCs in peripheral blood samples from a lung cancer patient, which suggested that this approach would be a promising tool for CTCs enrichment and detection in one step.

Magnetic nanoparticles (MNPs) have garnered increasing attention for use in the improvement and production of magnetic biosensing, magnetic isolation and purification, drug delivery, quantitative investigation, and magnetic imaging for numerous usages. The latest improvements in the exploitation of nanomagnetic particles for amending various levels of analytical procedures are reviewed. According to the results obtained in the field of analytical chemistry research, the exploitation of magnetic fields affords useful and beneficial techniques for manipulating and examining particles at the molecular level. Among these different applications of magnetic nanomaterials in analytical chemistry, this chapter considers the usages of MNPs in colorimetric sensors. Colorimetric techniques have acquired noteworthy consideration due to such benefits as selectivity; easy preparation; and fast, sensitive, and cost-effective recognition; and it does not require advanced and sophisticated devices.

International audience; Flow cytometry and magnetic bead technology enable the separation of cell populations with the highest degree of purity. Here, we describe protocols to sort bovine neutrophils from blood, the labeling and sorting, including gating strategies. We also provide advice to preserve neutrophil viability and detail a protocol to measure phagocytosis and oxidative species production.

A magnetically isolated MOSFET driving circuit based on a driving transformer has been widely used in aerospace secondary power supplies. Based on the analysis of two typical magnetically isolated driving circuits, a new type of driving circuit is proposed. This circuit has the characteristics of zero negative voltage, high reliability, and wide duty cycle operating range. It is very suitable for a new generation of aerospace high-efficiency high-power-density power supply topology, and it has important significance for improving the efficiency of space borne secondary power supplies.

Isolating and analyzing tumor-derived exosomes (TEX) can provide important information about the state of a tumor, facilitating early diagnosis and prognosis. Since current isolation methods are mostly laborious and expensive, we propose herein a fast and cost-effective method based on a magnetic nanoplatform to isolate TEX. In this work, we have tested our method using three magnetic nanostructures: (i) Ni magnetic nanowires (MNWs) (1500 &times, 40 nm), (ii) Fe3O4 nanorods (NRs) (41 &times, 7 nm), and (iii) Fe3O4 cube-octahedral magnetosomes (MGs) (45 nm) obtained from magnetotactic bacteria. The magnetic response of these nanostructures has been characterized, and we have followed their internalization inside canine osteosarcoma OSCA-8 cells. An overall depiction has been obtained using a combination of Fluorescence and Scanning Electron Microscopies. In addition, Transmission Electron Microscopy images have shown that the nanostructures, with different signs of degradation, ended up being incorporated in endosomal compartments inside the cells. Small intra-endosomal vesicles that could be precursors for TEX have also been identified. Finally, TEX have been isolated using our magnetic isolation method and analyzed with a Nanoparticle tracking analyzer (NanoSight). We observed that the amount and purity of TEX isolated magnetically with MNWs was higher than with NRs and MGs, and they were close to the results obtained using conventional non-magnetic isolation methods.

The Sn addition MnBi bulk magnets with high density and superior coercivity (Hc) were prepared by hot compaction. It was found that the added Sn was dispersed between the MnBi particles as an intergranular phase. The appropriate intergranular phase content could be beneficial in improving the Hc due to the strong magnetic isolation effects. The Hc of the MnBi/Sn bulk magnet was increased from 8.6 kOe to 11.5 kOe as the Sn addition contents were increased in the range of 0 wt.% to 5 wt.%. A relatively high squareness (Mr/Ms) of 0.91 was obtained for the 1 wt.% Sn addition Mn56Bi44 bulk magnet exhibiting the maximum energy product (BH)max of 7.8 MGOe with high Hc of 10 kOe at room temperature.

Human papillomavirus (HPV) and human immunodeficiency virus (HIV) are closely related infections; both diseases are mutually reinforcing due to the co-infection effects, which increase their clinical and epidemiological implications, raising urgent public health concerns. In the present study, a nano-system was developed for simultaneous detection of HPV and HIV targeting E6-HPV16 and Gag-HIV genes respectively. The target sequences from the samples of the patients suffering from head and neck cancer were isolated by magnetic glass particles (MGPs) and subsequently were hybridized with a mixture of the complementary probes attached to cadmium telluride quantum dots (CdTe QDs). Finally, the specific and successful binding was determined by an easy fluorescence analyses and was corroborated by PCR and electrochemistry. Red and green CdTe QDs were used for the dual-color detection of HPV and HIV respectively. Our nano-system can be applied as a proficient tool for the identification of HPV, HIV or both diseases simultaneously, getting a successful and high sensitivity system of detection.

The magnetic circuit environment is the most important factor restricting the normal operation of the magnetorheological fluid (MRF) device. A good magnetic circuit environment can make the MRF dynamometer more economical and practical. In this paper, a new MRF dynamometer is used as the research object. The magnetic field simulation is carried out by Ansoft Maxwell software to study the influence of magnetic isolation ring, magnetic permeability material, Working gap and Excitation current on the magnetic field performance of MRF dynamometer. The research shows that the magnetic induction line distribution in the working gap of the MRF dynamometer is relatively uniform, the magnetic induction intensity is in the range of 120-162 mT, For materials with higher permeability, the stronger the magnetic induction intensity in the working gap is. This research has certain guiding significance for the magnetic circuit optimization of MRF dynamometer.

A novel Cross-Over CMR transformer technology is proposed. The CMR (common-mode rejection) problem of magnetic isolation signal transmission is solved by the secondary Cross-Over differential coils and ANPTM (alternating narrow pulse transmission mode). Based on CSMC 1um BCD technology platform, the simulation results show that compared with the traditional magnetic isolation, the CODT (Cross-Over differential transformer) technique has a lower mismatch and smaller area. The circuit transmission delay reduces by 30% to 23ns with signal transmission frequency as high as 10MHz. The isolation voltage reaches 5kV while improving CMR (CMR greater than $50 \boldsymbol{kV}/\boldsymbol{\mu} \boldsymbol{s}$ ). The proposed technology can be applied in high voltage floating gate driver for IGBT, CoolMOS, and even GaN HMET devices.

Metallothioneins (MTs) are involved in heavy metal detoxification in a wide range of living organisms. Currently, it is well known that MTs play substantial role in many pathophysiological processes, including carcinogenesis, and they can serve as diagnostic biomarkers. In order to increase the applicability of MT in cancer diagnostics, an easy-to-use and rapid method for its detection is required. Hence, the aim of this study was to develop a fully automated and high-throughput assay for the estimation of MT levels. Here, we report the optimal conditions for the isolation of MTs from rabbit liver and their characterization using MALDI-TOF MS. In addition, we described a two-step assay, which started with an isolation of the protein using functionalized paramagnetic particles and finished with their electrochemical analysis. The designed easy-to-use, cost-effective, error-free and fully automated procedure for the isolation of MT coupled with a simple analytical detection method can provide a prototype for the construction of a diagnostic instrument, which would be appropriate for the monitoring of carcinogenesis or MT-related chemoresistance of tumors.

Italy is one of the main producers and exporters of cheese made from unpasteurized sheep milk. Since raw milk and its derived products are known sources of human infections, cheese produced from raw sheep milk could pose a microbiological threat to public health. Hence, the objectives of the study were: to characterize the potential risk of the presence of pathogens Escherichia coli O157, Listeria monocytogenes, and Salmonella in raw ovine milk destined for cheese production obtained from all the sheep farms (n = 24) in the Marches region (Central Italy) that directly transform raw milk into cheeses and to evaluate the equivalence between the analytical methods applied. A three-step molecular method (simultaneous culture enrichment, species-specific DNA magnetic isolation, and multiplex real-time polymerase chain reaction) was used for milk (n = 143) and cheese (n = 5) analysis over a 3-year period. L. monocytogenes was not detected on any of the farms, while E. coli O157 was found on three farms, although only using the molecular method. Four farms tested positive for Salmonella spp., and Salmonella enterica subsp. diarizonae serovar 61:k:1,5,7 was isolated in one of those cases. This information highlights the need to develop preventative measures to guarantee a high level of consumer safety for this specific product line, and the molecular method could be a time-saving and sensitive tool to be used in routine diagnosis.

Neutrophil migration and chemotaxis are fundamentally important biological processes and have direct relevance to various health problems. Microfluidic devices provide useful experimental tools for the quantitative analysis of neutrophil chemotaxis in controlled microenvironments. However, such experiments often require specialized research facilities and lengthy cell preparation from a large amount of blood. In this paper, we report a new, yet simple, all-on-chip method for the magnetic isolation of untouched neutrophils directly from small volumes of blood, followed by chemotaxis testing on the same microfluidic device. Furthermore, we incorporated a cell-docking structure to the microfluidic device for better control of the cells’ initial positions before the chemotaxis test and for improved data analysis. The whole experiment can be performed in less than 25 minutes. We successfully validated this method by testing neutrophil chemotaxis to both purified chemoattractant (i.e. fMLP) and clinical samples (sputum from patients with Chronic Obstructive Pulmonary Disease, COPD). Thus, the “all-on-chip” method can be a useful tool for research and clinical applications that require rapid and accurate chemotaxis testing of untouched neutrophils.

Iron-based hierarchical nanostructures with enhanced properties have attracted intense interest as sorbents for water treatment. It is important to design a facile and scalable procedure for the preparation of absorbents with hierarchical structures. Here, a green solid-state method for the fabrication of Fe/FeOOH hierarchical nanostructures (Fe-HNs) is developed. The obtained samples with sizes in the range of 500–800 nm are three-dimensional (3D) hierarchical structures assembled from ultrathin nanosheets. A plausible “reduction–oxidation” mechanism is proposed for the formation process of the Fe-HNs. The as-prepared Fe-HNs are utilized as an adsorbent for the removal of Congo red and Cr(VI) ions from aqueous solution. The results show that the adsorbent exhibits high adsorption capacity, fast adsorption rate, and superior magnetic isolation. These Fe-HNs prepared by a facile solid-state method display very promising potential application in water treatment as an adsorbent.

The noninvasive detection of tumor cells is significantly important for early diagnosis of cancers and monitoring of their progress. Herein, we have developed a novel aptamer-based isothermal exponential amplification reaction (EXPAR) for sensitive detection of tumor cells. In this new assay, biotinylated sgc8c DNA aptamers are immobilized on streptavidin-coated magnetic beads. After incubation with target CCRF-CEM cells and magnetic isolation, the tumor cells are detected by translating the structure-switching of an aptamer upon tumor cell binding into an input of DNA trigger for EXPAR. Since more and more specific aptamers towards different tumor cells would be identified in the future, our strategy can be easily extended to the detection of different tumor cells just by simply altering the corresponding aptamers. Therefore, this EXPAR-based strategy may serve as a generic platform for the accurate detection of tumor cells, which has great potential for the early diagnosis of cancers.