Atsushi Arakaki

Size-selective microcavity array for rapid and efficient detection of circulating tumor cells.

Circulating tumor cells (CTCs) are tumor cells circulating in the peripheral blood of patients with metastatic cancer. Detection of CTCs has clinical significance in cancer therapy because it would enable earlier diagnosis of metastasis. In this research, a microfluidic device equipped with a size-selective microcavity array for highly efficient and rapid detection of tumor cells from whole blood was developed. The microcavity array can specifically separate tumor cells from whole blood on the basis of differences in the size and deformability between tumor and hematologic cells. Furthermore, the cells recovered on the microcavity array were continuously processed for image-based immunophenotypic analysis using a fluorescence microscope. Our device successfully detected approximately 97% of lung carcinoma NCI-H358 cells in 1 mL whole blood spiked with 10-100 NCI-H358 cells. In addition, breast, gastric, and colon tumor cells lines that include EpCAM-negative tumor cells, which cannot be isolated by conventional immunomagnetic separation, were successfully recovered on the microcavity array with high efficiency (more than 80%). On an average, approximately 98% of recovered cells were viable. Our microfluidic device has high potential as a tool for the rapid detection of CTCs and can be used to study CTCs in detail.

Formation of magnetite by bacteria and its application

Magnetic particles offer high technological potential since they can be conveniently collected with an external magnetic field. Magnetotactic bacteria synthesize bacterial magnetic particles (BacMPs) with well-controlled size and morphology. BacMPs are individually covered with thin organic membrane, which confers high and even dispersion in aqueous solutions compared with artificial magnetites, making them ideal biotechnological materials. Recent molecular studies including genome sequence, mutagenesis, gene expression and proteome analyses indicated a number of genes and proteins which play important roles for BacMP biomineralization. Some of the genes and proteins identified from these studies have allowed us to express functional proteins efficiently onto BacMPs, through genetic engineering, permitting the preservation of the protein activity, leading to a simple preparation of functional protein–magnetic particle complexes. They were applicable to high-sensitivity immunoassay, drug screening and cell separation. Furthermore, fully automated single nucleotide polymorphism discrimination and DNA recovery systems have been developed to use these functionalized BacMPs. The nano-sized fine magnetic particles offer vast potential in new nano-techniques.

DNA extraction using bacterial magnetic particles modified with hyperbranched polyamidoamine dendrimer

A cascading hyperbranched polyamidoamine dendrimer was synthesized on the surface of bacterial magnetite from Magnetospirillum magneticum AMB-1 to allow enhanced extraction of DNA from fluid suspensions. Characterization of the synthesis revealed linear doubling of the surface amine charge from generations one through five starting with an amino silane initiator. Furthermore, transmission electron microscopy revealed clear dispersion of the single domain magnetite in aqueous solution. The dendrimer modified magnetic particles have been used to carry out magnetic separation of DNA. Binding and release efficiencies increased with the number of generations and those of bacterial magnetite modified with six generation dendrimer were 7 and 11 times respectively as many as those of bacterial magnetite modified with only amino silane.

MMS6 Protein Regulates Crystal Morphology during Nano-sized Magnetite Biomineralization in Vivo

Biomineralization, the process by which minerals are deposited by organisms, has attracted considerable attention because this mechanism has shown great potential to inspire bottom-up material syntheses. To understand the mechanism for morphological regulation that occurs during biomineralization, many regulatory proteins have been isolated from various biominerals. However, the molecular mechanisms that regulate the morphology of biominerals remain unclear because there is a lack of in vivo evidence. Magnetotactic bacteria synthesize intracellular magnetosomes that comprise membrane-enveloped single crystalline magnetite (Fe3O4). These nano-sized magnetite crystals (<100 nm) are bacterial species dependent in shape and size. Mms6 is a protein that is tightly associated with magnetite crystals. Based on in vitro experiments, this protein was first implicated in morphological regulation during nano-sized magnetite biomineralization. In this study, we analyzed the mms6 gene deletion mutant (Δmms6) of Magnetospirillum magneticum (M. magneticum) AMB-1. Surprisingly, the Δmms6 strain was found to synthesize the smaller magnetite crystals with uncommon crystal faces, while the wild-type and complementation strains synthesized highly ordered cubo-octahedral crystals. Furthermore, deletion of mms6 gene led to drastic changes in the profiles of the proteins tightly bound to magnetite crystals. It was found that Mms6 plays a role in the in vivo regulation of the crystal structure to impart the cubo-octahedral morphology to the crystals during biomineralization in magnetotactic bacteria. Magnetotactic bacteria synthesize magnetite crystals under ambient conditions via a highly controlled morphological regulation system that uses biological molecules.

Desulfovibrio magneticus sp. nov., a novel sulfate-reducing bacterium that produces intracellular single-domain-sized magnetite particles

A novel type of dissimilatory sulfate-reducing bacterium, designated strain RS-1T, capable of producing intracellular magnetite particles (magnetosomes) was isolated from freshwater sulfide-rich sediments. Phylogenetic analysis based on 16S rDNA sequences revealed that RS-1T is a member of the genus Desulfovibrio. Its closest known relative is Desulfovibrio burkinensis (sequence similarity of 98.7%). Strain RS-1T contains desulfoviridin, c-type cytochromes and, unlike other Desulfovibrio spp., it possesses menaquinone MK-7(H2) instead of MK-6 or MK-6(H2). Strain RS-1T is also unique compared with other members of Desulfovibrio in its ability to synthesize intracellular magnetite particles. A novel species, Desulfovibrio magneticus sp. nov., is proposed for RS-1T (= ATCC 700980T = DSM 13731T), a sulfate-reducing magnetotactic bacterium.

Control of the morphology and size of magnetite particles with peptides mimicking the Mms6 protein from magnetotactic bacteria.

Mms6 is a dominant protein that tightly associates with the surface of bacterial magnetites in Magnetospirillum magneticum AMB-1. The protein has previously been shown to mediate the formation of uniform magnetite crystals of cubo-octahedral morphology consisting of (1 1 1) and (1 0 0) crystal faces with a narrow size distribution during chemical magnetite synthesis. In order to understand the role of this protein in chemical magnetite synthesis, magnetite formation was investigated using synthetic peptides mimicking the Mms6 protein. Particles that were synthesized in the presence of short peptides harbouring the C-terminal acidic region of Mms6 exhibited a spherical morphology with circularities of 0.70-0.90 similar to those of bacterial magnetites and particles formed in the presence of the Mms6 protein. In contrast, a rectangular morphology with circularities of 0.60-0.85 were obtained when other peptides were used for synthesis. The results indicated that the C-terminal region of the Mms6 protein has significant control over the morphology of magnetite crystals in the chemical synthetic method. This method can, therefore, be useful as an alternative method of controlling the size and morphology of magnetite crystals under ambient conditions.

Fully automated DNA extraction from blood using magnetic particles modified with a hyperbranched polyamidoamine dendrimer

Bacterial and artificial magnetic particles were modified using a polyamidoamine (PAMAM) dendrimer and outer shell amines determined. Bacterial magnetic particles were the most consistently modified. Transmission electron microscopic (TEM) analysis showed that the artificial magnetic particles were structurally damaged by the modification process including sonication. Furthermore, laser particle analysis of the magnetite also revealed damage. Small quantities of dendrimer-modified bacterial magnetic particles were used to extract DNA from blood. The efficiency of DNA recovery was consistently about 30 ng of DNA using 2-10 microg of dendrimer-modified bacterial magnetite. This technique was fully automated using newly developed liquid handling robots and bacterial magnetic particles.

Whole genome sequence of Desulfovibrio magneticus strain RS-1 revealed common gene clusters in magnetotactic bacteria

Magnetotactic bacteria are ubiquitous microorganisms that synthesize intracellular magnetite particles (magnetosomes) by accumulating Fe ions from aquatic environments. Recent molecular studies, including comprehensive proteomic, transcriptomic, and genomic analyses, have considerably improved our hypotheses of the magnetosome-formation mechanism. However, most of these studies have been conducted using pure-cultured bacterial strains of alpha-proteobacteria. Here, we report the whole-genome sequence of Desulfovibrio magneticus strain RS-1, the only isolate of magnetotactic microorganisms classified under delta-proteobacteria. Comparative genomics of the RS-1 and four alpha-proteobacterial strains revealed the presence of three separate gene regions (nuo and mamAB-like gene clusters, and gene region of a cryptic plasmid) conserved in all magnetotactic bacteria. The nuo gene cluster, encoding NADH dehydrogenase (complex I), was also common to the genomes of three iron-reducing bacteria exhibiting uncontrolled extracellular and/or intracellular magnetite synthesis. A cryptic plasmid, pDMC1, encodes three homologous genes that exhibit high similarities with those of other magnetotactic bacterial strains. In addition, the mamAB-like gene cluster, encoding the key components for magnetosome formation such as iron transport and magnetosome alignment, was conserved only in the genomes of magnetotactic bacteria as a similar genomic island-like structure. Our findings suggest the presence of core genetic components for magnetosome biosynthesis; these genes may have been acquired into the magnetotactic bacterial genomes by multiple gene-transfer events during proteobacterial evolution.

High-density microcavity array for cell detection: Single-cell analysis of hematopoietic stem cells in peripheral blood mononuclear cells

Detection and isolation of specific cell types from limited biological samples have become a major challenge in clinical diagnosis and cell biology research. Here, we report a high-density microcavity array for target cell detection in which thousands of single cells were neatly arrayed onto 10,000 microcavities with high efficiency at approximately 90% of the loaded cells. Cell-specific immunophenotypes were exclusively identified at the single-cell level by measuring fluorescence intensities of cells labeled with antibodies targeting cell surface markers, and the purity of hematopoietic stem cells (HSCs) within human peripheral blood analyzed by this system was correlated with those obtained by conventional flow cytometry. Furthermore, gene expression of the stem cell marker, CD34, was determined from HSCs by isolating single cells using a micromanipulator. This technology has proven to be an effective tool for target cell detection and subsequent cellular analytical research at the single-cell level.

Magnetic bacterial protein Mms6 controls morphology, crystallinity and magnetism of cobalt-doped magnetite nanoparticles in vitro

Magnetic nanoparticles (MNPs) are in high demand within biomedical and nanotechnological industries. Size, shape, material and crystal quality directly affect the particles properties, namely their magnetic characteristics, and must be tuned and controlled to meet the specification of the application. A key challenge is to refine synthetic methods to tailor the MNP properties with precision, but using cheap, high-yield, industrially robust and environmentally friendly methods. In this study we compare simple high-yield precipitation methods of producing cobalt-doped magnetite MNPs. We explore the variation of magnetic coercivity and saturation with increasing Co-doping from 0–15% in magnetite MNPs, which increases coercivity from 5–62 mT, but decreases saturation from 91–28 emu g−1. An optimum of 6% was further investigated as this produced the greatest increase in coercivity to 34 mT with a relatively small reduction in saturation magnetisation to 79 emu g−1. The methods compared are refined with the addition of the recombinant biomineralisation protein Mms6 from a magnetic bacterium, as this has been shown to help control magnetite MNP morphology and grainsize distribution in vitro. Similar control is seen here over our Co-doped magnetite synthesis. Mms6 increases the size and decreases the size distribution of room temperature co-precipitated particles from 11.7 nm to 31.7 nm. The affinity tagged protein his6Mms6 also controls the size (23.2 nm) but less effectively than Mms6. Therefore the Mms6 mediated Co-doped MNP particles are found to be single domain and thus give very clear, square magnetic hysteresis with a coercivity of 48 mT at 10 K. Hysteresis of the smaller particles (Co-doped MNP with no protein and with his-tagged protein) clearly shows both superparamagnetic and single-domain magnetic behaviours. Powder X-ray diffraction shows that both the addition of Mms6 and cobalt increases the crystal quality of the MNP. Thus Mms6 protein mediated room temperature co-precipitation offers an environmentally friendly, industrially robust route towards tailored, uniform, single-domain, high-quality Co-doped magnetite MNPs.