Shohei Yamamura

Singlet-Oxygen-Sensitizing Near-Infrared-Fluorescent Multimodal Nanoparticles

Nanoprobes based on quantum clusters (QC) with near-infrared fluorescence, magnetic-resonance-imaging contrast, and singlet-oxygen-sensitized intracellular fluorescence are studied. The generation of singlet oxygen and singlet-oxygen-sensitized fluorescence uncaging by magnetic and NIR-emitting nanoparticles are exploited for multimodal bioimaging in vitro.

Rapid and Highly Sensitive Detection of Malaria-Infected Erythrocytes Using a Cell Microarray Chip

Background Malaria is one of the major human infectious diseases in many endemic countries. For prevention of the spread of malaria, it is necessary to develop an early, sensitive, accurate and conventional diagnosis system. Methods and Findings A cell microarray chip was used to detect for malaria-infected erythrocytes. The chip, with 20,944 microchambers (105 µm width and 50 µm depth), was made from polystyrene, and the formation of monolayers of erythrocytes in the microchambers was observed. Cultured Plasmodium falciparum strain 3D7 was used to examine the potential of the cell microarray chip for malaria diagnosis. An erythrocyte suspension in a nuclear staining dye, SYTO 59, was dispersed on the chip surface, followed by 10 min standing to allow the erythrocytes to settle down into the microchambers. About 130 erythrocytes were accommodated in each microchamber, there being over 2,700,000 erythrocytes in total on a chip. A microarray scanner was employed to detect any fluorescence-positive erythrocytes within 5 min, and 0.0001% parasitemia could be detected. To examine the contamination by leukocytes of purified erythrocytes from human blood, 20 µl of whole blood was mixed with 10 ml of RPMI 1640, and the mixture was passed through a leukocyte isolation filter. The eluted portion was centrifuged at 1,000×g for 2 min, and the pellet was dispersed in 1.0 ml of medium. SYTO 59 was added to the erythrocyte suspension, followed by analysis on a cell microarray chip. Similar accommodation of cells in the microchambers was observed. The number of contaminating leukocytes was less than 1 on a cell microarray chip. Conclusion The potential of the cell microarray chip for the detection of malaria-infected erythrocytes was shown, it offering 10–100 times higher sensitivity than that of conventional light microscopy and easy operation in 15 min with purified erythrocytes.

Simultaneous immunoassay analysis of plasma IL-6 and TNF-α on a microchip.

Sandwich enzyme-linked immunosorbant assay (ELISA) using a 96-well plate is frequently employed for clinical diagnosis, but is time-and sample-consuming. To overcome these drawbacks, we performed a sandwich ELISA on a microchip. The microchip was made of cyclic olefin copolymer with 4 straight microchannels. For the construction of the sandwich ELISA for interleukin-6 (IL-6) or tumor necrosis factor-α (TNF-α), we used a piezoelectric inkjet printing system for the deposition and fixation of the 1st anti-IL-6 antibody or 1st anti-TNF-α antibody on the surface of the each microchannel. After the infusion of 2 µl of sample to the microchannel and a 20 min incubation, 2 µl of biotinylated 2nd antibody for either antigen was infused and a 10 min incubation. Then 2 µl of avidin-horseradish peroxidase was infused; and after a 5 min incubation, the substrate for peroxidase was infused, and the luminescence intensity was measured. Calibration curves were obtained between the concentration and luminescence intensity over the range of 0 to 32 pg/ml (IL-6: R2u200a=u200a0.9994, TNF-α: R2u200a=u200a0.9977), and the detection limit for each protein was 0.28 pg/ml and 0.46 pg/ml, respectively. Blood IL-6 and TNF-α concentrations of 5 subjects estimated from the microchip data were compared with results obtained by the conventional method, good correlations were observed between the methods according to linear regression analysis (IL-6: R2u200a=u200a0.9954, TNF-α: R2u200a=u200a0.9928). The reproducibility of the presented assay for the determination of the blood IL-6 and TNF-α concentration was comparable to that obtained with the 96-well plate. Simultaneous detection of blood IL-6 and TNF-α was possible by the deposition and fixation of each 1st antibody on the surface of a separate microchannel. This assay enabled us to determine simultaneously blood IL-6 and TNF-α with accuracy, satisfactory sensitivity, time saving ability, and low consumption of sample and reagents, and will be applicable to clinic diagnosis.

Quantitative Analysis of Serum Procollagen Type I C-Terminal Propeptide by Immunoassay on Microchip

Background Sandwich enzyme-linked immunosorbent assay (ELISA) is one of the most frequently employed assays for clinical diagnosis, since this enables the investigator to identify specific protein biomarkers. However, the conventional assay using a 96-well microtitration plate is time- and sample-consuming, and therefore is not suitable for rapid diagnosis. To overcome these drawbacks, we performed a sandwich ELISA on a microchip. Methods and Findings The microchip was made of cyclic olefin copolymer with straight microchannels that were 300 µm wide and 100 µm deep. For the construction of a sandwich ELISA for procollagen type I C-peptide (PICP), a biomarker for bone formation, we used a piezoelectric inkjet printing system for the deposition and fixation of the 1st anti-PICP antibody on the surface of the microchannel. After the infusion of the mixture of 2.0 µl of peroxidase-labeled 2nd anti-PICP antibody and 0.4 µl of sample to the microchannel and a 30-min incubation, the substrate for peroxidase was infused into the microchannel; and the luminescence intensity of each spot of 1st antibody was measured by CCD camera. A linear relationship was observed between PICP concentration and luminescence intensity over the range of 0 to 600 ng/ml (r2u200a=u200a0.991), and the detection limit was 4.7 ng/ml. Blood PICP concentrations of 6 subjects estimated from microchip were compared with results obtained by the conventional method. Good correlation was observed between methods according to simple linear regression analysis (R2u200a=u200a0.9914). The within-day and between-days reproducibilities were 3.2–7.4 and 4.4–6.8%, respectively. This assay reduced the time for the antigen-antibody reaction to 1/6, and the consumption of samples and reagents to 1/50 compared with the conventional method. Conclusion This assay enabled us to determine serum PICP with accuracy, high sensitivity, time saving ability, and low consumption of sample and reagents, and thus will be applicable to clinic diagnosis.

Accurate Detection of Carcinoma Cells by Use of a Cell Microarray Chip

Background Accurate detection and analysis of circulating tumor cells plays an important role in the diagnosis and treatment of metastatic cancer treatment. Methods and Findings A cell microarray chip was used to detect spiked carcinoma cells among leukocytes. The chip, with 20,944 microchambers (105 µm width and 50 µm depth), was made from polystyrene; and the formation of monolayers of leukocytes in the microchambers was observed. Cultured human T lymphoblastoid leukemia (CCRF-CEM) cells were used to examine the potential of the cell microarray chip for the detection of spiked carcinoma cells. A T lymphoblastoid leukemia suspension was dispersed on the chip surface, followed by 15 min standing to allow the leukocytes to settle down into the microchambers. Approximately 29 leukocytes were found in each microchamber when about 600,000 leukocytes in total were dispersed onto a cell microarray chip. Similarly, when leukocytes isolated from human whole blood were used, approximately 89 leukocytes entered each microchamber when about 1,800,000 leukocytes in total were placed onto the cell microarray chip. After washing the chip surface, PE-labeled anti-cytokeratin monoclonal antibody and APC-labeled anti-CD326 (EpCAM) monoclonal antibody solution were dispersed onto the chip surface and allowed to react for 15 min; and then a microarray scanner was employed to detect any fluorescence-positive cells within 20 min. In the experiments using spiked carcinoma cells (NCI-H1650, 0.01 to 0.0001%), accurate detection of carcinoma cells was achieved with PE-labeled anti-cytokeratin monoclonal antibody. Furthermore, verification of carcinoma cells in the microchambers was performed by double staining with the above monoclonal antibodies. Conclusion The potential application of the cell microarray chip for the detection of CTCs was shown, thus demonstrating accurate detection by double staining for cytokeratin and EpCAM at the single carcinoma cell level.

Highly sensitive DNA detection with a combination of 2 DNA-intercalating dyes for microchip electrophoresis

A highly sensitive DNA detection method using a combination of ethidium bromide (EtBr) and SYBR Green II (SG II) for microchip electrophoresis was developed. By use of the combination of these intercalating DNA-staining dyes for microchip electrophoresis with Hitachi SV1100 system, the fluorescence intensities corresponding to DNA fragments were obviously increased over those obtained with EtBr only, with accuracy of DNA sizing and quantification. The detection limit with EtBr and the combination of EtBr and SG II were 0.048 and 0.007ng/μl, respectively. This highly sensitive DNA detection just using the combination of these dyes offering high resolution in a short time will be useful for various biological analyses.

Application of a cell microarray chip system for accurate, highly sensitive, and rapid diagnosis for malaria in Uganda.

Accurate, sensitive, rapid, and easy operative diagnosis is necessary to prevent the spread of malaria. A cell microarray chip system including a push column for the recovery of erythrocytes and a fluorescence detector was employed for malaria diagnosis in Uganda. The chip with 20,944 microchambers (105u2009μm width and 50u2009μm depth) was made of polystyrene. For the analysis, 6u2009μl of whole blood was employed, and leukocytes were practically removed by filtration through SiO2-nano-fibers in a column. Regular formation of an erythrocyte monolayer in each microchamber was observed following dispersion of an erythrocyte suspension in a nuclear staining dye, SYTO 21, onto the chip surface and washing. About 500,000 erythrocytes were analyzed in a total of 4675 microchambers, and malaria parasite-infected erythrocytes could be detected in 5u2009min by using the fluorescence detector. The percentage of infected erythrocytes in each of 41 patients was determined. Accurate and quantitative detection of the parasites could be performed. A good correlation between examinations via optical microscopy and by our chip system was demonstrated over the parasitemia range of 0.0039–2.3438% by linear regression analysis (R2u2009=u20090.9945). Thus, we showed the potential of this chip system for the diagnosis of malaria.

Detection of miRNA in cell cultures by using microchip electrophoresis with a fluorescence-labeled riboprobe.

The analysis of a microRNA (miRNA), miR-222 isolated from the PC12 cell line, was performed by use of the ribonuclease (RNase) protection assay, cyanine 5 (Cy5)-labeled miR-222 riboprobe, and a Hitachi SV1210 microchip electrophoresis system, which can be used to evaluate the integrity of total RNA. The fluorescence intensity corresponding to the protected RNA fragment increased in a dose-dependent manner with respect to the complementary-strand RNA. More highly sensitive detection of miRNA by microchip electrophoresis than by conventional method using fluorescence-labeled riboprobe could be obtained in 180 s. An obvious increase in miR-222 expression induced by nerve growth factor in PC12 cells could be observed. These results clearly indicate the potential of microchip electrophoresis for the analysis of miRNA using RNase protection assay with a fluorescence-labeled riboprobe.

Hydrophilic-treated plastic plates for wide-range analysis of Giemsa-stained red blood cells and automated Plasmodium infection rate counting

BackgroundMalaria is a red blood cell (RBC) infection caused by Plasmodium parasites. To determine RBC infection rate, which is essential for malaria study and diagnosis, microscopic evaluation of Giemsa-stained thin blood smears on glass slides (‘Giemsa microscopy’) has been performed as the accepted gold standard for over 100xa0years. However, only a small area of the blood smear provides a monolayer of RBCs suitable for determination of infection rate, which is one of the major reasons for the low parasite detection rate by Giemsa microscopy. In addition, because Giemsa microscopy is exacting and time-consuming, automated counting of infection rates is highly desirable.ResultsA method that allows for microscopic examination of Giemsa-stained cells spread in a monolayer on almost the whole surface of hydrophilic-treated cyclic olefin copolymer (COC) plates was established. Because wide-range Giemsa microscopy can be performed on a hydrophilic-treated plate, the method may enable more reliable diagnosis of malaria in patients with low parasitaemia burden. Furthermore, the number of RBCs and parasites stained with a fluorescent nuclear staining dye could be counted automatically with a software tool, without Giemsa staining. As a result, researchers studying malaria may calculate the infection rate easily, rapidly, and accurately even in low parasitaemia.ConclusionBecause the running cost of these methods is very low and they do not involve complicated techniques, the use of hydrophilic COC plates may contribute to improved and more accurate diagnosis and research of malaria.

Prognostic Impact of Circulating Tumor Cell Detected Using a Novel Fluidic Cell Microarray Chip System in Patients with Breast Cancer

Various types of circulating tumor cell (CTC) detection systems have recently been developed that show a high CTC detection rate. However, it is a big challenge to find a system that can provide better prognostic value than CellSearch in head-to-head comparison. We have developed a novel semi-automated CTC enumeration system (fluidic cell microarray chip system, FCMC) that captures CTC independently of tumor-specific markers or physical properties. Here, we compared the CTC detection sensitivity and the prognostic value of FCMC with CellSearch in breast cancer patients. FCMC was validated in preclinical studies using spike-in samples and in blood samples from 20 healthy donors and 22 breast cancer patients in this study. Using spike-in samples, a statistically higher detection rate (p = 0.010) of MDA-MB-231 cells and an equivalent detection rate (p = 0.497) of MCF-7 cells were obtained with FCMC in comparison with CellSearch. The number of CTC detected in samples from patients that was above a threshold value as determined from healthy donors was evaluated. The CTC number detected using FCMC was significantly higher than that using CellSearch (p = 0.00037). CTC numbers obtained using either FCMC or CellSearch had prognostic value, as assessed by progression free survival. The hazard ratio between CTC + and CTC − was 4.229 in CellSearch (95% CI, 1.31 to 13.66; p = 0.01591); in contrast, it was 11.31 in FCMC (95% CI, 2.245 to 57.0; p = 0.000244). CTC detected using FCMC, like the CTC detected using CellSearch, have the potential to be a strong prognostic factor for cancer patients.