Shunsuke Furutani

Use of a novel microfluidic disk in the analysis of single-cell viability and the application to Jurkat cells.

Jurkat cells were trapped in the microchambers of a novel disk-shaped cell separation device and stained with Cellstain. Approximately 90% of the cells were living. Single cells were isolated with a branching microchannel after rotation at 4500rpm for 30s, demonstrating that a living single cell could be trapped in the microchambers.

Rapid Detection of Salmonella enterica in Food Using a Compact Disc-Shaped Device

Rapid detection of food-borne pathogens is essential to public health and the food industry. Although the conventional culture method is highly sensitive, it takes at least a few days to detect food-borne pathogens. Even though polymerase chain reaction (PCR) can detect food-borne pathogens in a few hours, it is more expensive and unsatisfactorily sensitive relative to the culture method. We have developed a method to rapidly detect Salmonella enterica by using a compact disc (CD)-shaped device that can reduce reagent consumption in conventional PCR. The detection method, which combines culture and PCR, is more rapid than the conventional culture method and is more sensitive and cheaper than PCR. In this study, we also examined a sample preparation method that involved collecting bacterial cells from food. The bacteria collected from chicken meat spiked with S. enterica were mixed with PCR reagents, and PCR was performed on the device. At a low concentration of S. enterica, the collected S. enterica was cultured before PCR for sensitive detection. After cultivation for 4 h, S. enterica at 1.7 × 104 colony-forming units (CFUs)·g−1 was detected within 8 h, which included the time needed for sample preparation and detection. Furthermore, the detection of 30 CFUs·g−1 of S. enterica was possible within 12 h including 8 h for cultivation.

Hot Cell-Direct PCR Aimed at Specific Cell Detection

Since the polymerase chain reaction (PCR) was proposed, it has become an essential method in the field of biological gene analysis, providing a method to amplify DNA sequences of interest. To detect and/or analyze genes in cells, the gene or expressed gene must first be extracted before PCR. This procedure takes time and may result in the loss of samples. In order to avoid such drawbacks, two methods, hot cell-direct PCR and reverse transcription-PCR (RT-PCR), were invented, to detect genes in cells. Using hot cell-direct PCR, specific genes in microbial cells such as invA in Salmonella enterica have been easily detected and applied to discriminate Archaea from bacteria. As hot celldirect PCR and RT-PCR are fairly simple processes, they can be applied to detect genes in single cells. We developed an original compact disc (CD)-shaped microfluidic device with microchambers for single-cell isolation and a detection system for expressed genes in isolated single cells in a microchamber on the device. We succeeded in the detection of PCR and RT-PCR products in individual cells and successfully detected S. enterica cells by hot cell-direct PCR. Expressed genes in Jurkat cells—human leukemia T cells—were analyzed by this method.

4.2.1 Bisphenol A Sensing Device Utilizing Antibody Modified Beads on a Microfluidic Disk

Bisphenol A (BPA) is a major material of polycarbonate, which is used as a food container in our life and known as endocrine disruptor. In order to investigate the effect to health animal experiments are necessary. To determine the intake of BPA with food in animals, we need a sensing system of BPA with a small amount of biological sample in short reaction time. We have developed a disk-shaped microfluidic device for ELISA with 32 microchannels and chambers. In order to establish a rapid and sensitive assay system of BPA in biological sample, anti BPA antibody was immobilized on micro beads and introduced into microchambers on the microchannels on the device, in this study. Competitive immunoassay was performed using HRP conjugated BPA with a small amount of sample solution with in 20 min. BPA was determined on the microfluidic disk at the concentration range between 3.9250 ng ml. BPA spiked rat serum was determined on the disk.