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Jang K Xu Y Tanaka Y Sato K Mawatari K Konno T Ishihara K Kitamori T 《Biomicrofluidics》2010,4(3):32208
Recently, interest in single cell analysis has increased because of its potential for improving our understanding of cellular processes. Single cell operation and attachment is indispensable to realize this task. In this paper, we employed a simple and direct method for single-cell attachment and culture in a closed microchannel. The microchannel surface was modified by applying a nonbiofouling polymer, 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer, and a nitrobenzyl photocleavable linker. Using ultraviolet (UV) light irradiation, the MPC polymer was selectively removed by a photochemical reaction that adjusted the cell adherence inside the microchannel. To obtain the desired single endothelial cell patterning in the microchannel, cell-adhesive regions were controlled by use of round photomasks with diameters of 10, 20, 30, or 50 μm. Single-cell adherence patterns were formed after 12 h of incubation, only when 20 and 30 μm photomasks were used, and the proportions of adherent and nonadherent cells among the entire UV-illuminated areas were 21.3%±0.3% and 7.9%±0.3%, respectively. The frequency of single-cell adherence in the case of the 20 μm photomask was 2.7 times greater than that in the case of the 30 μm photomask. We found that the 20 μm photomask was optimal for the formation of single-cell adherence patterns in the microchannel. This technique can be a powerful tool for analyzing environmental factors like cell-surface and cell-extracellular matrix contact. 相似文献
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Kihoon Jang Yo Tanaka Jun Wakabayashi Reina Ishii Kae Sato Kazuma Mawatari Mats Nilsson Takehiko Kitamori 《Biomicrofluidics》2012,6(4)
Demand for analysis of rare cells such as circulating tumor cells in blood at the single molecule level has recently grown. For this purpose, several cell separation methods based on antibody-coated micropillars have been developed (e.g., Nagrath et al., Nature 450, 1235–1239 (2007)). However, it is difficult to ensure capture of targeted cells by these methods because capture depends on the probability of cell-micropillar collisions. We developed a new structure that actively exploits cellular flexibility for more efficient capture of a small number of cells in a target area. The depth of the sandwiching channel was slightly smaller than the diameter of the cells to ensure contact with the channel wall. For cell selection, we used anti-epithelial cell adhesion molecule antibodies, which specifically bind epithelial cells. First, we demonstrated cell capture with human promyelocytic leukemia (HL-60) cells, which are relatively homogeneous in size; in situ single molecule analysis was verified by our rolling circle amplification (RCA) method. Then, we used breast cancer cells (SK-BR-3) in blood, and demonstrated selective capture and cancer marker (HER2) detection by RCA. Cell capture by antibody-coated microchannels was greater than with negative control cells (RPMI-1788 lymphocytes) and non-coated microchannels. This system can be used to analyze small numbers of target cells in large quantities of mixed samples. 相似文献
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Immunoassay is one of the important applications of microfluidic chips and many methodologies were reported for decreasing sample∕reagent volume, shortening assay time, and so on. Micro-enzyme-linked immunosorbent assay (micro-ELISA) is our method that utilizes packed microbeads in the microfluidic channel and the immunoreactions are induced on the beads surface. Due to the large surface-to-volume ratio and small analytical volume, excellent performances have been verified in assay time and sample∕reagent volume. In order to realize the micro-ELISA, one of the important processes is the immobilization of antibody on the beads surface. Previously, the immobilization process was performed in a macroscale tube by physisorption of antibody, and long time (2 h) and large amount of antibody (or high concentration) were required for the immobilization. In addition, the processes including the reaction and washing were laborious, and changing the analyte was not easy. In this research, we integrated the immobilization process into a microfluidic chip by applying the avidin-biotin surface chemistry. The integration enabled very fast (1 min) immobilization with very small amount of precious antibody consumption (100 ng) for one assay. Because the laborious immobilization process can be automatically performed on the microfluidic chip, ELISA method became very easy. On-demand immunoassay was also possible just by changing the antibodies without using large amount of precious antibodies. Finally, the analytical performance was investigated by measuring C-reactive protein and good performance (limit of detection <20 ng∕ml) was verified. 相似文献
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Malawi and Ghana are among the numerous Sub-Saharan Africa countries that have in recent years introduced Free Primary Education (FPE) policy as a means to realizing the 2015 Education for All and Millennium Development Goals international targets. The introduction of FPE policy is, however, a huge challenge for any national government that has experienced declining or slow economic growth and heavily relied on charging fees to parents and other sources to finance the education system. It follows, therefore, that the approach taken in implementing the FPE policy has implications for equity and efficiency in the education sector. Malawi and Ghana have differently implemented FPE policy. In this article we assess the impact of the implementation approach taken by each of the two countries on equity and efficiency in their education systems. 相似文献
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