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Real-time and on-site monitoring of bacterial cells in freshwater environments by using a microfluidic system

利用微流控系统实时现场监测淡水环境中的细菌细胞

基本信息

批准号：
21256002
负责人：
NASU Masao
金额：
$ 30.78万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2009
资助国家：
日本
起止时间：
2009 至 2012
项目状态：
已结题

项目摘要

“Real-time” and “on-site” microbiological methods are required for prevention of waterborne diseases. We fabricated a portable microfluidic system (36 cm × 54 cm × 23 cm, 15 kg) for rapid monitoring of bacterial cells in freshwater at a density in the order of 104- 106/ml.A microfluidic device (size: 48 mm × 23 mm) was designed and fabricated using polydimethylsiloxane (PDMS) and a thin glass sheet. Escherichia coli O157:H7 and Legionella pneumophila cells were spiked in purified freshwater (101- 106cells/ml). Low numbers of targeted cells were collected on a filter and then resuspended in purified freshwater for a 100 to 1,000 -fold concentration. Fluorescent dye was used for nucleic acid stain and direct counting of total bacterial cells. Fluorescent antibody was used for specific detection of E. coli O157:H7 and L. pneumophila cells in the samples. The portable microfluidic system was used for the detection of fluorescently stained cells flowing i n the microchannel, and number of the cells in each sample was determined using original image analysis software. Bacterial cells in the same samples were counted by fluorescence microscopy for comparison. The numbers of bacterial cells determined using the microfluidic system was highly correlated with the microscopic counts. We could count targeted cells in the samples within 2 hours with this microfluidic system. The microfluidic device and portable counting system fabricated in this study will contribute to the microbiological quality control of freshwater.

预防水传播疾病需要“实时”和“现场”微生物方法。设计并制作了一种便携式微流控系统（36 cm × 54 cm × 23 cm，15 kg），用于淡水中104 ~ 106个/ml细菌细胞的快速检测。将大肠杆菌O 157：H7和嗜肺军团菌细胞加标于纯化淡水中（101- 106个细胞/ml）。在过滤器上收集少量的靶细胞，然后再悬浮于纯化的淡水中，浓度为100至1，000倍。荧光染料用于核酸染色和细菌总数的直接计数。荧光抗体用于特异性检测大肠杆菌。coliO 157：H7和L. pneumophila细胞在样品中。使用便携式微流控系统检测在微通道中流动的荧光染色的细胞，并且使用原始图像分析软件确定每个样品中的细胞数量。通过荧光显微镜对相同样品中的细菌细胞进行计数以进行比较。使用微流控系统确定的细菌细胞的数量与显微镜计数高度相关。我们可以用这个微流控系统在2小时内计数样品中的靶细胞。本研究所制作的微流控装置及便携式计数系统将有助于淡水微生物质量控制。