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

预防水传播疾病需要“实时”和“现场”微生物方法。我们制作了一种便携式微流体系统（36 cm × 54 cm × 23 cm, 15 kg），用于淡水中细菌细胞的快速监测，密度为104- 106/ml。以聚二甲基硅氧烷（PDMS）和薄玻璃片为材料，设计并制作了尺寸为48 mm × 23 mm的微流控装置。在纯化的淡水中加入大肠杆菌O157:H7和嗜肺军团菌细胞（101- 106个细胞/ml）。在过滤器上收集少量目标细胞，然后将其重悬在净化淡水中，浓度为100至1000倍。采用荧光染料进行核酸染色和直接计数。采用荧光抗体特异性检测样品中的大肠杆菌O157:H7和嗜肺乳杆菌细胞。使用便携式微流控系统检测在微通道中流动的荧光染色细胞，并使用原始图像分析软件确定每个样品中的细胞数量。用荧光显微镜对同一样品中的细菌细胞计数进行比较。微流控系统测定的细菌细胞数量与显微镜计数高度相关。利用该微流控系统，我们可以在2小时内对样品中的靶细胞进行计数。本研究制作的微流控装置和便携式计数系统将有助于淡水微生物质量控制。

“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.