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

防治水源性疾病，需要采用“实时”和“现场”的微生物学方法。我们研制了一种便携式微流控系统(36 cm×54 cm×23 cm，15 kg)，用于快速监测淡水中细菌细胞，密度在104-106个/毫升之间。将大肠杆菌O157：H7和嗜肺军团菌细胞添加到纯净水(101-106个/ml)中。少量的靶细胞被收集在过滤器上，然后再悬浮在净化的淡水中，浓度达到100到1000倍。用荧光染料进行核酸染色，直接计数细菌总数。用荧光抗体对样品中的O157：H7和嗜肺乳杆菌进行特异性检测。使用便携式微流控系统检测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本中的细胞数量。在荧光显微镜下对同一样本中的细菌细胞进行计数，以进行比较。用微流控系统测定的细菌细胞数与显微镜计数高度相关。利用这个微流控系统，我们可以在2小时内对样品中的靶细胞进行计数。本研究研制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.