In this work, we introduce an asymmetric membrane as a simple and robust nanofluidic platform for digital detection of single pathogenic bacteria directly in 10 mL of unprocessed environmental water samples. The asymmetric membrane, consisting of uniform micropores on one side and a high density of vertically aligned nanochannels on the other side, was prepared within 1 min by a facile method. The single membrane covers all the processing steps from sample concentration, purification, and partition to final digital loop-mediated isothermal amplification (LAMP). By simple filtration, bacteria were enriched and partitioned inside the micropores, while inhibitors typically found in the environmental samples ( i.e., proteins, heavy metals, and organics) were washed away through the nanochannels. Meanwhile, large particles, indigenous plankton, and positively charged pollutants in the samples were excluded by using a sacrificial membrane stacked on top. After initial filtration, modified LAMP reagents, including NaF and lysozyme, were loaded onto the membrane. Each pore in the asymmetric membrane functioned as an individual nanoreactor for selective, rapid, and efficient isothermal amplification of single bacteria, generating a bright fluorescence for direct counting. Even though high levels of inhibitors were present, absolute quantification of Escherichia coli and Salmonella directly in an unprocessed environmental sample (seawater and pond water) was achieved within 1 h, with sensitivity down to single cell and a dynamic range of 0.3-10000 cells/mL. The simple and low-cost analysis platform described herein has an enormous potential for the detection of pathogens, exosomes, stem cells, and viruses as well as single-cell heterogeneity analysis in environmental, food, and clinical research.

中文翻译：

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数字化检测复杂水样中的单个细菌的不对称膜。

在这项工作中，我们介绍了一种不对称膜，它是一种简单而健壮的纳米流体平台，可直接在10 mL未经处理的环境水样本中对单个病原菌进行数字检测。通过简便的方法在1分钟内制备了不对称膜，该膜由一侧的均匀微孔和另一侧的高密度垂直排列的纳米通道组成。单个膜覆盖了从样品浓缩，纯化和分配到最终数字环介导的等温扩增（LAMP）的所有处理步骤。通过简单的过滤，细菌在微孔中富集和分配，而环境样品中通常存在的抑制剂（即蛋白质，重金属和有机物）则通过纳米通道被洗去。同时，大颗粒，原生浮游生物，并使用堆叠在顶部的牺牲膜排除样品中带正电荷的污染物。初步过滤后，将修饰的LAMP试剂（包括NaF和溶菌酶）加载到膜上。不对称膜中的每个孔都充当单个纳米反应器，用于选择性，快速和有效地等温扩增单个细菌，从而产生明亮的荧光以便直接计数。即使存在高水平的抑制剂，也可以在1小时内直接在未处理的环境样品（海水和池塘水中）中对大肠杆菌和沙门氏菌进行绝对定量，灵敏度低至单细胞，动态范围为0.3-10000细胞/毫升 本文所述的简单且低成本的分析平台在检测病原体，外来体，