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.
Keywords: asymmetric membrane; digital LAMP; nanofluidics; pathogen detection; single-molecule counting.