Asymmetric Membrane for Digital Detection of Single Bacteria in Milliliters of Complex Water Samples

Abstract

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.

New strategies for the enumeration of enteric pathogens in water

“…After filtration the membrane is incubated sequentially in lysis and then LAMP reagents and positive reactions are scored microscopically by fluorescence. LOD varied from 0.3 to 3 CFU ml −1 depending primarily on the gene target used for design of the LAMP reactions (Lin et al ., ).…”

Rapid Detection Methods for Bacterial Pathogens in Ambient Waters at the Point of Sample Collection: A Brief Review

“…Lin et al further reported the development and validation of the simpler and more robust double-layer membrane for dLAMP of bacterial pathogens in complex environmental waters. Absolute quantification of E. coli and S. Typhi spiked in unprocessed pond water and seawater could be completed within 1 hour with the sensitivity down to single cell [ 27 ]. Huang et al developed a gLAMP system enabling absolute quantification of microbial pathogens in environmental waters within 30 minutes at a very low cost of $5 per test.…”

A critical review: Recent advances in “digital” biomolecule detection with single copy sensitivity

“…(D) The scheme of the asymmetric system integrated with modified LAMP signal production. Reprinted with permission from ( Lin et al, 2018 ). Copyright 2018 American Chemical Society.…”

“…The current gold standard method, culture-based colonies counting, is cumbersome and time-consuming. In 2018, a new platform utilizing an asymmetric membrane system with an integrated digital LAMP was developed to successfully detected bacteria in a digital manner ( Lin et al, 2018 , 2019 ).…”