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.

Cell heterogeneity, such as antibiotic heteroresistance and cancer cell heterogeneity, has been increasingly observed. To probe the underlying molecular mechanisms in the dynamically changing heterogeneous cells, a high throughput platform is urgently needed to establish single cell genotype-phenotype correlations. Herein, we report a platform combining single-cell viability phenotypic analysis with digital molecular detection for bacterial cells. The platform utilizes polyethylene glycol hydrogel that cross-links through a thiol-Michael addition, which is biocompatible, fast, and spontaneous. To generate uniform nanoliter-sized hydrogel beads (Gelbeads), we developed a convenient and disposable device made of needles and microcentrifuge tubes. Gelbead-based single cell viability and molecular detection assays were established. Enhanced thermal stability and uncompromised efficiency were achieved for digital polymerase chain reaction (PCR) and digital loop-mediated isothermal amplification (LAMP) within the Gelbeads. Reagent exchange for in situ PCR following viability phenotypic analyses was demonstrated. The combined analyses may address the genotypic differences between cellular subpopulations exhibiting distinct phenotypes. The platform promises unique perspectives in mechanism elucidation of environment-evolution interaction that may be extended to other cell types for medical research.

Waterborne microbial pathogen detection via nucleic acid analysis on portable microfluidic devices is a growing area of research, development, and application. Traditional polymerase chain reaction (PCR)-based nucleic acid analysis detects total extracted DNA, but cannot differentiate live and dead cells. A propidium monoazide (PMA) pretreatment step before PCR can effectively exclude DNA from nonviable cells, as PMA can selectively diffuse through compromised cell membranes and intercalate with DNA to form DNA–PMA complex upon light exposure. The complex strongly inhibits the amplification of the bound DNA in PCR, and thus, only cells with intact cell membranes are detected. Herein, this study reports the development of a microfluidic device to carry out PMA pretreatment ‘on-chip’. Chip design was guided by computer simulations, and prototypes were fabricated using a high-resolution 3D printer. The optimized design utilizes split and recombine mixers for initial PMA-sample mixing and a serpentine flow channel containing herringbone structures for dark and light incubation. On-chip PMA pretreatment to differentiate live and dead bacterial cells in buffer and natural pond water samples was successfully demonstrated.

While highly reactive cerium oxide nanoparticles (CeO₂ NPs) are widely used in industry, their transport in aquatic systems is not well understood. To fill this knowledge gap, the interactions of CeO₂ NPs with arsenite (As³⁺), a toxic metalloid and potential co-present contaminant, were investigated with respect to CeO₂ NP colloidal stability, dissolution, and surface redox reactions. Arsenite showed distinctive effects at different concentrations, with a high As³⁺ concentration (10⁻⁴ M) inducing 90% of CeO₂ NPs to settle from solution after 8 hours, while lower As³⁺ concentrations (10⁻⁵ or 10⁻⁶ M) led to only 20% of CeO₂ NPs settling. The dissolution of NPs was most significant in the 10⁻⁵ M As³⁺ system owing to a lesser extent of aggregation, exposing more CeO₂ surface for dissolution. In the three As³⁺ concentration systems, >97% of aqueous arsenic remained as As³⁺ over 6 hours. On the NP surface, adsorbed As^III was oxidized to As^V, resulting in 58–70% of the adsorbed arsenic remaining as As^III. Simultaneously Ce^IV was reduced to Ce^III, increasing Ce^III on the CeO₂ NP surface from 17% (without arsenite) to 21–25% (with arsenite). Further mechanistic analyses revealed that the adsorption of arsenite was the main contributor to neutralizing the CeO₂ NP surface potential, enhancing particle sedimentation. These findings suggest that the fate and transport of CeO₂ NPs in our experimental systems are strongly affected by arsenite concentration and its adsorption on NPs. The results also highlight the importance of the interplay between NP aggregation, oxidation, and dissolution in predicting the behaviors of CeO₂ NPs and associated toxic elements in aquatic systems.

Speech generates droplets averaged 6 μm in size. Their settling time and SARS-CoV-2 viral viability inside those droplets, both calculated from temperature and relative humidity, predict COVID transmission rates within one-sigma interval.