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Isolation of Single Intracellular Bacterial Communities Generated from a Murine Model of Urinary Tract Infection for Downstream Single-cell Analysis

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected ...

National University of Singapore

A droplet-based microfluidic method for the preparation of anisotropic gold nanocrystal dispersions is presented. Gold nanoparticle seeds and growth reagents are dispensed into monodisperse picoliter droplets within a microchannel. Confinement within small droplets prevents contact between the growing nanocrystals and the microchannel walls. The critical factors in translating macroscale flask-based methods to a flow-based microfluidic method are highlighted and approaches are demonstrated to flexibly fine tune nanoparticle shapes into three broad classes: spheres/spheroids, rods, and extended sharp-edged structures, thus varying the optical resonances in the visible-near-infrared (NIR) spectral range.

Droplet-based microfluidic synthesis of anisotropic metal nanocrystals.

Microfluidic wet-chemical synthesis of nanoparticles is a growing area of research in chemical microfluidics, enabling the development of continuous manufacturing processes that overcome the drawbacks of conventional batch-based synthesis methods. The synthesis of ultra-small (<5 nm) metallic nanocrystals is an interesting area with many applications in diverse fields, but is typically very challenging to accomplish in a microfluidics-based system due to the use of a strong gas-evolving reducing agent, aqueous sodium borohydride (NaBH(4)), which causes uncontrolled out-gassing and bubble formation, flow disruption and ultimately reactor failure. Here we present a simple method, rooted in the concepts of multiphase mass transfer that completely overcomes this challenge-we simply inject a stream of inert gas bubbles into our channels that essentially capture the evolving gas from the reactive aqueous solution, thereby preventing aqueous dissolved gas concentration from reaching the solubility threshold for bubble nucleation. We present a simple model for coupled mass transfer and chemical reaction that adequately captures device behaviour. We demonstrate the applicability of our method by synthesizing ultra-small gold nanocrystals (<5 nm); the quality of nanocrystals thus synthesized is further demonstrated by their use in an off-chip synthesis of high-quality gold nanorods. This is a general approach that can be extended to a variety of metallic nanomaterials.

Controlling bubbles using bubbles--microfluidic synthesis of ultra-small gold nanocrystals with gas-evolving reducing agents.

Urinary tract infections (UTIs) are a major infection of humans, particularly affecting women. Recurrent UTIs can cause significant discomfort and expose patients to high levels of antibiotic use, which in turn contributes to the development of higher antibiotic resistance rates. Most UTIs are caused by uropathogenic , which is able to form intracellular collections (termed intracellular bacterial communities [IBCs]) within the epithelial cells lining the bladder lumen. IBCs are seen in both infected mice and humans and are a potential cause of recurrent UTI. Genetic and molecular studies of IBCs have been hampered both by the low number of bacteria in IBCs relative to the number extracellular bacteria and by population bottlenecks that occur during IBC formation. We now report the development of a simple and rapid technique for isolating pure IBCs from experimentally infected mice. We verified the specificity and purity of the isolated IBCs via microscopy, gene expression, and culture-based methods. Our results further demonstrated that our isolation technique practically enables specific molecular studies of IBCs. In the first such direct measurement, we determined that a single epithelial cell containing an early IBC typically contains 10 viable bacteria. Our isolation technique complements recent progress in low-input, single-cell genomics to enable future genomic studies of the formation of IBCs and their activation pathways during recurrent UTI, which may lead to novel strategies to eliminate them from the bladder.

Suhanya Duraiswamy

Department of Chemical and Biomolecular Engineering

National University of Singapore

Isolation of Single Intracellular Bacterial Communities Generated from a Murine Model of Urinary Tract Infection for Downstream Single-cell Analysis

Suhanya Duraiswamy

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Chemical and Biomolecular Engineering

National University of Singapore

Isolation of Single Intracellular Bacterial Communities Generated from a Murine Model of Urinary Tract Infection for Downstream Single-cell Analysis

Department of Chemical and Biomolecular Engineering