A multiplex Taqman PCR assay for MRSA detection from whole blood.

Methicillin-resistant Staphylococcus aureus (MRSA) causes a wide range of hospital and community-acquired infections worldwide. MRSA is associated with worse clinical outcomes that can lead to multiple organ failure, septic shock, and death, making timely diagnosis of MRSA infections very crucial. In the present work, we develop a method that enables the positive enrichment of bacteria from spiked whole blood using protein coated magnetic beads, followed by their lysis, and detection by a real-time multiplex PCR directly. The assay targeted bacterial 16S rRNA, S. aureus (spa) and methicillin resistance (mecA). In addition, an internal control (lambda phage) was added to determine the assay's true negative. To validate this assay, staphylococcal and non-staphylococcal bacterial strains were used. The three-markers used in this study were detected as expected by monomicrobial and poly-microbial models of the S. aureus and coagulase-negative staphylococci (CoNS). The thermal cycling completed within 30 mins, delivering 100% specificity. The detection LoD of the pre-processing step was ∼ 1 CFU/mL from 2-5mL of whole blood and that of PCR was ∼ 1pg of NA. However, the combined protocol led to a lower detection limit of 100-1000 MRSA CFUs/mL. The main issue with the method developed is in the pre-processing of blood which will be the subject of our future study.

Current commercial dPCR platforms: technology and market review.

Digital polymerase chain reaction (dPCR) technology has provided a new technique for molecular diagnostics, with superior advantages, such as higher sensitivity, precision, and specificity over quantitative real-time PCRs (qPCR). Eight companies have offered commercial dPCR instruments: Fluidigm Corporation, Bio-Rad, RainDance Technologies, Life Technologies, Qiagen, JN MedSys Clarity, Optolane, and Stilla Technologies Naica. This paper discusses the working principle of each offered dPCR device and compares the associated: technical aspects, usability, costs, and current applications of each dPCR device. Lastly, up-and-coming dPCR technologies are also presented, as anticipation of how the dPCR device landscape may likely morph in the next few years.

Dean migration of unfocused micron sized particles in low aspect ratio spiral microchannels.

We present an analysis of the microfluidic Dean migration of 2.5 µm particles, which do not meet focus criterion, in tall and low aspect ratio microchannels. We demonstrate the use of such low aspect ratio and tall spirals (h > 50 µm) for isolating high concentration (> 106 particles or cells/mL) micron sized particles without an initial off-chip dilution step. We specifically show the need for a sheath fluid for isolation and systematically analyze the particle stream profile (i.e. thickness and distance from the channel wall) as a function of downstream channel length and curvature ratio, with changes in the fluid velocity and the flow rate ratio of particles to sheath fluid (FRR). We also show that the width of the particle stream can control the particle migration and that a threshold stream width and Dean drag is necessary to initiate the particle stream migration from the channel wall. We then propose a design guide based on the selection of optimum curvatures, flow velocities and the FRRs required for achieving a narrow particle stream through a particular outlet. Finally, we use the design guide to demonstrate the isolation of bacteria from bladder epithelial cells.

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 in the urinary tract. The protocol can be broadly divided into three sections: the infection, bladder epithelial cell harvesting, and mouth micropipetting to isolate individual infected epithelial cells. The isolated epithelial cell contains viable bacterial cells and is nearly free of contaminating extracellular bacteria, making it ideal for downstream single-cell analysis. The time taken from the start of infection to obtaining a single intracellular bacterial community is about 8 h. This protocol is inexpensive to deploy and uses widely available materials, and we anticipate that it can also be utilized in other infection models to isolate single infected cells from cell mixtures even if those infected cells are rare. However, due to a potential risk in mouth micropipetting, this procedure is not recommended for highly infectious agents.

Purification of Intracellular Bacterial Communities during Experimental Urinary Tract Infection Reveals an Abundant and Viable Bacterial Reservoir.

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 Escherichia coli, 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 103 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.

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

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.

Plasmonic nanoshell synthesis in microfluidic composite foams.

The availability of robust, scalable, and automated nanoparticle manufacturing processes is crucial for the viability of emerging nanotechnologies. Metallic nanoparticles of diverse shape and composition are commonly manufactured by solution-phase colloidal chemistry methods, where rapid reaction kinetics and physical processes such as mixing are inextricably coupled, and scale-up often poses insurmountable problems. Here we present the first continuous flow process to synthesize thin gold "nanoshells" and "nanoislands" on colloidal silica surfaces, which are nanoparticle motifs of considerable interest in plasmonics-based applications. We assemble an ordered, flowing composite foam lattice in a simple microfluidic device, where the lattice cells are alternately aqueous drops containing reagents for nanoparticle synthesis or gas bubbles. Microfluidic foam generation enables precisely controlled reagent dispensing and mixing, and the ordered foam structure facilitates compartmentalized nanoparticle growth. This is a general method for aqueous colloidal synthesis, enabling continuous, inherently digital, scalable, and automated production processes for plasmonic nanomaterials.

Ionic liquid-based compound droplet microfluidics for 'on-drop' separations and sensing.

We present a new and general scheme for analytical applications of droplet-based microfluidics in which flowing droplets function not only as isolated reaction flasks, but are also capable of on-drop separation and sensing. To demonstrate this, we choose ionic liquids as designer fluids whose chemical and physical properties can be tailored in task-specific fashion. We create aqueous-ionic liquid compound droplets with tunable structures using an imidazolium-based ionic liquid, and present two analytical applications-separation of a binary aqueous mixture of organic dyes and dynamic pH sensing-to highlight the salient features of this scheme. By combining designer fluids with designer microfluidic emulsions, our work opens up a rich space of exploration for analytical microfluidics.

Droplet-based microfluidic synthesis of anisotropic metal nanocrystals.

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.

Microfluidic emulsions with dynamic compound drops.

We demonstrate a new class of microfluidic emulsion where the 'drops' of the emulsion are dynamic reversible bubble-drop pairs, with potential applications in microfluidic technology for chemical synthesis, molecular separations and screening.