Automated processing integrated with a microflow cytometer for pathogen detection in clinical matrices

Abstract

A spinning magnetic trap (MagTrap) for automated sample processing was integrated with a microflow cytometer capable of simultaneously detecting multiple targets to provide an automated sample-to-answer diagnosis in 40 min. After target capture on fluorescently coded magnetic microspheres, the magnetic trap automatically concentrated the fluorescently coded microspheres, separated the captured target from the sample matrix, and exposed the bound target sequentially to biotinylated tracer molecules and streptavidin-labeled phycoerythrin. The concentrated microspheres were then hydrodynamically focused in a microflow cytometer capable of 4-color analysis (two wavelengths for microsphere identification, one for light scatter to discriminate single microspheres and one for phycoerythrin bound to the target). A three-fold decrease in sample preparation time and an improved detection limit, independent of target preconcentration, was demonstrated for detection of Escherichia coli 0157:H7 using the MagTrap as compared to manual processing. Simultaneous analysis of positive and negative controls, along with the assay reagents specific for the target, was used to obtain dose–response curves, demonstrating the potential for quantification of pathogen load in buffer and serum.

The intersection of flow cytometry with microfluidics and microfabrication

“…Though fiber systems will offer more robust performance due to their simplicity and rigid construction, the low numerical aperture of most such systems is currently highly limiting to fluorescence detection. Nonetheless, the robust alignment afforded by this approach has made it possible to create simple flow cytometers that effectively perform bead based detection of bacteria and toxins 148, 149 . However, the inherent optical limitations of the numerical aperture of most fibers has been recognized by the microfabrication community, which has fuelled the development of on chip lenses for light collection or use of lenses incorporated directly into fiber systems.…”

“…For example, in flow cytometers designed for routine use in the field where service is difficult and usage may be rough, it may be desirable to include a robustly aligned optical pathway or alignment free detection at the expense of sensitivity and resolution. This has been done both with patterned optical masks and fixed fiber optics 145, 148, 149 . Another common approach is to simplify an instrument through the use of field based or inertial particle focusing.…”

“…Their also have been several excellent systems created that use either microstructures or inertial forces to focus particles in one dimension, and hydrodynamic focusing to focus them in the other direction. This simplifies chip construction and has been coupled with embedded fiber optics to make simple to use robust flow cytometry prototypes 121, 148, 149, 164 . The use of microfluidics also makes it possible to consider parallel flow streams as a pathway to increased throughput analysis 74, 92, 102 .…”

A portable microfluidic platform for rapid determination of microbial load and somatic cell count in milk

“…Since the separation was based on hydrodynamic effects, it is mainly based on size, and cannot differentiate viable and non-viable bacteria. Golden et al (2013) and Wen et al (2014) proposed microfluidic platforms for identification of multiple pathogenic bacteria responsible for urinary tract infection with a coincidence rate above 90% as compared to the conventional methods. Metzger et al (2014) suggested automated dark field microscopy to simultaneously identify and quantify immobilized live pathogen bacteria directly from clinical samples within less than 4 h including sample preparation and data acquisition.…”

A portable centrifugal analyser for liver function screening

“…Recently, Vella et al(Vella et al 2012) demonstrated an innovative micropatterned paper device for measuring liver function markers from a finger prick of blood. Over the last two decades, various microfluidic "lab-on-a-disc" (LoaD) platforms have proven to facilitate full integration and automation of laboratory unit operations such as blood separation, metering, aliquoting, mixing, reagent storage and sequential reagent delivery Duford et al 2013;Honda et al 2005;Kim et al 2013;Robert et al 2013;Steigert et al 2007;van Oordt et al 2013) for applications in bioprocess, (Nwankire et al 2013) biomedical, (Godino et al 2013;Park et al 2012) food allergen (Tortajada-Genaro et al 2011), pathogen detection (Golden et al 2013) and environmental monitoring. (Czugala et al 2012;Hwang et al 2013) Nevertheless, few examples of portable sample-to-answer systems with optical read-out have been demonstrated.…”