Results are presented from an experimental proof of concept for a full-spectrum microfluidic flow cytometer. Existing flow cytometers operate on photomultiplier tubes (PMTs) and are restricted in the number of available fluorophores, with newer systems costing $250k to $500k. Current EMCCD technology paired with microfluidics promises to offer superior channel decomposition while allowing a significant reduction in price. A device was constructed from polydimethysiloxane (PDMS) with fiber optics for sample excitation and fluorescence collection, and with an 80 micron channel for sample flow. In the first experiment, we captured spectra of individual fluorescently labeled polystyrene beads and demonstrated classification of beads according to their labeling fluorophore, as well as characterization of anomalous beads. In the second experiment, two stoichiometric mixtures of six dyes were created with the successful decomposition of the mixtures into components, using reference spectra acquired on the system. We anticipate the capability of full spectra to enable the extension to twenty-one fluorophores with the microfluidic design creating a robust system with a total cost of under $50,000.
Direct ultra-sensitive detection of pathogen biomarkers in blood could provide a universal strategy for diagnosis of bacterial infections, which remain a leading cause of morbidity and mortality in many areas of the world. Many factors complicate diagnosis, including the presence of multiple co-infections in a given patient, and lack of infrastructure in rural settings. In some pediatric patients, such as those in areas with poor resources, an additional challenge exists with low sample volumes due to age and other health factors such as anemia and dehydration. Our team is working on developing novel diagnostic assays, with a waveguide-based biosensor platform, to rapidly and specifically identify pathogen biomarkers from small samples of serum or plasma, allowing for the timely and sensitive diagnosis of infection at the point of care. In addition to the platform, we have developed novel membrane insertion and lipoprotein capture assay methods, to capture lipidated pathogen biomarkers in aqueous blood, by virtue of their interactions with host lipoprotein carriers. Herein, we demonstrate our efforts to adapt the lipoprotein capture assay for the detection of small concentrations of pathogen-secreted lipopolysaccharides in aqueous blood, with the ultimate aim of diagnosing Gram-negative infections effectively.
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