Browsing by Author "Raja, Balakrishnan"
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Item A low-cost smartphone-based platform for highly sensitive point-of-care testing with persistent luminescent phosphors(Lab on a Chip, 2018-03) Paterson, Andrew S.; Raja, Balakrishnan; Mandadi, Vinay; Townsend, Blane; Lee, Miles; Buell, Alex; Vu, Binh V.; Brgoch, Jakoah; Willson, Richard C.Through their computational power and connectivity, smartphones are poised to rapidly expand telemedicine and transform healthcare by enabling better personal health monitoring and rapid diagnostics. Recently, a variety of platforms have been developed to enable smartphone-based point-of-care testing using imaging-based readout with the smartphone camera as the detector. Fluorescent reporters have been shown to improve the sensitivity of assays over colorimetric labels, but fluorescence readout necessitates incorporating optical hardware into the detection system, adding to the cost and complexity of the device. Here we present a simple, low-cost smartphone-based detection platform for highly sensitive luminescence imaging readout of point-of-care tests run with persistent luminescent phosphors as reporters. The extremely bright and long-lived emission of persistent phosphors allows sensitive analyte detection with a smartphone by a facile time-gated imaging strategy. Phosphors are first briefly excited with the phone's camera flash, followed by switching off the flash, and subsequent imaging of phosphor luminescence with the camera. Using this approach, we demonstrate detection of human chorionic gonadotropin using a lateral flow assay and the smartphone platform with strontium aluminate nanoparticles as reporters, giving a detection limit of ?45 pg mL?1 (1.2 pM) in buffer. Time-gated imaging on a smartphone can be readily adapted for sensitive and potentially quantitative testing using other point-of-care formats, and is workable with a variety of persistent luminescent materials.Item Development of a panel of recombinase polymerase amplification assays for detection of common bacterial urinary tract infection pathogens(Journal of Applied Microbiology, 8/1/2018) Raja, Balakrishnan; Goux, Heather J.; Marapadaga, Archana; Rajagopalan, Sri; Kourentzi, Katerina D.; Willson, Richard C.Aims: To develop and evaluate the performance of a panel of isothermal real?time recombinase polymerase amplification (RPA) assays for detection of common bacterial urinary tract infection (UTI) pathogens. Methods and Results: The panel included RPAs for Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa and Enterococcus faecalis. All five RPAs required reaction times of under 12 min to reach their lower limit of detection of 100 genomes per reaction or less, and did not cross?react with high concentrations of nontarget bacterial genomic DNA. In a 50?sample retrospective clinical study, the five?RPA assay panel was found to have a specificity of 100% (95% CI, 78�0%) and a sensitivity of 89% (95% CI, 75�%) for UTI detection. Conclusions:The analytical and clinical validity of RPA for the rapid and sensitive detection of common UTI pathogens was established. Significance and Impact of the Study: Rapid identification of the causative pathogens of UTIs can be valuable in preventing serious complications by helping avoid the empirical treatment necessitated by traditional urine culture's 48�?h turnaround time. The routine and widespread use of RPA to supplement or replace culture?based methods could profoundly impact UTI management and the emergence of multidrug?resistant pathogens.Item Flotation Immunoassay: Masking the Signal from Free Reporters in Sandwich Immunoassays(Scientific Reports, 2016-04) Chen, Hui; Hagstrom, Anna E. V.; Kim, Jinsu; Garvey, Gavin; Paterson, Andrew S.; Ruiz-Ruiz, Federico; Raja, Balakrishnan; Strych, Ulrich; Rito-Palomares, Marco; Kourentzi, Katerina D.; Conrad, Jacinta C.; Atmar, Robert L.; Willson, Richard C.In this work, we demonstrate that signal-masking reagents together with appropriate capture antibody carriers can eliminate the washing steps in sandwich immunoassays. A flotation immunoassay (FI) platform was developed with horseradish peroxidase chemiluminescence as the reporter system, the dye Brilliant Blue FCF as the signal-masking reagent, and buoyant silica micro-bubbles as the capture antibody carriers. Only reporters captured on micro-bubbles float above the dye and become visible in an analyte-dependent manner. These FIs are capable of detecting proteins down to attomole levels and as few as 106 virus particles. This signal-masking strategy represents a novel approach to simple, sensitive and quantitative immunoassays in both laboratory and point-of-care settings.