Development of a Fluorescent Enzyme-Linked DNA Aptamer-Magnetic Bead Sandwich Assay and Portable Fluorometer for Sensitive and Rapid Leishmania Detection in Sandflies
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A fluorescent peroxidase-linked DNA aptamer-magnetic bead sandwich assay is described which detects as little as 100 ng of soluble protein extracted from Leishmania major promastigotes with a high molarity chaotropic salt. Lessons learned during development of the assay are described and elucidate the pros and cons of using fluorescent dyes or nanoparticles and quantum dots versus a more consistent peroxidase-linked Amplex Ultra Red (AUR; similar to resazurin) fluorescence version of the assay. While all versions of the assays were highly sensitive, the AUR-based version exhibited lower variability between tests. We hypothesize that the AUR version of this assay is more consistent, especially at low analyte levels, because the fluorescent product of AUR is liberated into bulk solution and readily detectable while fluorophores attached to the reporter aptamer might occasionally be hidden behind magnetic beads near the detection limit. Conversely, fluorophores could be quenched by nearby beads or other proximal fluorophores on the high end of analyte concentration, if packed into a small area after magnetic collection when an enzyme-linked system is not used. A highly portable and rechargeable battery-operated fluorometer with on board computer and color touchscreen is also described which can be used for rapid (<1 h) and sensitive detection of Leishmania promastigote protein extracts (∼100 ng per sample) in buffer or sandfly homogenates for mapping of L. major parasite geographic distributions in wild sandfly populations.
KeywordsAmplex Red Aptamer Assay Leishmania Magnetic bead Portable fluorometer Resazurin Resorufin SELEX
Funding was provided by Phase 2 SBIR Contract No. W81XWH-10-C-0179. The authors are grateful to Texas State University (San Marcos, TX) and its faculty (Profs. Joseph Koke, Dana Garcia and Shannon Weigum) for advice and guidance related to confocal fluorescence microscopy. Additionally, the authors acknowledge the technical assistance of Alexander Carr at Texas State University for culture of Leishmania promastigotes. Finally, the authors express gratitude to Dr. Edgar Rowton of the Walter Reed Army Institute of Research (WRAIR) for guidance on culturing of Leishmania promastigotes and assistance in obtaining infected and uninfected sandflies.
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