Abstract
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.
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References
Kobets T, Grekov I, Lipoldova M (2012) Leishmaniasis: prevention, parasite detection and treatment. Curr Med Chem 19:1443–1474
O’Daly JA, Spinetti HM, Gleason J, Rodríguez MB (2013) Clinical and immunological analysis of cutaneous leishmaniasis before and after different treatments. J Parasitol Res 2013:657016. doi:10.1155/2013/657016
Palatnik-de-Sousa CB, Day MJ (2011) One Health: the global challenge of epidemic and endemic leishmaniasis. Parasit Vectors 4:197. doi:10.1186/1756-3305-4-197
Sundar S, Rai M (2002) Laboratory diagnosis of visceral leishmaniasis. Clin Diag Lab Immunol 9:951–958
Souza AP, Soto M, Costa JM, Boaventura VS, de Oliveira CI, Cristal JR, Barral-Netto M, Barral A (2013) Towards a more precise serological diagnosis of human tegumentary leishmaniasis using Leishmania recombinant proteins. PLoS One 8(6):e66110. doi:10.1371/journal.pone.0066110
Moreno EC, Gonçalves AV, Chaves AV, Melo MN, Lambertucci JR, Andrade AS, Negrão-Corrêa D, de Figueiredo Antunes CM, Carneiro M (2009) Inaccuracy of enzyme-linked immunosorbent assay using soluble and recombinant antigens to detect asymptomatic infection by Leishmania infantum. PLoS Negl Trop Dis. doi:10.1371/journal.pntd.0000536, 3e536
McAvin JC, Swanson KI, Chan AS, Quintana M, Coleman RE (2012) Leishmania detection in sand flies using a field-deployable real-time analytic system. Mil Med 177:460–466
Paiva BR, Secundino NF, Nascimento JC, Pimenta PF, Galati EA, Junior HF, Malafronte RS (2006) Detection and identification of Leishmania species in field-captured phlebotomine sandflies based on mini-exon gene PCR. Acta Trop 99:252–259
Bruno JG, Phillips T, Carrillo MP, Crowell R (2009) Plastic-adherent DNA aptamer-magnetic bead and quantum dot sandwich assay for Campylobacter detection. J Fluoresc 19:427–435
Wei B, Li F, Yang H, Yu L, Zhao K, Zhou R, Hu Y (2012) Magnetic beads-based enzymatic spectrofluorometric assay for rapid and sensitive detection of antibody against ApxIVA of Actinobacillus pleuropneumoniae. Biosens Bioelectron 35:390–393. doi:10.1016/j.bios.2012.03.027
Yolken RH, Stopa PJ (1979) Enzyme-linked fluorescence assay: ultrasensitive solid-phase assay for detection of human rotavirus. J Clin Microbiol 10:317–332
Duan N, Wu S, Zhu C, Ma X, Wang Z, Yu Y, Jiang Y (2012) Dual-color upconversion fluorescence and aptamer-functionalized magnetic nanoparticles-based bioassay for the simultaneous detection of Salmonella Typhimurium and Staphylococcus aureus. Anal Chim Acta 723:1–6
Wang FB, Rong Y, Fang M, Yuan JP, Peng CW, Liu SP, Li Y (2013) Recognition and capture of metastatic hepatocellular carcinoma cells using aptamer-conjugated quantum dots and magnetic particles. Biomaterials 34:3816–3827. doi:10.1016/j.biomaterials.2013.02.018
Ikanovic M, Rudzinski WE, Bruno JG, Allman A, Carrillo MP, Dwarakanath S, Bhahdigadi S, Rao P, Kiel JL, Andrews CJ (2007) Fluorescence assay based on aptamer-quantum dot binding to Bacillus thuringiensis spores. J Fluoresc 17:193–199
Dwarakanath S, Bruno JG, Shastry A, Phillips T, John AA, Kumar A, Stephenson LD (2004) Quantum dot-antibody and aptamer conjugates shift fluorescence upon binding bacteria. Biochem Biophys Res Commun 325:739–743
Fitzpatrick JA, Andreko SK, Ernst LA, Waggoner AS, Ballou B, Bruchez MP (2009) Long-term persistence and spectral blue shifting of quantum dots in vivo. Nano Lett 9:2736–2741. doi:10.1021/nl901534q
Generalov R, Kavaliauskiene S, Westrøm S, Chen W, Kristensen S, Juzenas P (2011) Entrapment in phospholipid vesicles quenches photoactivity of quantum dots. Int J Nanomed 6:1875–1888. doi:10.2147/IJN.S22953
Grabolle M, Ziegler J, Merkulov A, Nann T, Resch-Genger U (2008) Stability and fluorescence quantum yield of CdSe-ZnS quantum dots-influence of the thickness of the ZnS shell. Ann N Y Acad Sci 1130:235–241. doi:10.1196/annals.1430.021
Jamieson T, Bakhshi R, Petrova D, Pocock R, Imani M, Seifalian AM (2007) Biological application of quantum dots. Biomaterials 28(31):4717–4732
Ji X, Palui G, Avellini T, Na HB, Yi C, Knappenberger KL Jr, Mattoussi H (2012) On the pH-dependent quenching of quantum dot photoluminescence by redox active dopamine. J Am Chem Soc 134:6006–6017. doi:10.1021/ja300724x
Liu YS, Sun Y, Vernier PT, Liang CH, Chong SY, Gundersen MA (2007) pH-sensitive photoluminescence of CdSe/ZnSe/ZnS quantum dots in human ovarian cancer cells. J Phys Chem C Nanomater Interfaces 111:2872–2878
Riegler J, Ditengou F, Palme K, Nann T (2008) Blue shift of CdSe/ZnS nanocrystal-labels upon DNA-hybridization. J Nanobiotech 6:7. doi:10.1186/1477-3155-6-7
Summers HD, Holton MD, Rees P, Williams PM, Thornton CA (2010) Analysis of quantum dot fluorescence stability in primary blood mononuclear cells. Cytometry A 77:933–939. doi:10.1002/cyto.a.20932
Zarkowsky D, Lamoreaux L, Chattopadhyay P, Koup RA, Perfetto SP, Roederer M (2011) Heavy metal contaminants can eliminate quantum dot fluorescence. Cytometry A 79:84–89. doi:10.1002/cyto.a.20986
Zhang Y, He J, Wang PN, Chen JY, Lu ZJ, Lu DR, Guo J, Wang CC, Yang WL (2006) Time-dependent photoluminescence blue shift of the quantum dots in living cells: effect of oxidation by singlet oxygen. J Am Chem Soc 128:13396–13401
Mather IH, Keenan TW (1975) Studies on the structure of milk fat globule membrane. J Membr Biol 21:65–85
Bruno JG, Carrillo MP, Phillips T, Andrews CJ (2010) A novel screening method for competitive FRET-aptamers applied to E. coli assay development. J Fluoresc 20:1211–1223
Carothers JM, Goler JA, Kapoor Y, Lara L, Keasling JD (2010) Selecting RNA aptamers for synthetic biology: investigating magnesium dependence and predicting binding affinity. Nucleic Acids Res 38:2736–2747
Jayasena SD (1999) Aptamers: an emerging class of molecules that rival antibodies in diagnostics. Clin Chem 45:1628–1650
Bruno JG, Carrillo MP, Phillips T (2007) Effects of immobilization chemistry on enzyme-linked aptamer assays for Leishmania surface antigens. J Clin Ligand Assay 30:37–43
Gonzalez VM, Martin ME, Moreno M (2013) Aptamers targeting protozoan parasites. In: Bruno JG (ed) Biomedical applications of aptamers. Nova, New York, pp 73–88
Homann M, Lorger M, Engstler M, Zacharias M, Göringer HU (2006) Serum-stable RNA aptamers to an invariant surface domain of live African trypanosomes. Comb Chem High Throughput Screen 9:491–499
Moreno M, González VM (2011) Advances on aptamers targeting Plasmodium and trypanosomatids. Curr Med Chem 18:5003–5010
Moreno M, Rincón E, Piñeiro D, Fernández G, Domingo A, Jiménez-Ruíz A, Salinas M, González VM (2003) Selection of aptamers against KMP-11 using colloidal gold during the SELEX process. Biochem Biophys Res Commun 308:214–218
Ramos E, Moreno M, Martín ME, Soto M, Gonzalez VM (2010) In vitro selection of Leishmania infantum H3-binding ssDNA aptamers. Oligonucleotides 20:207–213. doi:10.1089/oli.2010.0240
Ramos E, Piñeiro D, Soto M, Abanades DR, Martín ME, Salinas M, González VM (2007) A DNA aptamer population specifically detects Leishmania infantum H2A antigen. Lab Invest 87:409–416
Ulrich H, Magdesian MH, Alves MJ, Colli W (2002) In vitro selection of RNA aptamers that bind to cell adhesion receptors of Trypanosoma cruzi and inhibit cell invasion. J Biol Chem 277:20756–20762
Burns JM, Shreffler WG, Benson DR, Ghalib HW, Badaro R, Reed SG (1993) Molecular characterization of a kinesin-related antigen of Leishmania chagasi that detects specific antibody in African and American visceral Leishmaniasis. Proc Natl Acad Sci U S A 90:775–779
Burchardt ER, Kroll W, Gehrmann M, Schroder W (2009) Monoclonal antibody and assay for detecting PIIINP. U.S. Patent No. 7,541,149
Bruno JG, Carrillo MP, Phillips T, Edge A (2011) Discrimination of recombinant from natural human growth hormone using DNA aptamers. J Biomolec Techn 22:27–36
Ivens AC, Peacock CS, Worthey EA et al (2005) The genome of the kinetoplastid parasite, Leishmania major. Science 309:436–442
Holzer TR, McMaster WR, Forney JD (2006) Expression profiling by whole-genome interspecies microarray hybridization reveals differential gene expression in procyclic promastigotes, lesion-derived amastigotes, and axenic amastigotes in Leishmania mexicana. Mol Biochem Parasitol 146:198–218
Acknowledgments
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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Bruno, J.G., Richarte, A.M., Phillips, T. et al. Development of a Fluorescent Enzyme-Linked DNA Aptamer-Magnetic Bead Sandwich Assay and Portable Fluorometer for Sensitive and Rapid Leishmania Detection in Sandflies. J Fluoresc 24, 267–277 (2014). https://doi.org/10.1007/s10895-013-1315-6
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DOI: https://doi.org/10.1007/s10895-013-1315-6