Ratiometric fluorescence transduction by hybridization after isothermal amplification for determination of zeptomole quantities of oligonucleotide biomarkers with a paper-based platform and camera-based detection |
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| Institution: | 1. Chemical Sensors Group, Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, ON L5L 1C6, Canada;2. Department of Cell and Systems Biology, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, ON L5L 1C6, Canada;3. Department of Biology, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, ON L5L 1C6, Canada;1. Dpto. Química Física y Analítica, Universidad de Oviedo, Julián Clavería 8, 33006 Oviedo, Spain;2. Servicio de Microbiología, Unidad de Referencia Regional de Micobacterias de Asturias. Hospital Universitario Central de Asturias, Avda Roma s/n, 33011 Oviedo, Spain;1. Department of Industrial Engineering, University of Trento, Via Sommarive 9, I-38123 Povo, Trento, Italy;2. INFN – TIFPA, Via Sommarive 14, I-38123 Povo, Trento, Italy;1. Instituto de Engenharia de Sistemas e Computadores - Microsistemas e Nanotecnologias (INESC MN) and IN - Institute of Nanoscience and Nanotechnology, Portugal;2. IBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal;3. Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal;1. Department of Chemical Engineering & Applied Chemistry, University of Toronto, 200 College Street, Toronto, Ontario M5S 3E5, Canada;2. Department of Cell & Systems Biology, University of Toronto, Mississauga, 3359 Mississauga Road North, Mississauga, Ontario L5L 1C6, Canada;1. Department of Agrifood, Environmental and Animal Science, University of Udine, via Cotonificio 108, I-33100 Udine, Italy;2. Department of Chemical and Geological Science, University of Modena and Reggio Emilia, via Campi 183, I-41125 Modena, Italy |
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| Abstract: | Paper is a promising platform for the development of decentralized diagnostic assays owing to the low cost and ease of use of paper-based analytical devices (PADs). It can be challenging to detect on PADs very low concentrations of nucleic acid biomarkers of lengths as used in clinical assays. Herein we report the use of thermophilic helicase-dependent amplification (tHDA) in combination with a paper-based platform for fluorescence detection of probe-target hybridization. Paper substrates were patterned using wax printing. The cellulosic fibers were chemically derivatized with imidazole groups for the assembly of the transduction interface that consisted of immobilized quantum dot (QD)–probe oligonucleotide conjugates. Green-emitting QDs (gQDs) served as donors with Cy3 as the acceptor dye in a fluorescence resonance energy transfer (FRET)-based transduction method. After probe-target hybridization, a further hybridization event with a reporter sequence brought the Cy3 acceptor dye in close proximity to the surface of immobilized gQDs, triggering a FRET sensitized emission that served as an analytical signal. Ratiometric detection was evaluated using both an epifluorescence microscope and a low-cost iPad camera as detectors. Addition of the tHDA method for target amplification to produce sequences of ～100 base length allowed for the detection of zmol quantities of nucleic acid targets using the two detection platforms. The ratiometric QD-FRET transduction method not only offered improved assay precision, but also lowered the limit of detection of the assay when compared with the non-ratiometric QD-FRET transduction method. The selectivity of the hybridization assays was demonstrated by the detection of single nucleotide polymorphism. |
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| Keywords: | Quantum dots Paper-based assays Ratiometric detection Nucleic acid hybridization Isothermal amplification Fluorescence resonance energy transfer |
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