title: 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.
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.
authors.affiliation: Chemical Sensors Group, Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, ON L5L 1C6, Canada.
authors.forename: M Omair
authors.initials: MO
authors.lastname: Noor
authors.affiliation: Chemical Sensors Group, Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, ON L5L 1C6, Canada.
authors.forename: David
authors.initials: D
authors.lastname: Hrovat
authors.affiliation: Department of Cell and Systems Biology, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, ON L5L 1C6, Canada.
authors.forename: Maryam
authors.initials: M
authors.lastname: Moazami-Goudarzi
authors.affiliation: Department of Cell and Systems Biology, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, ON L5L 1C6, Canada; Department of Biology, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, ON L5L 1C6, Canada.
authors.forename: George S
authors.initials: GS
authors.lastname: Espie
authors.affiliation: Chemical Sensors Group, Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, ON L5L 1C6, Canada. Electronic address: ulrich.krull@utoronto.ca.
authors.forename: Ulrich J
authors.initials: UJ
authors.lastname: Krull
mesh_terms: D015415:Biomarkers / Q000032:analysis; D002232:Carbocyanines / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D009693:Nucleic Acid Hybridization / Q000379:methods*; D009841:Oligonucleotides / Q000032:analysis* / Q000235:genetics; D010209:Paper; D020641:Polymorphism, Single Nucleotide; D045663:Quantum Dots / Q000737:chemistry*
chemical_list: D015415:Biomarkers; D002232:Carbocyanines; D009841:Oligonucleotides; C089187:cyanine dye 3
keywords: Fluorescence resonance energy transfer; Isothermal amplification; Nucleic acid hybridization; Paper-based assays; Quantum dots; Ratiometric detection