Oligonucleotide-templated lateral flow assays for amplification-free sensing of circulating microRNAs

Abstract

Herein we demonstrate the first example of oligonucleotidetemplated reaction (OTR) performed on paper, using lateral flow to capture and concentrate specific nucleic acid biomarkers on a test line. Quantitative analysis, using a low-cost benchtop fluorescence reader showed very high specificity down to the single nucleotide level and proved sensitive enough for amplification-free, on-chip, detection of endogenous concentrations of miR-150-5p, a recently identified predictive blood biomarker for preterm birth. † Electronic supplementary information (ESI) available: Including experimental information, PNA synthesis, coumarin probe concentration, optimization of the LFA parameters and information on the clinical samples. See Scheme 1 Schematic of the OTR-based lateral flow assay. Biotinylated PNA thiols (capture probe, green) are immobilised on a streptavidin test line (red) on a paper strip (panel 2). A mixture of PNA coumarin (sensing probe, red) and miRNA target (blue) is then added to the sample loading pad (dark grey, panel 1). Upon wicking, the miRNA of interest is retained on the test line via hybridisation to the capture thiol probe, whilst also hybridising to the sensing probe (panel 3). The excess of sensing probe and all non-complementary miRNAs are then eluted off. Fluorescence, resulting from the product of the Michael addition OTR, is then measured using either a benchtop reader or a fluorescence scanner (panel 4). Fig. 1Fluorescence scanned images (top) and traces (bottom) of LFA strips for OTR in the presence (blue, (+) DNA) and absence (green, (À) DNA) of 0.5 mM miR-150-5p DNA, and a blank strip with only buffer added (orange, Blank). The dark spot on the (À) DNA sample loading pad is background fluorescence from the excess of PNA coumarin which remains adsorbed to the paper unless hybridized to a complementary DNA. CommunicationChemComm

Maternal plasma miRNAs as potential biomarkers for detecting risk of small-for-gestational-age births

“…Our results highlight unique circulating miRNA expression profiles of SGA, and identify potential candidate plasma miRNAs as predictive markers for SGA births which can be utilised in future bedside tests based on lateral flow assays [43] . The ability to identify those women at risk of SGA early in pregnancy would allow earlier clinical interventions, such as administration of low dose aspirin, focused monitoring of foetal growth, avoidance of drugs/smoking, control of maternal disorders (eg.…”

Visible-light triggered templated ligation on surface using furan-modified PNAs

“…45 On the other hand, the possibility to perform templated reactions at the surface interface was barely explored over the past years. [72][73][74] We here report, for the rst time, the possibility to perform the ligation on a surface, templated by short 22mer oligonucleotides, exploiting the in situ activation of a stable proreactive moiety using a simple white light lamp. The proposed system, applied to a classical 96-well plate detection platform, was designed to not require delicate enzymatic steps and only involves functional groups with long shelf-lives.…”

“…The eld of oligonucleotide-templated reactions features many applications of PNA systems, as evidenced by the large number of templated reactions and ligations reported in literature (i.e., carrier release, [18][19][20] in cellulo and in vivo nucleic acid imaging, 17,21 developments of lateral ow devices for RNA detection, 22,23 uorogenic reactions, 10,12,[24][25][26][27] catalytic and autocatalytic systems, 14,[28][29][30][31][32] detection and targeting of oligonucleotides involved in genetic-based diseases, 15,33,34 articial aminoacyl transferases and peptide ligation [35][36][37][38][39] ). Oligonucleotide templated reactions, in a wider perspective, have also been explored in the eld of nanotechnology.…”

Integrating high-performing electrochemical transducers in lateral flow assay

“…Thus, monitoring the hybridization event of PNA with DNA or RNA based on electrochemical label-free techniques is feasible as demonstrated in previous studies where charge of the probe plays an important role for signal generation [48,50,51]. LFA combined with PNA probes has been successfully implemented to determine micro-RNA-150 in plasma samples for preterm birth screening test [52].…”