Structurally responsive oligonucleotide-based single-probe lateral-flow test for detection of miRNA-21 mimics

Abstract

A single-probe strip test for the rapid and sensitive detection of miRNA-21 mimics is reported herein. Highly specific structurally responsive bi-functional, thiol and biotin, DNA/LNA oligonucleotide probes (molecular beacons-MB) were designed and conjugated with gold nanoparticles (AuNPs) (i.e. biotin-MB-AuNPs). The proposed design had the ability to modulate the accessibility of the biotin group as a function of the presence of a miRNA target allowing the interaction of the boilable with the streptavidin test zone only in the presence of the miRNA-21 mimics. For quantitative evaluation, images of the strip tests were recorded using a flatbed scanner (Epson Perfection V370 Photo). The colour intensities of the test zones of the strip tests were analysed with the ImageJ software (Scion Corp., USA) and quantified as a function of pixel intensity. The response of the strip test was linear over the range 0.5 to 20 nM miRNA-21 (limit of detection of 115 pM) and showed good reproducibility (intra and inter CVs below 8%); furthermore, the assay was shown to be highly selective, discriminating other interference miRNAs mimics (e.g. miRNA-221 and miRNA-205). Finally, the proposed strip test was used for detection of miRNA-21 mimics in spiked serum samples, demonstrating its potential for point-of-care clinical applications. Main advantages of the single-probe strip test design are its versatility, simplicity and robustness, which can be easily extended to other miRNA targets by tuning the sequence of the single probe. Furthermore, the use of the structurally responsive single probe is particularly relevant in the case of short-length targets, such as miRNA, whereas a conventional sandwich approach might require a careful control of assay conditions such as hybridization temperature and salt concentration.

Neutravidin biosensor for direct capture of dual-functional biotin-molecular beacon-AuNP probe for sensitive voltammetric detection of microRNA

“…The peak current increased with increasing amount of biotin-MB-AuNPs from 1.56 to 3.12 mg/mL. When the concentration of the biotin-MB-AuNPs further increased from 3.12 to 3.68 mg/mL, the peak current decreased slightly, probably due to the use of higher concentrations of the biotin-MB-AuNPs, which seems to have a saturation effect on the signal response [22,25]. Subsequently, 3.12 mg/mL of the biotin-MB-AuNP was used as the optimised concentration.…”

“…All chemicals used in this study were of analytical reagent grade. All solutions were prepared with ultrapure (18.2 MΩ) water from a Millipore Milli-Q water purification system (Billerica, MA).The sequence of the DNA/LNA MBs was taken from previous reports [22]. The presence of a thiol group at the 3' end of the MB allowed its immobilisation onto the AuNPs, while at the other end the biotin at the 5' was use to facilitate the capture of the biotin-MB-AuNP/miRNA complex onto the transducer surface via interaction with an immobilised Neutravidin layer.…”

“…The biotin-MB-AuNP conjugate was synthesised in accordance with a previously published protocol [22]. Briefly; 250 µL of the AuNPs (OD 2.3) in 0.1 mM phosphate buffer at pH 7.4…”

Improvement in Detection Limit for Lateral Flow Assay of Biomacromolecules by Test-Zone Pre-enrichment

“…The synthesis and characterization of gold nanoparticles (AuNPs) according to published procedures 32,33 with some modifications, and preparation of AuNPs-detecting probe conjugates for miR-210 mimic detection were in the Supplementary File.…”

Generic Neutravidin Biosensor for Simultaneous Multiplex Detection of MicroRNAs via Electrochemically Encoded Responsive Nanolabels

“…The DNA/LNA MBs sequences were available from previous studies. 32,33 The MB consisted of a 3′ end thiol group for immobilization onto the metal-NPs and the presence of the biotin at the 5′ end, which interacts with the immobilized neutravidin on the transducer surface forming the biotin-MB-AuNP/miRNA complex upon the presence of analyte In order to facilitate the handling of the sample, the RNA sequences were selected according to miRBase (http://www.mirbase. org) and synthesized by biomers.net (Ulm, Germany).…”