Freezing-assisted nano-sweeping of short oligonucleotides using spherical nucleic acids: A fluorometric platform for quantification of microRNAs

Abstract

MicroRNAs (miRNAs) have been identified as biomarkers for early cancer screening and diagnosis. As a result, the development of simple methods for quantifying these small RNA species is of great interest. This study describes a new biosensing platform termed nano-sweeping for quantitative detection of miRNA-30a. The sweeping process is driven by a combination of gold nanoparticle-core spherical nucleic acid (AuNP-core SNA) hybridization and gravitational separation. Two modified oligonucleotides, i.e., a capture DNA strand for attachment to the surface of AuNPs and a fluorescent reporter strand for fluorescence signal detection, were designed to be semi-complementary to the miRNA-30a. MiRNA-30a is hybridized with the two semi-complementary oligonucleotides using freezing-assisted hybridization, yielding a three-segment sandwich-type duplex. A centrifugation step causes the nanoparticles to settle, allowing the fluorescent reporter strands involved in sandwich hybridization to coprecipitate with the nanoparticles and be removed from the solution. The decrease in fluorescence intensity of the supernatant is proportional to the miRNA-30a concentration. Calibration curves were constructed using data from both traditional fluorometry and smartphone-based fluorescence imaging. The obtained quantification limit for miRNA-30a was 0.22 and 0.42 ng/µL for fluorometric and smartphone-based calibration models, respectively and a linear response established for the range of 0–3.55 ng/µL. Unlike fluorescence resonance energy transfer (FRET)-based methods, this approach is fluorophore independent, allowing for attachment of any fluorescent dye on the reporter oligonucleotide strand.