DNA functionalized double quantum dots-based fluorescence biosensor for one-step simultaneous detection of multiple microRNAs

Abstract

The disease diagnosis by detecting single microRNAs (miRNAs) can produce high false positive rate. Herein, a novel fluorescence biosensor method for one-step simultaneous detection of multiple miRNAs was proposed by using single-stranded DNA (ssDNA) functionalized double quantum dots (QDs) and black hole quencher (BHQ)-decorated magnetic nanobeads (MNs). MNs were linked with two black hole quenchers (BHQ1 and BHQ3) via a complementary DNA (cDNA). The ssDNA/cDNA hybridization contributed to the fluorescence quenching of double QDs due to the fluorescence resonance energy transfer (FRET) between double QDs and BHQ. In the presence of target miRNA-33 (miR-33) and miRNA-125b (miR-125b), the ssDNA₁ and ssDNA₂ were respectively hybridized with miR-33 and miR-125b to form more stable duplexes. Thus, the double QDs were released into supernatant after the magnetic separation, leading to the fluorescence signals recovery at 537 nm and 647 nm. A wide linear range (0.5 nM-320 nM for miR-33 and 0.1 nM-250 nM for miR-125b) and low limits of detection (0.09 nM for miR-33 and 0.02 nM for miR-125b) were achieved. Moreover, our approach has been demonstrated to simultaneously detect miR-33 and miR-125b in cell extracts. With advantages of high sensitivity, strong specificity, low background and low cost, the strategies show great potentials for the detection of various targets in bioanalysis and disease diagnosis.

Keywords: DNA hybridization; Double quantum dots; Magnetic nanobeads; Simultaneous detection; microRNA.