DNA hydrogel-based nanoprobe for enzyme-free ultra-sensitive nucleic acid assay

Abstract

Rapid, sensitive, specific, and the user-friendly nucleic acid assay is a key technique for the next generation point-of-care molecular diagnosis. However, a high-cost, labor-intensive, and complicated enzymatic amplification step is essential to achieve high sensitivity. Herein, I suggested 3-dimensional (3D) self-assembled DNA hydrogel as a cascade amplification platform for rapid and ultra-sensitive nucleic acid assay. I utilized the in situ functionalization of QDs with pt-ssDNA as an effective means to achieve both desirable luminescent properties and colloidal stability, which are required for practical FRET-based DNA assay. Furthermore, QD-DNA hydrogel was prepared via self-assembly between target-catalyzed formed multiple Y-DNAs and DNA-QDs. The QD-DNA hydrogel was DNA-programmed to be continuously formed in the presence of target DNA, and efficient FRET signals were observed. The cascade amplification, which includes target-catalyzed multiple formations of Y-DNA, self-assembly with a multivalent donor into DNA hydrogel, and efficient and hierarchical energy transfer within DNA hydrogel, improved the sensitivity from nanomolar to femtomolar level even without enzymatic amplification, and the assay time was only 1 hour. In addition, by introducing the base-pair mismatches to the stem of hairpin DNA, I successfully facilitated the TMSD kinetics specifically for target miRNA, enabling DNA-hydrogel-based miRNA. The universalness of this strategy was investigated with three different miRNA targets, and the detection limits of DNA hydrogel were all improved from nanomolar to picomolar level. Since the DNA hydrogel-based nucleic acid assay enables rapid, ultra-sensitive, and specific detection of the target without enzymatic amplification, it is expected that the proposed system would be a potential sensing tool in the field of point-of-care testing.