A DNA Tetrahedron Assembly-Based Nanoshell-MediatedNanoflares for Intracellular MiRNA Imaging: Reducing False Positivityand Improving Reaction Kinetics and Efficiency

Nanoflares (NFs) are extensively used for the analysis of biomarkers within living cells. However, the severe false positives and low reaction efficiency and kinetics have restricted the further application of NF. To address these issues, we have developed an aptamer-modified DNA tetrahedron-assembled 3D nucleic acid nanoshell-mediated nanoflares (Ap-TDN-NF) for highly sensitive and accurate analysis of miRNA in living cells. Compared to the traditional NF system, the developed Ap-TDN-NF system exhibits enhanced stability and improved cellular uptake efficiency due to the incorporation of the 3D nucleic acid nanoshell, reducing false positives. Because the 3D nucleic acid nanoshell hybridizes with the double-stranded probe attached on the surface of gold nanoparticles (AuNPs), it extends the distance between the target recognition sequence of double-stranded and the surface of AuNPs, resulting in improved reaction efficiency and kinetics of the hybridization between the target analyte and the target recognition sequence on the AuNP surface. The Ap-TDN-NF is capable of detecting miR-21 down to as low as 1.2 pM, which is about 780 times more sensitive than the traditional NF system. The Ap-TDN-NF system can also be effectively used for imaging miRNA in living cells with high accuracy. This proposed strategy is expected to become an important tool for nucleic acid imaging and play a significant role in disease diagnosis, treatment, and pharmaceutical research.