Localized hybridization chain reaction amplifier utilizing three-dimensional DNA nanostructures for efficient and reliable microRNA imaging in living cells.

MicroRNAs (miRNAs) are pivotal in regulating biological processes such as cell proliferation and disease progression. Traditional miRNA detection methods like qRT-PCR and Northern blotting do not allow for monitoring dynamic changes in living cells and typically require invasive sample collection. This research presents a robust, non-enzymatic technique known as the Localized Hybridization Chain Reaction Amplifier (LHCRA), designed for real-time, in vivo miRNA imaging. This method addresses these limitations by providing rapid and sensitive detection of miRNAs, crucial for progressing clinical diagnostics and research. The LHCRA utilizes hairpin chain reaction probes embedded within self-assembled DNA micelles (DM), significantly amplifying miRNA signals. LHCRA demonstrated exceptional performance, with a detection limit of 100 pM and a response time of 10 min. Importantly, it effectively differentiated between cancerous and normal cells using clinical peripheral blood samples, showcasing high specificity and stability under physiological conditions. Additionally, LHCRA maintained consistent performance in complex biological media, including 10 % serum and 2 U/mL DNase I, confirming its broad applicability in various diagnostic scenarios. This performance highlights LHCRA's potential as a transformative tool in miRNA-based diagnostics. The introduction of LHCRA marks a significant advancement in miRNA detection technology. By enabling accurate, non-invasive, and real-time monitoring of miRNA dynamics within living cells, this method offers substantial potential to enhance diagnostic accuracy and deepen our understanding of disease mechanisms, particularly in cancer. Thus, it could greatly improve therapeutic strategies and patient outcomes in clinical environment.