Dual-tetrahedral DNA-enabled strand displacement amplification biosensor for cell imaging and clinical detection of miR-34a-5p in NAFLD.

The clinical diagnosis of nonalcoholic fatty liver disease (NAFLD) remains challenging due to the invasiveness of liver biopsy and the lack of reliable serum biomarkers, particularly for detecting low-abundance, highly homologous microRNAs such as miR-34a-5p. Here, we constructed a dual-tetrahedral DNA nanostructure biosensor (named DTDN) that couples a target-triggered strand displacement reaction with a catalytic feedback amplification circuit within a rigid tetrahedral scaffold. Unlike conventional molecular beacon or linear probe designs, this spatial architecture accelerates recognition kinetics and suppresses false-positive signals from complex biological matrices, allowing the DTDN probe to discriminate miR-34a-5p from single-base mismatched family members and to achieve a detection limit of 500 fM directly in untreated serum. Importantly, the DTDN accurately quantifies circulating miR-34a-5p levels in NAFLD individuals, which correlate significantly with lipid profiles and transaminase levels. Beyond clinical detection, the same design enables high-contrast live-cell imaging for real-time tracking of miR-34a-5p dynamics in hepatocytes under metabolic stress. By overcoming key barriers-low abundance, sequence homology, matrix interference, and lack of non-invasive spatial readouts-this dual-tetrahedral platform offers a translational route for early NAFLD diagnosis and broader miRNA-based liquid biopsy applications.