Electrochemical detection of miRNA-21 coupling catalytic hairpin assembly with photo-ATRP amplification

Target-triggered CHA generates azide-bearing duplexes on the GCE surface. • CuAAC links CHA-derived DNA assemblies with ATRP initiator sites. • Py-mediated photo-ATRP forms electroactive poly(FMMA) for signal amplification. • The platform shows a 100 aM–100 pM range and a blank-based LOD of 60 aM. • Matrix-matched calibration supports preliminary analysis in 10% diluted NHS. The electrochemical conversion of nucleic acid recognition events into amplified current signals remains important for developing sensitive miRNA biosensors. Herein, we propose a target-programmed and light-triggered interfacial signal-amplification strategy for miRNA-21 detection by coupling catalytic hairpin assembly (CHA), CuAAC click chemistry, and pyrene-mediated photo-induced atom transfer radical polymerization (photo-ATRP). In this design, miRNA-21 first triggers CHA on the H1-modified glassy carbon electrode surface, generating surface-bound H1–H2 duplexes carrying terminal azide groups. These azide-bearing duplexes are subsequently coupled with the alkyne-containing ATRP initiator PBIB through CuAAC, thereby converting the target-triggered DNA assembly product into interfacial polymerization-related sites. Under UV irradiation, pyrene activates the photo-ATRP of ferrocenylmethyl methacrylate (FMMA), leading to the in situ formation of electroactive poly(FMMA) and an enhanced square-wave voltammetry response. The stepwise sensing mechanism was supported by agarose gel electrophoresis, FTIR, component-deletion SWV experiments, EIS/CV characterization, and interfacial morphology analysis. Under optimized conditions, the biosensor showed a linear response toward miRNA-21 from 100 aM to 100 pM, with a blank-based limit of detection of 60 aM. The method also exhibited acceptable selectivity, reproducibility, short-term storage stability, and preliminary recovery performance in 10% diluted normal human serum spiked samples with matrix-matched calibration. This work demonstrates a proof-of-concept electrochemical amplification strategy that links target-catalytic DNA assembly with light-controlled interfacial polymer growth for sensitive miRNA detection. Download: Download high-res image (210KB) Download: Download full-size image