Conductive Cu-MOF synergizing with enzyme-free catalytic cascade amplification for ultrasensitive electrochemical detection of miRNA-21.

BACKGROUND: MicroRNA-21 (miRNA-21) is an oncogenic biomarker overexpressed in various early-stage tumors, making its accurate detection vital for diagnosis. However, its concentration in clinical samples is extremely low, posing a significant analytical challenge. Conventional electrochemical sensors often rely on exogenous enzymes for amplification, which suffer from high cost, instability, and complex immobilization procedures. Moreover, the poor conductivity of common electrode materials limits signal transduction efficiency. Therefore, developing an enzyme-free sensing platform with a highly conductive interface that enables robust signal amplification remains an urgent unmet need for trace miR-21 quantification.

RESULTS: In this work, a laser-induced graphene (LIG) electrode modified with conductive Cu-MOF is developed to mediate an enzyme-free cascade toehold-mediated strand displacement reaction (CTSDR). When miRNA-21 is recognized, the CTSDR is successfully initiated to form numerous G-quadruplexes (G4), which subsequently bind hemin to create a peroxidase-like G4/hemin complex. This complex efficiently catalyzes the hydroquinone (HQ)/H2O2 redox system, generating a significantly amplified current. The sensor delivers a wide linear range from 101 to 107 fM, with a detection limit reaching down to 1.94 fM. It also demonstrates outstanding selectivity, readily discriminating single-base mismatched sequences, and good reproducibility. Importantly, this strategy successfully detects miRNA-21 in thyroid cancer cell lines and clinical patient serum samples, proving its excellent practical viability.

SIGNIFICANCE AND NOVELTY: This work is novel in synergistically integrating a conductive LIG electrode enhanced with Cu-MOF and an enzyme-free catalytic cascade amplification for the first time, and in revealing that CTSDR-generated fuel strands undergo a previously unreported transition to G4 structures induced by K+, enabling ultrasensitive miRNA-21 detection without external enzymes or intricate labeling. Given its facile Cu-MOF synthesis, straightforward electrode modification, and excellent clinical concordance, this strategy provides a highly accessible, affordable, and reliable tool for early tumor diagnostics and routine point-of-care biomarker screening.