On-off-on electrochemiluminescence biosensor with dual-cycle amplification for ultrasensitive prenatal detection of thalassemia mutations using lanthanide europium nanorods

Herein, we develop an on-off-on electrochemiluminescence (ECL) biosensor with dual-cycle amplification for ultrasensitive prenatal detection of thalassemia mutations. The biosensor utilizes hydrothermally synthesized lanthanide europium nanorods (EuBTC), which possess abundant active sites and exhibit a 3.2-fold improvement in electron transfer efficiency compared to conventional spherical MOFs. By integrating catalytic hairpin assembly and exonuclease-driven target recycling amplification, the biosensor achieves sequential detection of CD122 (β-globin IVS-II-654 mutation) and CD17 (β-globin codon 17 mutation) in a single assay. The CHA-triggered ferrocene quenching (signal-off) enables CD122 quantification with 0.739 fM detection limit, while T7 exonuclease-mediated strand displacement restores ECL signals (signal-on) for CD17 detection at 0.301 fM sensitivity, representing 2-3 orders of magnitude improvement over existing PCR-based methods. Validation using actual maternal serum samples showed recoveries ranging from 97.7 % to 108.0 %, with relative standard deviation (RSD) < 4.1 %, confirming its reliability in non-invasive prenatal diagnosis. This dual-signal modulation strategy, combines with the antenna effect of EuBTC, provides a universal platform for multiplexed gene analysis through programmable DNA sequence replacement.