A double methylene blue labeled single-stranded DNA and hairpin DNA coupling biosensor for the detection of Fusarium oxysporum f. sp. cubense race 4.

The DCL gene in Fusarium oxysporum f. sp. cubense Race 4 (Foc4) is a pivotal pathogenic factor causing banana fusarium wilt. Precise DCL detection is crucial for Foc4 containment. Here, we present a novel ssDNA-hDNA coupling electrochemical biosensor for highly specific DCL detection. The sensing interface was formed via electrodeposition of a composite containing reduced graphene oxide (rGO) and gold nanoparticles (AuNPs) onto a carbon screen-printed electrode (SPE), followed by thiol-modified ssDNA functionalization. Additionally, the incorporation of hDNA, with methylene blue (MB) at both ends, binds to ssDNA through base complementarity, forming an ssDNA-hDNA coupling probe with bismethylene blue. This sensing strategy relies on DCL recognition by the hDNA probe, leading to DNA hairpin unfolding and detachment of hDNA bearing two MBs from ssDNA, generating a robust "on-off" signal. Empirical results demonstrate the sensor's amplified electrical signals, reduced background currents, and an extended detection range (6.02 × 106-3.01 × 1010 copies/µL) with a limit of detection (3.01 × 106 copies/µL) for DCL identification. We applied this sensor to analyze soil, banana leaves, and fruit samples, confirming its high specificity and stability. Moreover, post-sample detection, the sensor exhibits reusability, offering a cost-effective and rapid approach for banana wilt detection.