Rapid label-free colorimetric ssDNA biosensor based on gold nanoparticles: Application to fusarium oxysporum detection in soil

Rapid and reliable detection of specific DNA sequences is essential for environmental and agricultural monitoring, yet amplification-based assays remain limited by equipment demand, multistep workflows, and susceptibility to matrix inhibitors. Here, we present a label-free colorimetric biosensor based on gold nanoparticles (AuNPs) and a freeze-mediated complementary-probe anti-aggregation mechanism (CPAA) for fast detection of Fusarium oxysporum ssDNA. The assay operates through a two-step hybridization process completed in 5 min, achieving a limit of detection of 2 nM (12 pg/μL). Compared with reported AuNP-based DNA sensors, which typically achieve LODs in the pM–nM range, the present platform provides moderate sensitivity but combines amplification-free operation, 5-min assay time and preliminary compatibility with diluted soil-derived DNA extracts, which are rarely achieved simultaneously. It exhibits discrimination against tested non-complementary sequences, both individually and in complex mixtures, while tolerating a single central mismatch without loss of signal. The biosensor preserved a measurable response in diluted soil-derived DNA extracts despite residual matrix interference. The platform is rapid, low-cost, and easily reconfigurable through probe substitution. Its simplicity and compatibility with automated spectral interpretation support its potential use as a proof-of-concept amplification-free sensing platform for soil-derived DNA extracts.