Sensitive homogeneous electrochemical aptasensor for aflatoxin B1 detection enabled by integrating ultrasmall nanochannels with 2D nanoprobe and DNase I-assisted target recycling

A homogeneous electrochemical aptasensor was developed for sensitive detection of aflatoxin B1 (AFB1) by integrating ultrasmall silica nanochannel film (SNF)-modified electrode, two-dimensional (2D) nanoprobes, and DNase I-assisted target recycling. The 2D nanoprobes (Ru(NH 3 ) 6 3+ /AFB1 Apt@GO) were prepared by co-loading cationic electrochemical probe Ru(NH 3 ) 6 3+ and AFB1 specific aptamers (AFB1 Apt) onto graphene oxide (GO) via π–π stacking and electrostatic interaction. SNF was rapidly formed on indium tin oxide (ITO) electrodes within 10 s. The resulting structure provided 2–3 nm nanochannels, which enabled the electrostatic enrichment of Ru(NH 3 ) 6 3+ . In addition, the 2D nanoprobes were unable to enter the ultrasmall nanochannels of the SNF due to size exclusion and thus could not access the underlying electrode surface. In the presence of AFB1, aptamer–target binding caused conformational changes and aptamer detachment, releasing Ru(NH 3 ) 6 3+ . The detached aptamer was digested by DNase I, triggering further Ru(NH 3 ) 6 3+ release as AFB1 participated in additional recognition cycles. The released Ru(NH 3 ) 6 3+ was electrostatically enriched at the electrode surface, amplifying the ECL signal. DNase I-assisted recycling enhanced the ECL response by 3-fold compared to the non-enzymatic system. Under optimized conditions, the aptasensor exhibited a linear detection range for AFB1 detection from 3.12 × 10 −4 to 312 ng/mL and a limit of detection of 302 fg/mL. The method was successfully applied to detect AFB1 in corn and peanut samples, offering a simple, rapid, and highly sensitive strategy for electrochemical toxin detection in food safety analysis.