G-quadruplex/hemin DNAzyme mediated biosensor for colorimetric detection of trace aflatoxin B1 using entropy-driven catalytic reaction-facilitated catalytic hairpin assembly

G-quadruplex/hemin DNAzyme (G-DNAzyme) is widely utilized in biosensors due to its ability to catalyze peroxidase-like reactions. However, its relatively low catalytic efficiency can limit detection sensitivity. To address this limitation and enable trace detection, this study presents a G-DNAzyme-mediated biosensor that combines entropy-driven catalytic reactions (EDC) and catalytic hairpin assembly (CHA) to enhance sensitivity and broaden its applicability for detecting trace levels of aflatoxin B1 (AFB1). In this biosensor, AFB1 specifically binds to aptamer, triggering the release of a complementary initiator chain, which activates both EDC and CHA amplification processes. This dual amplification results in the generation of numerous free G-quadruplex sequences, which then bind to hemin, catalyzing a color change in the chromogenic substrate and significantly amplifying the signal. The biosensor demonstrated excellent linearity between the absorbance intensity and logarithm of AFB1 concentrations, with a detection limit (LOD) of 0.47 pM. Furthermore, spiked recovery tests of AFB1 in actual samples showed recovery rates between 96.12 % and 108.50 %, indicating strong accuracy. The method is simple, rapid, and does not require complex instrumentation, making it an efficient tool for detecting AFB1 in food safety applications.