A novel fluorescent aptasensor for aflatoxin M₁ detection using rolling circle amplification and g-C₃N₄ as fluorescence quencher

Aflatoxin M₁ is a prevailing natural carcinogenic mycotoxin, found in milk. Herein, we reported a sensitive fluorescent aptasensor for AFM₁, based on rolling circle amplification (RCA) technology to improve the sensitivity of the assay. The detection of AFM₁ was achieved using KYF₄: Eu³⁺ time-resolved fluorescent nanoparticles (KYF₄: Eu³⁺ TRFNPs) as signal probe and graphitic carbon nitride (g-C₃N₄) nanosheet as a quencher. The AFM₁ aptamer act as a recognition element and as a primer for DNA synthesis. TRFNPs attached to complementary DNA (TRFNPs-cDNA) acts as a signal probe. In the absence of target, a rolling circle template (RCT) ligated to aptamer and RCA reaction initiated. The TRFNPs-cDNA was strengthened to amplified RCA product (RCAP) to make RCAP/TRFNPs-cDNA duplex. This duplex cannot be adsorbed onto g-C₃N₄, and ultimately no fluorescence quenched. In the presence of AFM₁, aptamer recognized the target and aptamer-target complex was formed. Hence, no aptamer hybridized with RCT, therefore, no RCA and duplex formation. Consequently, free TRFNPs-cDNA adsorbed onto g-C₃N₄ and resulted in quenched fluorescence. The assay provides a lower detection limit (0.0194 pg/mL) than the previously reported assays and has good recoveries between 92–99.8 %. Hence, the present method would be a suitable method for the quantification of small molecules.