Visual detection of aflatoxin B1 based on specific aptamer recognition combining with triple amplification strategy

A highly sensitive and specific visual detection method for aflatoxin B1 (AFB1) based on the target specificity of aptamer, rolling circle amplification (RCA) and enzyme catalysis biological amplification effect has been established. In this work, AFB1 aptamer immobilized on the surface of magnetic beads (MB) serves as a molecular recognition probe. In the absence of AFB1, the aptamer and auxiliary linking probe (LP) maintain a double stranded state due to partial base pair complementarities. By contrast, in the presence of AFB1, the aptamer preferentially binds to AFB1 specifically, and the LP later restores to a single stranded state. Subsequently, the RCA reaction is triggered by above-mentioned single stranded LP to generate long DNA strands, which are employed to capture amounts of signal probes (SP) and horse radish peroxidases (HRP). Finally, amounts of HRP catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by H₂O₂ and leads to a dramatic color change of the solution from colorlessness to deep blue as a signal indicator, obtaining a high sensitivity, high specificity and visual detection of AFB1. Under optimal conditions, a good linear detection range (0.5-40 pg·mL^-1) was achieved, and the limit of detection (LOD) was 0.13 pg·mL^-1. Besides, the proposed aptasensor showed excellent specificity for AFB1 compared with five other mycotoxins. More than that, all reactions occur on the surface of the magnetic beads, which not only facilitates the detection operation process including the efficient isolation and collection of AFB1 from sample matrix, but also gets better selectivity and stronger resistibility to target analyte in complex sample matrix, adequately indicating its potential application in AFB1 practical detection.