Sensitive colorimetric assay for the detection of aflatoxin B1 via target-induced generation of multivalent DNA nanowires accelerates the catalytic hairpin assembly reaction.

Background: Aflatoxin B1 (AFB1) is a highly carcinogenic mycotoxin frequently found in contaminated food products, posing a significant threat to public health and food safety. Therefore, the development of rapid, sensitive, and reliable detection methods for AFB1 is critical for early warning and prevention. However, traditional detection techniques often require expensive equipment, skilled personnel, and complex procedures, limiting their suitability for on-site applications. This study introduces a novel colorimetric assay based on DNA nanotechnology to enhance sensitivity, reduce cost, and simplify the detection process for broader applicability.

Results: The colorimetric assay for AFB1 detection that utilizes a target-induced generation of multivalent DNA nanowires, which in turn accelerate a catalytic hairpin assembly (CHA) reaction. Upon binding to AFB1, the DNA nanowires are formed and act as multivalent scaffolds that significantly enhance the speed of the CHA process. Compared to the use of free T strand and monovalent DNA nanowires, the multivalent design increased the initial reaction rate by 7.6-fold and 32.3-fold, respectively. This enhanced reaction kinetics, driven by the multivalent effect, led to improved signal amplification in the target-induced cascade reaction. The assay demonstrated a strong linear correlation between absorbance and the logarithm of AFB1 concentration over a dynamic range of 10 pM to 1 nM. Furthermore, the method achieved a low detection limit of 4.26 pM. Recovery rates in spiked peanut samples ranged from 95.96 % to 107.75 %, confirming its reliability in complex food matrices.

Significance: This assay provides a highly sensitive method for detecting AFB1 without the need for sophisticated instruments or specialized training. Its simple visual readout and portability make it particularly advantageous for on-site testing. By leveraging multivalent DNA nanowires and CHA amplification, this assay holds great promise not only for AFB1 detection but also for broader applications in food safety monitoring and mycotoxin control.