Low Temperature Greatly Enhancing Responses of Aptamer Electrochemical Sensor for Aflatoxin B1 Using Aptamer with Short Stem.

Aflatoxin B1 (AFB1), one of the most toxic mycotoxins, poses great health risks. Rapid and sensitive detection of AFB1 is important for food safety, environment monitoring, and health risk assessment. We report here the development of a simple and reusable electrochemical aptasensor for rapid and sensitive detection of AFB1. Main improvements were achieved through engineering an aptamer containing a short stem-loop structure and enhancing the binding affinity at a lower temperature. The DNA aptamer with a methylene blue (MB) label at one end was immobilized on a gold electrode. Upon AFB1 binding, the aptamer folded into a stem-loop structure and brought MB close to the electrode surface, resulting in increases in electric current. The aptamer having a shorter stem (2-4 bp) underwent a larger conformation change upon target binding. The sensors built with the aptamer containing a 2 bp stem generated much higher signal-on responses to AFB1 at 4 °C than at room temperature (25 °C). The improvements resulted in a detection limit of 6 pM, enabling the determination of trace AFB1 in a complex sample matrix. This study demonstrates that low temperature greatly enhances the performance of aptamer electrochemical sensors. This aptasensor is simple to construct and readily regenerated by washing with deionized water for reuse. This aptasensor strategy could be applied to the development of an electrochemical aptasensor for other targets.