A reagentless electrochemical sensor for aflatoxin B1 with sensitive signal-on responses using aptamer with methylene blue label at specific internal thymine

Aptamer electrochemical sensors using immobilized aptamers with redox tag rely on the target binding-induced changes of current signal on electrode, offering advantages in operation convenience, no separation, rapidity, and sensitivity. Usually, the redox tag is placed on aptamer terminal, however, sometimes the terminal label may be insensitive to target-binding and fail to generate sensitive responses. The redox tag methylene blue (MB) labeled on different sites of aptamer may experience distinct changes in local environment, distance to electrode, or interactions with aptamer bases during affinity binding, which affect the current signal. Thus, it is possible to construct aptamer electrochemical sensors with sensitive and significant responses to targets by screening a series of sites (e.g., internal thymine T) of the aptamer and placing MB tag on a specific site of the aptamer. With this strategy, we successfully fabricated an electrochemical sensor on gold electrode for rapid, reagentless, and sensitive detection of aflatoxin B1 (AFB1), an important mycotoxin causing great health risks, by using a 26-mer DNA aptamer with MB on an internal T site (e.g., 18th T) and a thiol moiety at 5′ terminal. This sensor generated remarkable signal-on responses to AFB1, allowed a detection limit of 6 pM, and enabled detection of AFB1 in wine, milk and corn flour samples. This sensor can be well regenerated by rinsing with deionized water and reused, and shows good stability. This sensor and the demonstrated strategy are promising in wide applications.