Temperature and pH tolerance ratiometric aptasensor: Efficiently self-calibrating electrochemical detection of aflatoxin B1.

Ratiometric electrochemical aptasensors based on the two different redox reporters can hardly achieve the efficient self-calibration, as a result of variations between reporters. In this work, we reported a ratiometric electrochemical aptasensor using ferrocene as a single redox reporter (Fc) to achieve self-calibrating electrochemical detection of aflatoxin B1 (AFB1). The hybrid duplex of Fc-labeled AFB1 aptamer (Fc-Apt) and Fc-labeled assistant DNA (Fc-aDNA) was designed to construct the sensing interface. Such DNA structure adopted the fixed molar ratio (1:1) and locations (the distances to electrode) of these two types of Fc (i.e., Fc-Apt and Fc-aDNA). In this way, the ratio of redox currents from Fc-Apt (IFc-Apt) and Fc-aDNA (IFc-aDNA) kept stable under the varied temperatures (15-40 °C) and pH values (4-10). Moreover, values of IFc-Apt and IFc-aDNA under the different conditions can be calculated simply by recording the total current of Fc (IFc-total). For analysis, the recognition of AFB1 by Fc-Apt led to the striping of their complex from the aptasensor, resulting in a decrease in IFc-Apt while IFc-aDNA remained stable. Consequently, the developed aptasensor using the ratio of IFc-Apt/IFc-aDNA as a yardstick offered a linear range of 0.1-10000 pg mL-1 with a detection limit of 0.012 pg mL-1 for the detection of AFB1. The applicability of aptasensor was validated by corn sample analysis, which exhibited comparable reliability and accuracy of gold standard method, i.e., HPLC-MS/MS. Our work provided an efficient strategy to fabricate high-performance ratiometric electrochemical aptasensors.