A disposable paper-based electrochemical biosensor for detection of aflatoxin B1 based on a structure-switching aptamer

Aflatoxin B₁ (AFB₁) is a highly toxic mycotoxin that readily contaminates major food crops. This study presents a novel ratiometric electrochemical paper-based aptasensor for AFB₁ detection that utilizes a structure-switching aptamer. The sensor detects AFB₁ by folding the DNA aptamer, reducing the electron transfer efficiency between the methylene blue (MB) molecules attached to the DNA and the electrode surface. An inner reference element modified with ferrocene (Fc) is co-assembled on the electrode surface of the paper-based biosensor to enhance the accuracy of AFB₁ detection. In the presence of target AFB₁, a folded target–aptamer complex is formed, distancing the MB molecules from the electrode and generating a low current signal in a differential pulse voltammetry mode. The ratio of oxidation currents for MB and Fc serves as the metric for monitoring AFB₁ levels. The detection range spans from 20.0 to 1000.0 ng mL⁻¹, with a limit of detection of 7.6 ng mL⁻¹. The paper-based aptasensor exhibits excellent specificity and stability. Additionally, its high reliability and applicability are validated by the strong correlation observed between the sensor results and the results obtained using UPLC−MS/MS analysis of real samples. This study offers a low-cost, readily available approach for portable detection of foodborne hazards.