A Simple Paper-Based Dual-Analyte Electrochemical Nanobiosensor for Mycotoxins Using Phosphorene-Gold Nanocomposites

In this study, a double-layer structured paper-based electrochemical biosensor was developed for the simultaneous detection of aflatoxin B1 (AFB1) and ochratoxin A (OTA). Microelectrodes were screen-printed on the paper sheet substrate, containing two working electrodes (WEs) and a pair of the shared counter electrode (CE) and reference electrode (RE). Two sheets of paper were overlapped to form two three-electrode setups with common CE and RE. Aptamers that were specific to AFB1 and OTA served as the sensing probes. The incorporation of black phosphorus-gold (BP-Au) nanocomposites facilitated the sensing probe immobilization and detection sensitivity enhancement due to the high surface-to-volume ratio and aptamer-capturing efficiency. To achieve superior performance, critical design parameters, namely the concentration and the self-assembly time of immobilized aptamer probes, as well as the assay time were investigated and optimized. The results demonstrate that under the optimized conditions, the detection limits of AFB1 and OTA sensing were calculated to be 0.023 and 0.039 ng/mL, respectively, with a dynamic range between 0.05 and 10 ng/mL. The applicability of the proposed biosensor was demonstrated by testing spiked wine samples. The resulting recovery was in the range of 94.1%–108.6% for simultaneous detection of AFB1 or OTA with relative standard deviations (RSDs) less than 6.9%. Hence, the constructed paper-based electrochemical nanobiosensor would be a promising method for dual-analyte detection in food safety.