Programmable Analytical Feature of Ratiometric Electrochemical Biosensor by Alternating the Binding Site of Ferrocene to DNA Duplex for the Detection of Aflatoxin B1†

Comprehensive Summary Ratiometric biosensor is among the promising strategies for the advanced analysis; its applicability in multiple scenarios, however, is seriously limited for the fixed analytical feature. Here, we reported ratiometric electrochemical biosensors with programmable analytical feature by alternating the binding site of ferrocene (Fc) to DNA duplex for the detection of aflatoxin B1 (AFB1). DNA duplex was synthesized with AFB1 aptamer and its complementary DNA (cDNA) with Fc molecular tagged on aptamer or cDNA. For analysis, aptamer-AFB1 complex stripped from the electrode, and free cDNA triggered hybridization chain reaction to produce double-stranded DNA for methylene blue (MB) absorption. In this way, redox currents of Fc (I-Fc) and absorbed MB (I-MB) at the electrode were collected to produce the ratiometric signal of I-MB/I-Fc as a yardstick for AFB1. When Fc was tagged on aptamer, I-Fc decreased with higher concentration of AFB1 (c(AFB1)), the resultant biosensor allowed detection with an On/Off (I-MB/I-Fc) mode; when Fc was tagged on cDNA, I-Fc kept constant as c(AFB1) varying, offering an On/Constant (I-MB/I-Fc) mode for the detection. Consequently, biosensor with On/Off mode showed a linear range of 0.1-100 pg/mL with a limit of detection (LOD) of 0.0650 pg/mL, while biosensor with On/Constant mode offered a wider linear rang of 1-10000 pg/mL but a relatively poor LOD of 0.330 pg/mL. Our work revealed the vital importance of binding site of probes to DNA, and this principle can endow ratiometric biosensor with programmable analytical feature (e.g., linear range, LOD), allowing detections in various scenarios.