Highly sensitive and aptamer-based electrochemical fipronil biosensor based on primer exchange reaction and catalytic strand displacement dual recycling amplifications

Background: The indiscriminate use of pesticides in agriculture has caused substantial harm to both the environment and human health. Therefore, it is crucial to develop methods for evaluating trace amounts of pesticide residues in food products. This study presents a highly sensitive electrochemical aptasensor to detect fipronil utilizing a new primer exchange reaction (PER)/catalytic strand displacement reaction (CSDR)-integrated dual signal amplification strategy. Results: Upon binding of fipronil to its specific aptamer in the template hairpin/aptamer complex probe, the template hairpin is released and hybridizes with primer to initiate PER for generating a substantial amount of assistant ssDNAs, which trigger subsequent CSDR for cyclically confining numerous methylene blue (MB)-conjugated signal probes onto sensing surface with existence of Klenow (exo-) fragment polymerase (KF) and deoxynucleotide triphosphates (dNTPs). Such many MB labels thus yield considerably enhanced electrochemical current signals, resulting in detection limit of 0.054 nM for fipronil within the dynamic range of 0.1 nM-1 mu M. Moreover, the sensing method is highly selective and also effective for detecting low concentrations of fipronil in cabbage samples. Significance: With the successful demonstration of the significant signal amplification capability of our PER/CSDR methodology for fipronil, such electrochemical biosensor can be further developed as a robust sensing system for sensitive detection of various small molecules and other biomarkers by substituting the corresponding aptamers for different applications.