Hairpin DNA-enabled ratiometric electrochemical aptasensor for detection of malathion

A hairpin DNA-enabled ratiometric electrochemical aptasensor is reported for sensitive and reliable detection of malathion (MAL). The approach employs hairpin DNA (ferrocene-labeled, Fc-hDNA) as a carrier to hybridize MAL aptamers (methylene blue-labeled, MB-Apt) to form double-stranded DNA structures on an electrode. The presence of MAL induces the removal of aptamers, and hDNA re-forms hairpin structures, causing a decrease in the oxidation current of MB (I_MB) and an increase in the oxidation current of Fc (I_Fc). The ratiometric signal of I_Fc/I_MB responds quantitatively to MAL concentrations. To compare analytical performances, a linear single-stranded DNA (ssDNA) is also used to construct the ssDNA-based aptasensor. We demonstrate that hairpin DNA possessing a rigid two-dimensional structure can improve the assembly efficiency of aptamers and the stability of redox probes. The approach combines the advantages of the ratiometric electrochemical method with hairpin DNA-based conformational switching probes, enabling hDNA-based aptasensor with enhanced sensitivity and reliability, offering a linear range of 0.001 to 1.0 ng mL^-1. The platform was applied to detect MAL in lettuce, and the statistical analysis indicated that no significant differences were found between the developed platform and HPLC-MS.