DNA Electrochemical Sensor Based on Exonuclease III-Assisted Cycling Signal Amplification for Ultrasensitive...

The safety of genetically modified crops, particularly the commercial cultivation of glyphosate-resistant genetically modified soybeans, has given rise to significant public concern. Consequently, there is an urgent need to develop efficient and precise methods for detecting genetically modified components. The present study constructed a novel electrochemical biosensor based on nucleic acid exonuclease III (Exo III)-assisted cyclic signal amplification and hairpin probe recognition for the highly sensitive and specific detection of the CP4-EPSPS gene in genetically modified soybeans. The sensor achieves exponential signal amplification by triggering Exo III to cyclically cleave the hairpin probe (H1) upon target DNA binding. Subsequent to this, the released DNA fragments hybridize with the methylene blue-labeled signal probe (HS-MB) that has been immobilized on the electrode surface. This process induces conformational changes and a decrease in the current signal, thereby enabling quantitative analysis of the target gene. The experimental phase of the study successfully validated the sensor's mechanism and systematically optimized key parameters such as Exo III concentration and reaction time. In optimal conditions, the sensor demonstrated excellent linearity with target DNA concentrations ranging from 100 fM to 10 nM, achieving a detection limit as low as 0.1072 pM. Furthermore, it exhibited remarkable repeatability and stability. This study provides an analytical tool with broad application prospects for the rapid and precise detection of genetically modified crops.