A dual-switch electrochemical aptasensor for label-free detection of thrombin and ATP based on split aptamers

Background: Aptamers, consisting of specialized single-stranded nucleic acids, are engineered through the SELEX technique to recognize specific targets with strong affinity. Aptamers are exceptionally useful in various sensor technologies, such as fluorescence-based sensors, electrochemical sensors, and colorimetric detection systems. Due to its high sensitivity, specificity and fast response, electrochemical aptasensor shows great application prospects in analytical detection, food safety, and environmental monitoring. However, one aptasensor can usually detect only one type of target, limiting its universality in practical applications. Results: Here, we constructed a dual-switch and label-free electrochemical aptasensor based on split aptamer and nuclease. The feasibility, specificity, and sensitivity of the aptasensor were investigated by using thrombin and adenosine triphosphate (ATP) as targets. Split aptamer can not only capture target specifically but also form a stable sandwich structure with the target. In the presence of thrombin, it triggered a hydrolysis reaction of exonuclease I, leading to a decrease in the impedance signal. Differently, the presence of ATP could form a sandwich structure with split aptamers, leading to an increase in output signals. The aptasensor achieved sen- sitive and specific detection of thrombin and ATP, with low detection limits of 0.76 pM and 0.27 pM, respectively. Significance and novelty: The aptasensor realized the detection of two targets without replacing any reagents or equipment, which greatly saved time and cost. Furthermore, electrochemical impedance spectroscopy (EIS) uses impedance as an output signal, showing great application prospects in electrochemical aptasensors as label-free and simple methods. Because of its simplicity, label-free, and sensitivity in complex samples, the split aptamer- assisted aptasensor provides new ideas and methods in early diagnosis of diseases.