High-density aptamer synthesis based on primer exchange reaction for label-free electrochemical biosensing

We report an electrochemical aptamer-based (E-AB) sensor for label-free quantitative detection of thrombin. In-situ synthesis of high-density aptamer replicas on the electrode is accomplished based on primer exchange reaction (PER), by which a DNA primer is continuously extended with the presence of a catalytic hairpin template and DNA polymerase. The PER enables the synthesis of long single-stranded DNA with replicated aptamer sequence for thrombin binding. The PER products on the surface of a gold electrode increase the density of aptamers on a limited electrode area, which can provide more effective binding sites for thrombin and therefore improve the performance of the sensor based on electrochemical impedance spectroscopy. Using our method, thrombin is quantitatively detected from 1.0 pM − 50 nM with a detection limit of 0.91 pM. Compared with the conventional E-AB sensor based on a 15-mer aptamer immobilized on the electrode, the detection limit and linear range of our method is improved. Therefore, we believe our method is promising for label-free electrochemical detection of biomarkers.