A simple electrochemical aptasensor for ultrasensitive protein detection using cyclic target-induced primer extension

A simple electrochemical aptasensor was developed for ultrasensitive protein detection by combining a novel strategy of cyclic target-induced primer extension (CTIPE) with an aptamer-hairpin probe and enzyme-amplified electrochemical readout. In the presence of protein target, the immobilized aptamer-hairpin probe recognized the protein to trigger primer extension reaction by target-induced conformational transition, which released the protein from replicated DNA duplex. The released target could cyclically bind with other aptamer-hairpin probes and trigger new primer extension, leading to formation of numerous biotin-tagged DNA duplex, which significantly amplified the protein recognition event and facilitated the subsequent enzymatic signal enhancement, leading to an ultrasensitive electrochemical aptasensor. Using human vascular endothelial growth factor as a model protein, the designed aptasensor could detect protein down to 0.82 pg mL−1 with a linear range from 1 pg mL−1 to 1 ng mL−1. The proposed aptasensor was amenable to quantification of protein in complex biological matrixes, and would become a simple and powerful tool for bioanalysis and clinic diagnostic application.

► This work constructs an aptamer-hairpin probe for target binding of protein on gold electrode. ► Cyclic target-induced primer extension and enzymatic reaction are used for signal amplification. ► Single VEGF/aptamer binding event is transited to many biotin-tagged DNA duplex. ► Enzymatic product produces the amplified electrochemical signal. ► The aptasensor shows ultra-high sensitivity and acceptable reproducibility.