Highly sensitive electrochemical thrombin aptasensor based on peptide-enhanced electrocatalysis of hemin/G-quadruplex and nanocomposite as nanocarrier

In this work, we first conjugated a short peptide to thrombin binding aptamer (TBA) and bond hemin to the hybrid, effectively rendering hemin/G4-peptide more active over the original hemin/G4, so that a highly sensitive electrochemical thrombin (TB) aptasensor was developed based on it and PtNTs@rGO nanocomposite. It was the first report on the application of hemin/G4-peptide in electrochemical aptasensor. PtNTs@rGO with large surface area served as excellent nanocarrier for high loading of hemin/G4-peptide hybrids, resulting in the formation of hemin/G4-peptide-PtNTs@rGO bioconjugate as the secondary aptamer and further signal enhancement. The specific affinity of aptamer for target TB made the secondary aptamer go into the sensing interface, and then a noticeable current signal was obtained from hemin without additional redox mediators. Due to the collaborative electrocatalysis of hemin/G4-peptide and PtNTs toward H2O2, which was formed in situ during the process of hemin/G4-peptide-catalyzed oxidation of NADH with dissolved O2, the current intensity increased dramatically. Such an electrochemical aptasensing system could be used to detect TB with a linear range of 0.05 pM-60 nM and very lower detection limit of 15 fM. Notably, this method exhibited a higher sensitivity than that of many hemin/G4-based electrochemical strategies for TB detection due to the improvement of the catalytic activity of hemin/G4-peptide. The present works opened a new way for expanding the application of hemin/G4 in biological detection. With the mediator-free, proteinous enzyme-free yet high-sensitivity advantages, this electrochemical aptasensor held great promise for other biomarker detections in clinical diagnostics.