Target-triggered catalytic hairpin assembly and TdT-catalyzed DNA polymerization for amplified electronic detection of thrombin in human serums

Specific and sensitive detection of protein biomarkers is of great importance in biomedical and bioanalytical applications. In this work, a dual amplified signal enhancement approach based on the integration of catalytic hairpin assembly (CHA) and terminal deoxynucleotidyl transferase (TdT)-mediated in situ DNA polymerization has been developed for highly sensitive and label-free electrochemical detection of thrombin in human serums. The presence of the target thrombin leads to the unfolding and capture of a significant number of hairpin signal probes with free 3′-OH termini on the sensor electrode. Subsequently, TdT can catalyze the elongation of the signal probes and formation of many G-quadruplex sequence replicates with the presence of dGTP and dATP at a molar ratio of 6:4. These G-quadruplex sequences bind hemin and generate drastically amplified current response for sensitive detection of thrombin in a completely label-free fashion. The sensor shows a linear range of 0.5 pM-10.0 nM and a detection limit of 0.12 pM for thrombin. Moreover, the developed sensor can selectively discriminate the target thrombin against other non-target proteins and can be employed to monitor thrombin in human serum samples.