A pseudo triple-enzyme cascade amplified aptasensor for thrombin detection based on hemin/G-quadruplex as signal label

In this work, a pseudo triple-enzyme cascade amplified electrochemical aptasensor based on hemin/G-quadruplex as signal label for thrombin (TB) was constructed and the amplified electrochemical signal was achieved by the corporate catalysis of alcohol dehydrogenase-graphene sheets (ADH-GSs) bionanocomposite and hemin/G-quadruplex, which simultaneously acted as NADH oxidase and HRP-mimicking DNAzyme. Through “sandwich” reaction, hemin/G-quadruplex labeled gold nanoparticles-ADH-GSs bionanocomposite (AuNPs-ADH-GSs) was captured on electrode surface and thus obtained the electrochemical signal. After the addition of ethanol into the electrolytic cell, ADH availably catalyzed the oxidation of ethanol with the reduction of NAD+ to NADH. Then, hemin/G-quadruplex as NADH oxidase catalyzed the oxidization of NADH, accompanying with the production of H2O2. Simultaneously, hemin/G-quadruplex as HRP-mimicking DNAzyme catalyzed the reduction of the generated H2O2. Such a catalysis strategy greatly promoted the electron transfer of hemin and resulted in the specific enhancement of electrochemical signal. The proposed TB aptasensor achieved a linear range of 1 pM-50 nM with a detection limit of 0.3 pM (defined as S/N=3). In addition, it showed satisfying stability and reproducibility, good specificity and sensitivity, indicating promising application for the detection of various proteins in clinical analysis.