A pseudo triple-enzyme electrochemical aptasensor based on the amplification of Pt–Pd nanowires and hemin/G-quadruplex

•Pt–Pd nanowires were combined with pseudo triple-enzyme electrochemical aptasensor.•Pt–Pd nanowires served as signal enhancer.•Labeling process, deactivation and spatial distribution of enzymes were solved.

Our present work aimed at developing a pseudo triple-enzyme cascade electrocatalytic electrochemical aptasensor for determination of thrombin with the amplification of alcohol dehydrogenase (ADH)-Pt–Pd nanowires bionanocomposite and hemin/G-quadruplex structure that simultaneously acted as NADH oxidase and HRP-mimicking DNAzyme. With the addition of ethanol to the electrolyte, the ADH immobilized on the Pt–Pd nanowires catalyzed ethanol to acetaldehyde accompanied by NAD+ being converted to NADH. Then the hemin/G-quadruplex firstly served as NADH oxidase, converting the produced NADH to NAD+ with the concomitant local formation of high concentration of H2O2. Subsequently, the hemin/G-quadruplex acted as HRP-mimicking DNAzyme, bioelectrocatalyzing the produced H2O2. At the same time, the Pt–Pd nanowires employed in our strategy not only provided a large surface area for immobilizing thrombin binding aptamer (TBA) and ADH, but also served as HRP-mimicking DNAzyme which rapidly bioelectrocatalyzed the reduction of the produced H2O2. Thus, such a pseudo triple-enzyme cascade electrochemical aptasensor could greatly promote the electron transfer of hemin and resulted in the dramatic enhancement of electrochemical signal. As a result, a wide dynamic concentration linear range from 0.2 pM to 20 nM with a low detection limit of 0.067 pM for thrombin (TB) determination was obtained. The excellent performance indicated that our strategy was a promising way for ultrasensitive assays in electrochemical aptasensors.

Graphical abstractFigure optionsDownload full-size imageDownload as PowerPoint slide