A sensitive electrochemical aptasensor for thrombin detection based on exonuclease-catalyzed target recycling and enzyme-catalysis

• A thrombin aptasensor based on target recycling and ADH catalysis was developed.
• ADH was abundantly encapsulated into the MPTS sol with a 3-D network.
• The ADH effectively improved the electrochemical signal via the enzyme-catalysis.
• The employment of the RecJf exonuclease achieved the target-recycling.
• The strategy realized high sensitivity, good stability and reproducibility.

In the present study, a sensitive electrochemical aptasensor based on exonuclease-catalyzed target recycling and enzyme-catalysis was developed for thrombin (TB) detection. Firstly, the alcohol dehydrogenase (ADH) was abundantly embedded in the 3-(mercaptopropyl)trimethoxysilane (MPTS) sol with a 3-D network that exhibited tunable porosity and high thermal stability. ADH, as an alcohol oxidase, catalyzed the conversation of alcohol into acetaldehyde coupling with the production of NADH in the presence of NAD+. Then the immobilized gold nanoparticles (AuNPs) could electrocatalyze the oxidation of NADH, finally promoting the redox reaction of the electroactive material methylene blue (MB) labeled on the hybrid double strand DNA (dsDNA). Furthermore, when the mixture of TB and RecJf exonuclease was introduced, TB combined with the thrombin aptamer II (TBA II) and the aptamer–TB complex was formed. And then, the RecJf exonuclease selectively degraded the TBA II from 5′→3′, releasing the target TB into the solution. The free TB was reused to combine with other TBA II to accomplish the target recycling and realize the electrochemical signal amplification. In this way, excellent sensitivity of the aptasensor was obtained. The thrombin aptasensor achieved a detection limit of 1.7 pM (defined as S/N=3) with a linear range from 5 pM to 100 nM. In addition, the proposed aptasensor had good stability and sensitivity, and would become a promising choice for the protein diagnostics in clinical analysis.