A novel label-free and enzyme-free electrochemical aptasensor based on DNA in situ metallization

In this work, we presented a novel label-free and enzyme-free electrochemical aptasensor based on DNA in situ silver metallization as effective electrochemical label. Molecular beacon 2 (MB2, Peptide nucleic acid) was first immobilized on the gold electrode (AuE) through Au-S bond. In the presence of thrombin, the thrombin binding aptamer (MB1) preferred to form thrombin/aptamer complex in lieu of aptamer-DNA duplex, resulting in the 8-17 DNAzyme liberating from the caged structure and hybridization with the MB2, the MB2 will replace and free the target thrombin when it hybridizes with MB1. The released target thrombin can participate in the next hybridization process with MB1. Eventually, each target thrombin went through many cycles, resulting in numerous MB1 confining close to the AuE, which leaded to the surface became negatively charged and allowed the absorption of silver ions on the DNA skeleton. After chemical reduction by hydroquinone, the formed silver nanoparticles could be afforded a signal trace for electrochemical stripping analysis of target thrombin. Through introducing a hybridization chain reaction to increase the DNA length, the current signal was further amplified, achieved the detection of thrombin with a linear range from 1.0×10−16 to 1.0×10−11 M and a detection limit of 37 aM. In addition, the signal amplification is realized without using any enzymes or sophisticated label process, and the sensing strategy is completely non-labeled. The success in the present biosensor served as a significant step towards the development of monitoring ultratrace thrombin in clinical detection.