Exonuclease-Catalyzed Methylene Blue Releasing and Enriching onto a Dodecanethiol Monolayer for an Immobilization-Free and Highly Sensitive Electrochemical Nucleic Acid Biosensor.

Herein, a unique and versatile immobilization-free electrochemical nucleic acid biosensor architecture is proposed for the first time based on the catalyzed release of a methylene blue (MB)-tagged mononucleotide by exonuclease III (Exo III) and the successive enrichment onto a dodecanethiol monolayer, which can be attributed to the hydrophobic force between the alkyl chain of the dodecanethiol monolayer and the hydrophobic part of the MB-tagged mononucleotide. The fabricated biosensor demonstrates considerable advantages including assay simplicity, rapidness, and high sensitivity owing to its immobilization-free and homogenous operation for the biorecognition and amplification process. A low detection limit of approximately 1 pM toward the target DNA could be achieved with an excellent selectivity. The proposed immobilization-free electrochemical biosensing strategy was also extended for the assay of Exo I and III activity. Furthermore, it might be easily extended for the detection of a wide spectrum of targets and thus provide a promising avenue for the development of immobilization-free and sensitive electrochemical biosensors.