Electrochemical detection of DNA 3'-phosphatases based on surface-extended DNA nanotail strategy

Determination of DNA dephosphorylation is of great value due to its vital role in many cellular processes. Here we report a surface-extended DNA nanotail strategy for simple and ultrasensitive detection of DNA 3'-phosphatases by terminal deoxynucleotidyl transferase (TdT) mediated signal amplification. In this work, DNA probes labeled with thiols at their 5' terminals and phosphoryls at 3' terminals are immobilized on gold electrode and are used as substrates for DNA 3'-phosphatases, taking T4 polynucleotide kinase phosphatase (T4PNKP) as an example. T4PNKP can catalyze the dephosphorylation reaction of the substrate DNA, followed by the formation of a long DNA strand by TdT on its 3' terminal hydroxyl, leading to an evident chronocoulometry signal enhancement. The proposal presents a considerable analytical performance with low detection limit and wide linear range, making it promise to be applied in the fields of DNA dephosphorylation related processes, drug discovery, and clinical diagnostics.