A signal-on electrochemical strategy for protease detection based on the formation of ATCUN-Cu(II)

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Amino terminal Cu(II)- and Ni(II)-binding (ATCUN) motif is included in a protein or peptide with a structural characterization comprising of a free NH2-terminus, a histidine residue in the third position and two intervening peptide nitrogens. We reported a signal-on electrochemical strategy for detection of protease based on the formation of ATCUN-Cu(II) on electrode surface. In the method, an amino residue in the native peptide substrate was replaced with a histidine. Cleavage of the sequence-specific peptide pre-immobilized on an electrode by the protease led to the presence of ATCUN motif and the formation of ATCUN-Cu(II) in the presence of Cu(II). Consequently, a voltammetric signal was observed. Contrarily, inhibition of protease by its potential inhibitor decreased the voltammetric response. To demonstrate the feasibility and sensitivity of our strategy, β-secretase was tested as a model protease. As a result, a detection limit of 0.08nmolL−1 was achieved. The half-maximum inhibition value (IC50) of a well-known β-secretase inhibitor was found to be 22.6nmolL−1, which is in agreement with the reported value.