A reference electrode-free electrochemical biosensor for detecting MMP-9 using a concentric electrode device

Matrix metalloproteinase-9 (MMP-9) is a core protease of cancer invasion and metastasis. In this paper, we report an electrochemical biosensor integrated with a redox reporter labeled peptide for the detection of MMP-9. The peptide substrate conjugated with methylene blue (MB) specific to MMP-9 was immobilized on Au electrodes using a thiol group in cysteine. The peptide cleavage by MMP-9 caused the release of electroactive MB from the electrodes and resulted in the decrease of the electrical tunneling current. The concentric electrode device consisted of an island electrode and an enclosing electrode which is ca. 1000 folds larger than the island electrode. The self-gating effect from the concentric electrode device was able to provide a reliable electrical measurement without a reference electrode. From the electrochemical biosensor, a linear correlation between the concentration of MMP-9 and the electrical signal reduction was found in a range from 1 pM to 1 nM and the limit of detection was determined at 7 pM.