Label-free detection of target proteins using peptide molecular wires as conductive supports

We report on the electrochemistry of a peptide molecular wire as a conductive support for ligand immobilization in biosensing assays. The helical 9-mer peptide, tagged at N-terminus with Methylene Blue (MB) and thiolfunctionalized at C-terminus was anchored on a gold surface via gold/thiol chemistry. The helical peptide acts as molecular wire, mediating the two-step electron transfer (ET) from MB to the gold surface. The forward and backward square wave voltametric (SWV) components recorded in the presence of peptide wire were used to estimate the kinetic parameters of the electrode reaction. The simulated data matched the experimental ones for the two-step sequential surface mechanism (EE), with the rate constant of the first step k(sur,1) = 20 s(-1) and the cathodic ET coefficient alpha(1) = 0.55. The kinetic parameters of the second step were k(sur,2) > 1000 s(-1), and alpha(2) = 0.5. Small ligands for high-molecular weight targets can be grafted on the peptide wire between the MB tag and the surface through covalent bonding. The binding of the target to the peptide-anchored ligand hampers the ET transfer from MB to the electrode surface, causing a decrease of the peak current. These findings were used further to develop an electrochemical peptide-based biosensor with signaling-off interrogation. The biosensor was tested against two relevant targets for medical diagnosis: the anti-tumor-associated carbohydrate antigen (alpha-Tn) antibody and the growth hormone secretagogue receptor (GHS-R1a), after ligand grafting. Both targets were detected in the nanomolar range with an overall assay time of 10 min.