Nanostructured functional peptide films and their application in C-reactive protein immunosensors

Peptides with an active redox molecule are incorporated into nanostructured films for electrochemical biosensors with stable and controllable physicochemical properties. In this study, we synthesized three ferrocene (Fc)-containing peptides with the sequence Fc-Glu-(Ala)_n-Cys-NH₂, which could form self-assembled monolayers on gold and be attached to antibodies. The peptide with two alanines (n = 2) yielded the immunosensor with the highest performance in detecting C-reactive protein (CRP), a biomarker of inflammation. Using electrochemical impedance-derived capacitive spectroscopy, the limit of detection was 240 pM with a dynamic range that included clinically relevant CRP concentrations. With a combination of electrochemical methods and polarization-modulated infrared reflection-absorption spectroscopy, we identified the chemical groups involved in the antibody-CRP interaction, and were able to relate the highest performance for the peptide with n = 2 to chain length and efficient packing in the organized films. These strategies to design peptides and methods to fabricate the immunosensors are generic, and can be applied to other types of biosensors, including in low cost platforms for point-of-care diagnostics.