A bioorganometallic approach for the electrochemical detection of proteins: A study on the interaction of ferrocene-peptide conjugates with papain in solution and on Au surfaces

In this paper, a new bioorganometallic approach for the detection of proteins using surface-bound ferrocene-peptide conjugates is presented. Specifically, a series of peptide conjugates of l′-aminoferrocene-l- carboxylic acid (ferrocene amino acid, Fca) is synthesized: Boc-Fca-Gly- GlyTyr(Bzl)-Arg(NO₂)-OMe (2), Thc-FcaGly-Gly-Tyr(Bzl)-Arg(NO ₂)-OMe (3), Thc-Fca-Gly-Gly-Tyr(Bzl)-Arg(NO₂)OH (4), Boc-Fca-Gly-Gly-Arg(Mtr)Tyr-OMe (7), Thc-Fca-Gly-Gly-Arg(Mtr)-Tyr-OMe (8), Thc-Fca-Gly-GlyArg(Mtr)-Tyr-OH (9), Thc-Fca-GlyGly-Arg-Tyr-OH (10). The peptide is conjugated to the C-terminal side of Fca and compounds 4, 7-10 possess a thiostic acid linked to the N-terminal side of Fca in order to facilitate formation of thin films on gold substrates. Competitive inhibition towards papain was determined for Thc-Fca-Gly-GlyTyr(Bzl)-Arg(NO₂)-OH (4), Thc-FcaGly-Gly-Arg(Mtr)-Tyr-OH (9) and Thc-Fca-Gly-Gly-Arg-Tyr-OH (10). The binding interaction between the peptide modified substrates and papain enzyme was studied using real-time surface plasmon resonance (SPR) imaging. Peptide 10 showed the strongest binding affinity to papain. Adsorption/desorption rate constants were k_a = 1.75±0.05 × 10⁵M ^-1s^-1 and k_d = 2.90 ± 0.05 × 10^-2 s^-1 Interactions of papain with Fca-peptide 10 were investigated by cyclic voltammetry. The interaction results were also verified by measuring the electrochemical response of the peptide-papain interaction as function of increasing enzyme concentration. A linear relationship was observed for papain concentration of up to 80 nM. Shifting to higher potentials as well as decrease in the overall signal intensity was observed. The detection limit was 4 × 10^-9 M.