HaloTag-Modified, Ferrocene Labeled Self-Assembled Monolayers for Protein Sensing

Ferrocene (Fc)-based disulfide molecules of various lengths with amino acid scaffolds and alkane or oligo(phenylene-ethynylene) (OPE) bridges are used in a mixed SAM with a di-(ethylene oxide) terminal mercaptoundecanol diluent (PEG₂). The relative height of the Fc redox reporter in the SAM is compared to determine if there are protective effects like antifouling and specific detection. The HaloTag-binding motif is used as a proof-of-concept to investigate the electrochemical response to the HaloTag protein due to its known covalent and fast linkage. When the Fc-SAMs are exposed to the HaloTag protein, there are an antifouling nature and more specific detection for the engulfed Fc-based molecules (C₆tBu/Halo). The further out the Fc is from the SAM layer, the more nonspecific adsorption is detected. The double layer capacitance (CD_L) has the smallest change for the C₆tBu control (ΔCD_L = -0.1 μF cm^-2) showing antifouling properties and produces a large change (ΔCD_L = 0.9 μF cm^-2) as well as a shift in oxidation potential when the active C₆Halo is exposed to the HaloTag protein (ΔE_1/2 = 50 ± 10 mV). The remaining Fc molecules are partially in or outside the PEG₂ layer, allowing more ion penetration/mobility even when the HaloTag protein is bound. Generally, a more disordered environment was observed for the Fc-based molecules when adding the HaloTag ligand, which is evident from a larger E_fwhm and higher CD_L. Desorption of the SAMs with sodium iodide (NaI) showed retention of the HaloTag protein bound with the corresponding ligand, whereas negative controls did not. Self-assembled monolayers for MALDI mass spectrometry (SAMDI-MS) were used as an orthogonal detection technique to show the qualitative binding of the HaloTag protein to the electrode. Together, these results provide insight into the antifouling and detection methods of engulfing the redox molecules in the SAM diluent.