HaloTag-Modified,\nFerrocene Labeled Self-Assembled\nMonolayers for Protein Sensing

Ferrocene (Fc)-based disulfide molecules of various lengths\nwith\namino acid scaffolds and alkane or oligo­(phenylene-ethynylene) (OPE)\nbridges are used in a mixed SAM with a di-(ethylene oxide) terminal\nmercaptoundecanol diluent (PEG<sub>2</sub>). The relative height of\nthe Fc redox reporter in the SAM is compared to determine if there\nare protective effects like antifouling and specific detection. The\nHaloTag-binding motif is used as a proof-of-concept to investigate\nthe electrochemical response to the HaloTag protein due to its known\ncovalent and fast linkage. When the Fc-SAMs are exposed to the HaloTag\nprotein, there are an antifouling nature and more specific detection\nfor the engulfed Fc-based molecules (<b>C</b><sub><b>6</b></sub><b>tBu/Halo</b>). The further out the Fc is from the\nSAM layer, the more nonspecific adsorption is detected. The double\nlayer capacitance (CD<sub>L</sub>) has the smallest change for the <b>C</b><sub><b>6</b></sub><b>tBu</b> control (ΔCD<sub>L</sub> = −0.1 μF cm<sup>–2</sup>) showing antifouling\nproperties and produces a large change (ΔCD<sub>L</sub> = 0.9\nμF cm<sup>–2</sup>) as well as a shift in oxidation potential\nwhen the active <b>C</b><sub><b>6</b></sub><b>Halo</b> is exposed to the HaloTag protein (Δ<i>E</i><sub>1/2</sub> = 50 ± 10 mV). The remaining Fc molecules are partially\nin or outside the PEG<sub>2</sub> layer, allowing more ion penetration/mobility\neven when the HaloTag protein is bound. Generally, a more disordered\nenvironment was observed for the Fc-based molecules when adding the\nHaloTag ligand, which is evident from a larger <i>E</i><sub>fwhm</sub> and higher CD<sub>L</sub>. Desorption of the SAMs with\nsodium iodide (NaI) showed retention of the HaloTag protein bound\nwith the corresponding ligand, whereas negative controls did not.\nSelf-assembled monolayers for MALDI mass spectrometry (SAMDI-MS) were\nused as an orthogonal detection technique to show the qualitative\nbinding of the HaloTag protein to the electrode. Together, these results\nprovide insight into the antifouling and detection methods of engulfing\nthe redox molecules in the SAM diluent.