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

Abstract

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.

Figure 1.

Ferrocene SAM molecules and the PEG₂ diluent used in the study were adsorbed on a gold electrode from their homodisulfide counterparts. The inset labels correspond to the type of thiol-redox reporter connector. C_n is used to designate the number of carbons (n = 6, 11, 16) in an alkane bridge or Con for the conjugated OPE bridge. The HaloTag ligand is Halo, while tBu corresponds to the tert-butyl protecting group connected to the molecules by the attachment point icon. The Con group has a Boc instead of a tBu protecting group, and the alkane linkers do not have the succinic acid (CO(CH₂)CO); see supplemental for more detailed structures. All SAM molecules were oxidized to homodisulfides for chemical stability and ease of handling.

Figure 2.

CVs of the C_ntBu (Top) and C_nHalo (Bottom) when free (Blue) and with HaloTag protein (Red) (ΔE_1/2 ± 10 mV, n ≥ 3). The further out the Fc is from the SAMs layer the more nonspecific adsorption is detected whereas the more engulfed it is there is less nonspecific adsorption detected and more specific adsorption detection. The rigidity of Con-tBu/Halo did not make a substantial difference as it produced almost the same shifts as C₁₆-tBu/Halo (Figure S3 and Table S1).

Figure 3.

SAMDI-MS spectra of SAMs formed from 10 mol % of C₁₁Halo treated with or without HaloTag. (left) Mass spectra obtained in linear high-mass positive mode for measurement of immobilized HaloTag and (right) mass spectra obtained in reflector positive mode for measurement of the diluent and C₁₁Halo. (top) The red spectra were obtained from monolayers after applying a solution of HaloTag for 6 h. (bottom) The black spectra were obtained from monolayers not treated with HaloTag.