Effects of Composition of Oligo(ethylene glycol)-Based Mixed Monolayers on Peptide Grafting and Human Immunoglobulin Detection

Alkanethiols carrying ethylene glycol units (EG_n, n = 3 or 6) with amine termini (EG₃NH₂ or EG₆NH₂) were coadsorbed with a “diluent”, hydroxyl-terminated alkanethiol (EG₃OH), to form mixed self-assembled monolayers (SAMs). The mixed SAMs were characterized, and hexameric peptide ligand His-Trp-Arg-Gly-Trp-Val (HWRGWV), which shows affinity binding toward the Fc (constant fragment) of human immunoglobulin (IgG), was grafted onto different dilutions of EG₆NH₂–EG₃OH mixed SAMs for preparation of IgG detection surfaces. The specificity toward IgG was optimal for peptides grafted on SAMs prepared from 10% EG₆NH₂ precursor solution, even though this surface did not have the highest number of peptides per unit area. Surface plasmon resonance (SPR) experiments showed that IgG bound to the peptides on the mixed SAM with a dissociation constant K_d of 9.33 × 10^–7, maximum binding capacity Q_m of 3.177 mg m^–2, and adsorption rate constant k_a of 1.99 m³ mol^–1 s^–1. IgG binding from complex mixtures of Chinese Hamster Ovary supernatant (CHO) was investigated on peptides grafted to mixed and pure SAMs. Regeneration of the surfaces was achieved by treatment with 10% acetonitrile in 0.1 M NaOH solution. Overall, the use of peptides grafted on mixed SAMs improved the effectiveness of detection and had an impact on specificity and regeneration of biosensors.