Synthesis of grafted phosphorylcholine polymer layers as specific recognition ligands for C-reactive protein focused on grafting density and thickness to achieve highly sensitive detection

Abstract

We studied the effects of layer thickness and grafting density of poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) thin layers as specific ligands for the highly sensitive binding of C-reactive protein (CRP). PMPC layer thickness was controlled by surface-initiated activators generated by electron transfer for atom transfer radical polymerization (AGET ATRP). PMPC grafting density was controlled by utilizing mixed self-assembled monolayers with different incorporation ratios of the bis[2-(2-bromoisobutyryloxy)undecyl] disulfide ATRP initiator, as modulated by altering the feed molar ratio with (11-mercaptoundecyl)tetra(ethylene glycol). X-ray photoelectron spectroscopy and ellipsometry measurements were used to characterize the modified surfaces. PMPC grafting densities were estimated from polymer thickness and the molecular weight obtained from sacrificial initiator during surface-initiated AGET ATRP. The effects of thickness and grafting density of the obtained PMPC layers on CRP binding performance were investigated using surface plasmon resonance employing a 10 mM Tris-HCl running buffer containing 140 mM NaCl and 2 mM CaCl₂ (pH 7.4). Furthermore, the non-specific binding properties of the obtained layers were investigated using human serum albumin (HSA) as a reference protein. The PMPC layer which has 4.6 nm of thickness and 1.27 chains per nm² of grafting density showed highly sensitive CRP detection (limit of detection: 4.4 ng mL⁻¹) with low non-specific HSA adsorption, which was improved 10 times than our previous report of 50 ng mL⁻¹.