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 nm(2) 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).

A plasmonic chip-based bio/chemical hybrid sensing system for the highly sensitive detection of C-reactive protein

“…S8, ESI †). The obtained LOD was much lower than those of previously reported PMPC-based SPR, LSPR and RIfS sensing systems 10,[12][13][14][15][16][17][18] (Table S2, ESI †).…”

“…15,16 We recently demonstrated the highly sensitive detection of CRP based on non-immunological interactions. 17,18 The phosphorylcholine group is a specific ligand for CRP in the presence of Ca 2+ ions [19][20][21] and thus our methodology utilizes protein-ligand interactions between a polymeric ligand, poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC), and CRP. 17,18,22 PMPC has been reported to exhibit a low non-specific protein binding [23][24][25] which can be decreased further.…”

“…17,18 The phosphorylcholine group is a specific ligand for CRP in the presence of Ca 2+ ions [19][20][21] and thus our methodology utilizes protein-ligand interactions between a polymeric ligand, poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC), and CRP. 17,18,22 PMPC has been reported to exhibit a low non-specific protein binding [23][24][25] which can be decreased further. The selective binding capability of PMPC toward CRP compared with other proteins has been investigated.…”

“…The selective binding capability of PMPC toward CRP compared with other proteins has been investigated. 17,18,22 A highly sensitive detection of CRP (440 pM and 39 pM) was achieved using localized surface plasmon resonance (LSPR) based sensing using PMPC-grafted-gold nanoparticles 17 and a PMPC-grafted-SPR sensor chip, 18 respectively. Plasmonic chips are prepared by coating thin metal films on a periodic structure and have been developed as a powerful sensing tool for grating-coupled surface plasmon field-enhanced fluorescence (GC-SPF).…”

Post‐Imprinting‐Modified Molecularly Imprinted Nanocavities with Two Synergetic, Orthogonal, Glycoprotein‐Binding Sites to Transduce Binding Events into Fluorescence Changes

“…Dithioethyl methacrylate groups were covalently conjugated on AFP by coupling 2-(2-pyridyl)dithioethyl methacrylate with the thiolated AFP prepared by 2-immunothiolane treatment. [18] This yielded methacryloyl-SS-AFP as a designed template molecule for subsequent molecular imprinting with a functional pyrrolidyl methacrylate (PyM) monomer capable of electrostatically interacting with AFP. According to matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/ MS), the estimated number of dithioethyl methacrylate groups introduced onto AFP was approximately 4.4 (Supplementary Figure S1), and circular dichroism spectra suggested a secondary structure similar to that of native AFP (Supplementary Figure S2).…”