Magnetic Nanoparticles Stabilized by Phosphorylcholine-Containing Polymer for Label-Free C-Reactive Protein Detection

This research aims to develop a simple yet effective assay for detecting C-reactive protein (CRP), based on magnetic nanoparticles functionalized with a phosphorylcholine-containing polymer. Magnetic nanoparticles stabilized with poly[(methacrylic acid)-ran-(methacryloyloxyethyl phosphorylcholine)] (PMAMPC-MNPs), were prepared by coprecipitation of ferric and ferrous salts in the presence of PMAMPC. Carboxyl groups in the methacrylic acid (MA) repeat units chelate with Fe atoms during MNPs formation, while the methacryloyloxyethyl phosphorylcholine (MPC) repeat units provide specifically binding sites and conjugate with CRP in the presence of Ca²⁺, leading to the aggregation of PMAMPC-MNPs. The PMAMPC-MNPs were characterized by attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), transmission electron microscopy (TEM), dynamic light scattering (DLS), thermogravimetric analysis (TGA), and X-ray diffraction (XRD). To determine CRP detection with the naked eye, the precipitation of PMAMPC-MNPs in the presence of CRP and Ca²⁺ was monitored without an external magnetic field. Additionally, by taking advantage of the peroxidase-like activity of MNPs, the addition of 3,3',5,5'-tetramethylbenzidine (TMB) and H₂O₂ to the supernatant of unbound PMAMPC-MNPs after magnetic separation allows for the colorimetric determination of CRP. This measurement is inversely proportional to the amount of CRP and is detected in an antibody-free system, with a linear range of 1-5 μg/mL and an experimental limit of detection (LOD) of 1.0 μg/mL. Moreover, 3 μg/mL CRP can also be detected in 50% diluted rabbit serum, covering the CRP cutoff level associated with the risk threshold for cardiovascular disease.