Abstract:
This research aims to develop a simple, yet effective assay for C-reactive protein (CRP) detection based on a combination of magnetic separation and antibody-free colorimetric assay. Magnetic nanoparticles stabilized with phosphorylcholine-containing polymer, poly[methacrylic acid)-ran-(methacryloyloxyethyl phosphorylcholine)] (PMAMPC-MNPs) were prepared by co-precipitation 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 presence of Ca2+. The PMAMPC-MNPs were characterized by ATR-FTIR, TEM, DLS, TGA and XRD. To determine the CRP binding, the PMAMPC-MNPs were mixed with CRP in presence of Ca2+ and then precipitation of PMAMPC-MNPs conjugated with CRP was induced by magnet. Taking advantage of peroxidase-like activity of MNPs, an addition of 3,3′,5,5′-tetramethylbenzidine (TMB) and H2O2 in 0.1 M acetate buffer, pH 3.8 to the supernatant allows colorimetric determination for a short period of time of unbound PMAMPC-MNPs which is inversely proportional to the amount of CRP and detects in antibody-free system. A linear range of 0 – 5 µg/ml and detection limit of 1.39 µg/mL. Moreover, 3 µg/ml CRP in undiluted rabbit serum can be detected as well.
