Label-free dynamic light scattering assay for C-reactive protein detection using magnetic nanoparticles

In this work, we present a simple method for label-free detection of C-reactive protein (CRP) in diluted saliva samples without the use of specific molecules against CRP. We use the dynamic light scattering (DLS) technique and silica-coated Fe3O4 nanoparticles (similar to 50 nm in diameter) functionalized with amino carboxylate moieties (Fe3O4@SiO2/COOH) as probes. After contact with the sample, the particles could be easily separated with a handy magnet and redispersed for DLS analysis simply by vortex shaking. The variation of the hydrodynamic diameter of the nanoparticles (Z-average size) could be correlated with the concentration of CRP up to concentrations of 10 mg L-1. The detection limit (LOD) in diluted saliva samples that were spiked with CRP was 0.205 mg L-1, which is below salivary levels of CRP detected in unhealthy individuals. The coefficient of variation was found to be less than 1.5% in the entire detection range. The variation of Z-average size of nonfunctionalized silica coated nanoparticles (Fe3O4@SiO2) also correlated well with CRP concentration. Nevertheless, the Fe3O4@SiO2/COOH nanoparticles were less susceptible to interference from other biomolecules present in saliva and adsorbed more CRP, indicating higher selectivity toward CRP than nonfunctionalized nanoparticles. This higher affinity was attributed to the chelating interaction between the aminocarboxylate groups of the organosilane N-[3-(trimethoxysilyl)propyl]ethylenediaminetriacetic acid trisodium salt (EDTA-TMS) grafted onto the surface of the Fe3O4@SiO2/COOH nanoparticles and the Ca2+ ions of CRP. LC-MS/MS analyses allowed identification of the proteins adsorbed on the nanoparticles and confirmation of the presence of CRP, which is involved in several biological processes, including immune response, response to stress and transport.