Ultrasensitive detection of anti-p53 autoantibodies based on nanomagnetic capture and separation with fluorescent sensing nanobioprobe for signal amplification.

The detection of biomarkers expressed at early onset of carcinogenesis, hold promise for the identification of subjects with a preclinical malignant tumor. However, these biomarkers exist in bodily fluids at an ultra-low concentration. Therefore, ultrasensitive techniques are required for their detection. This work investigated the detection of anti-p53 autoantibodies (anti-p53aAbs) using nanomagnetic beads capture probe and anti-IgG functionalized-fluorescence nanoparticles as the detection probe. Specificity was achieved by the use of human p53 protein (p53Ag) immobilized onto nanomagnetic beads, blocked with BSA (MB-p53Ag/BSA) for capture and separation. Anti-IgG antibody conjugated FITC-doped silica nanoparticles (FITC@SiO2-NH2-anti-IgGNPs) used as the sensing nanobioprobe. The target anti-p53aAbs from human serum samples is selectively isolated and purified using the MB-p53Ag/BSA. A sandwich-type immunoreaction was achieved via the Fc-specific FITC@SiO2-NH2-anti-IgG binding to the captured anti-p53aAbs. The alkali hydrolysis of the FITC@SiO2-NH2-anti-IgG released FITC molecules, leading to an amplified fluorescence detection signal. The analytical performance evaluated using the FITC@SiO2-NH2-anti-IgGNPs as sensing nanobioprobe, MB-p53Ag/BSA as a nanomagnetic bead, and microwell ELISA plate, MTP-p53Ag/BSA were compared. The proposed immunosensor exhibited linear correlation in two concentration ranges from 1.50 to 500 pg mL-1 and from 0.50 to 100 ng mL-1. The limit of detection (LoD) and limit of quantification were calculated from the lower linear concentration range close to zero (1.50-500 pg mL-1) following a method reported in literature. The LoD was found to be 1.49 pg mL-1 and the limit of quantification was 3.81 pg mL-1. For the microwell ELISA plate assay, the LoD was 42.0 pg mL-1 and the linear range was 1.60-100 ng mL-1. The nanomagnetic capture-based assay time was 50 min, which is quicker than 3 h needed for the microwell ELISA plate assay.