An immunomagnetic separation based fluorescence immunoassay for rapid myoglobin quantification in human blood

Abstract

Magnetic nanoparticle (NPs) and fluorescent NP pairs were designed for myoglobin (Myo) detection in human blood. Magnetic NPs MNP@SiO₂@BSA@Au with about 200 nm diameter (by TEM) were prepared through a layer by layer technique following by adsorption of anti-Myo antibody 4E2 on a Au surface. The MNPs were well dispersed in an aqueous solution. Fluorescent NPs were prepared by conjugating fluorescent Rhodamine Red-X (RhX) and another Myo antibody 7C3 on silica NPs with a final diameter of 80 nm. In the presence of Myo, magnetic NPs bearing 4E2 and fluorescent NPs bearing 7C3 form a sandwich structure, which can be separated from blood under a magnetic field. The concentration of Myo is quantified by the fluorescence intensity of SNP@RhX NPs in the sandwich structure. The calibration curve of the Myo showed a linear range between 0 and 250 ng mL⁻¹ (R² = 0.993). The minimum detectable concentration was 0.28 ng mL⁻¹. The immunomagnetic separation based fluorescence immunoassay offered a simple and accurate way to determine Myo in human blood in 30 min.