Vinyl sulfonamide/PEG co-immobilized on the surface of magnetic beads for the chemiluminescent immunoassay

Chemiluminescent immunoassays utilizing magnetic nanoparticles exhibit critical importance in in vitro diagnostics, attributed to their remarkable superparamagnetic properties, high specificity, and uniform monodispersity. Current surface functionalization strategies for antibody conjugation predominantly rely on carboxyl or amino groups, requiring pre-activation steps for both amino- and carboxyl-modified nanoparticles prior to antibody labeling. However, the complex activation protocols and persistent challenges in developing efficient chemiluminescent detection methods highlight significant technical obstacles. To address these limitations, we developed vinyl sulfone-functionalized magnetic beads (VSF-GMP), enabling rapid antibody conjugation via specific reactions between vinyl sulfone groups and lysine residues exposed on the surface of creatine kinase MB isoenzyme antibodies (CK-MB). This approach facilitates highly efficient chemiluminescent immunoassays. The engineered VSF-GMP magnetic beads demonstrated molecular weight-dependent adsorption profiles, achieving maximal binding capacity (86.32 μg/mg) for mouse IgG (150 kDa) compared to β-lactoglobulin (18.2 kDa) and bovine serum albumin (66.4 kDa). Moreover, compared with the protein coupling method using EDC/NHS-activated carboxylated magnetic beads, our VSF-GMP beads exhibited superior coupling efficiency. Additionally, bis-functionalized poly(ethylene glycol) (NH 2 -PEG-NH 2 ) was employed as a blocking agent to optimize the reactivity and stability of antibody conjugation, reducing non-specific protein labeling on the magnetic beads by 97.8 %. The analytical performance of the proposed chemiluminescent immunoassay (CLIA) was rigorously evaluated. Finally, the practical utility of VSF-GMP magnetic beads for CK-MB determination in plasma samples was investigated. These engineered magnetic beads showcase exceptional analytical sensitivity, specificity, and operational simplicity, establishing a robust platform for advanced chemiluminescent diagnostics with enhanced throughput capabilities.