Stable and Controllable Magnetic Functionalization of Polymer Microspheres via Covalent Layer-by-Layer Assembly

Magnetic polymer microspheres have attracted significant attention due to their wide applications in bioanalysis. However, achieving stable and controllable magnetic functionalization remains a critical challenge. Here, covalent layer-by-layer (LBL) self-assembly technique was first applied to the magnetic functionalization of polymer microspheres, preparing P(GMA-co-tBMA)@(Fe₃O₄@APTES/Fe₃O₄@GPTMS)_n (PG@(FeA/FeG)_n) microspheres with controllable magnetic content, suppressed detachment of magnetic particles, and surface functionalization. The alternating loading of Fe₃O₄@APTES nanoparticles and Fe₃O₄@GPTMS nanoparticles onto the porous P(GMA-co-tBMA) microsphere templates was achieved through covalent bonds formed between amino groups and epoxy groups. The results indicated that the loading capacity of magnetic particles in a single-layer assembly on the porous microspheres reached its upper limit when the mass ratio ω (M_Fe3O4@APTES/M_{P(GMA-co-tBMA)}) was 0.05:1, at which point the saturation magnetization value of PG@(FeA/FeG)₁ microspheres was 3.00 emu/g. PG@(FeA/FeG)_n microspheres were prepared under this condition, and their saturation magnetization value were proportionally with the count of layers, reaching 13.90 emu/g at n = 7. Compared to electrostatic self-assembly, the covalent self-assembly strategy reduced magnetic leakage by approximately 83.4%. PGMA was coated onto magnetic PG@(FeA/FeG)₃ microspheres utilizing the amino groups on their surface, and subsequent surface carboxylation was developed. Finally, the carboxylated magnetic microspheres were employed as carriers for chemiluminescence immunoassay to detect creatine kinase-MB (CKMB), a biomarker for acute myocardial infarction (AMI), and exhibited higher chemiluminescence intensity compared to commercially available magnetic beads from JSR Corporation. This method provides a novel approach for achieving stable, controllable, and facile loading of other metals and their compounds, organic compounds, and biomolecules onto polymer substrate.