The surface characteristics of magnetic microspheres critically determine their performance in bioseparation

applications including protein purification, enzyme immobilization, nucleic acid extraction, and immunoassays.

To optimize microsphere performance, ATRP-synthesized polymer brushes can be grafted onto their surfaces to

introduce high-density functional groups (e.g., amino, carboxyl, and tosyl groups). This strategy significantly

enhances functional group density by constructing interfaces with extended polymer brushes. This study employs

polymer chains grafting strategy to substantially increase functional group density through long-chain polymer

interfaces on magnetic microspheres, thereby optimizing surface properties and expanding application potential.

Initially, carboxylated polymer chains with controlled molecular weights (5.2–51.2 kDa) are synthesized via

atom transfer radical polymerization (ATRP), followed by strain-promoted azide-alkyne cycloaddition (SPAAC)

for surface grafting. To further suppress non-specific adsorption associated with protein conjugation, we also

design and synthesis binary copolymer brushes, P(GMA-NH-COOH-co-GAMA), incorporating gradient glucose-

based monomers (GAMA). Experimental results demonstrate that ZY-P(G)-1 magnetic microspheres modified

with 10.2 kDa PGMA-NH-COOH polymer brushes exhibit a surface carboxyl group density of 1.384 mmol g

1

.

The bovine serum albumin (BSA) coupling capacity increases to 44.21 mg g

1

, with the chemiluminescence high-

value signal-to-background (S/B) ratio showing a 7.97-fold enhancement compared to conventional carboxyl

microspheres (ZY-COOH). ZY-P(GG)-100:2 microspheres modified with amphiphilic polymers containing 2 mol

% GAMA maintain superior detection sensitivity with 1.74-fold higher S/B ratio than ZY-P(G)-1 while signifi

cantly reducing nonspecific protein adsorption. This study establishes a synergistic engineering approach for

precise surface modification through coordinated optimization of spacer dimensions and functional monomer

ratios. The developed platform significantly enhances the capture efficiency of low-abundance biomarkers,

providing a novel material basis for high-precision in vitro diagnostic reagent development.