Fabrication of Polyamidoamine Dendrimer-Grafted Magnetic Beads for Enhanced Protein Immobilization and Signal-to-Noise Ratio in Chemiluminescent Immunoassays

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Abstract
This study aims to enhance the signal-to-noise ratio (SNR) in chemiluminescence immunoassays (CLIA) by increasing the protein immobilization capacity on magnetic bead surfaces. Proteins are macromolecules with three-dimensional conformations where merely increasing the density of surface functional groups on magnetic beads is insufficient to substantially enhance protein immobilization. To address this limitation, we grafted polyamidoamine (PAMAM) dendrimers with three-dimensional architectures onto magnetic beads, simultaneously augmenting the functional group density and expanding the spatial distribution to improve protein loading capacity. PAMAMs were synthesized via iterative Michael addition and amidation cycles between ethylenediamine and methyl acrylate, with its grafting density and behavior on magnetic beads characterized by nuclear magnetic resonance (NMR) spectroscopy. Dynamic light scattering (DLS) analysis revealed an increased hydrodynamic radius of the PAMAM-grafted beads, confirming the successful expansion of protein-binding spatial domains. We investigated the impact of PAMAM grafting on the immobilization efficiency of bovine serum albumin (BSA) and mouse immunoglobulin G (IgG). Experimental results demonstrated that PAMAM grafting enhanced the protein immobilization capacity from 11.5 to 33.5 mg/g for BSA and from 8.3 to 19.2 mg/g for IgG, representing 2.9-fold and 2.3-fold improvements, respectively. The SNR improvement was evaluated by using a CLIA-based detection system for the creatine kinase-MB (CK-MB) biomarker. PAMAM grafting achieved 7.21-fold and 4.79-fold improvements in low-to-background and high-to-background ratios and reduced the limits of detection (LOD) from 52.58 to 7.73 pg/mL, respectively, and the optimized LOD fully meets the clinical sensitivity requirements for detecting CK-MB. This work demonstrates that grafting three-dimensional PAMAM dendrimer effectively enhances protein immobilization on magnetic beads, significantly improving CLIA SNR, thereby providing a novel strategy for optimizing immunoassay performance.

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