Chitosan hydrogel membrane-integrated ELISA platform for high-sensitive detection of alpha-fetoprotein in human serum

Enzyme-linked immunosorbent assay (ELISA) is widely employed. Enhancing the surface of ELISA plate wells to increase the number of immobilized antibody molecules per unit area represents a viable strategy for improving the assay's detection sensitivity. Chitosan is particularly suitable for biomolecule attachment due to its porous structure and high density of amine groups. We aimed to enhance ELISA performance by applying a chitosan hydrogel layer onto polystyrene (PS) surfaces. The primary challenge was the instability of the hydrogel membrane formed on the PS surface, which was susceptible to detachment and cracking. To address this, a standardized three-step protocol was developed for the immobilization of chitosan hydrogel onto PS wells, comprising O 2 plasma pretreatment, vacuum drying for 3 h to enhance membrane adhesion, and glutaraldehyde-based chemical activation. The modified PS microplates exhibited stability for at least 1 year. The chitosan hydrogel membrane-based ELISA for alpha-fetoprotein (AFP) detection (AFP-CHM-ELISA) demonstrated excellent linearity (R 2  = 0.9992), with a limit of detection (LOD) of 1.98 ng/mL—comparable to the 1.00 ng/mL LOD of chemiluminescence—achieving a sensitivity approximately 2.5 times higher than that of conventional ELISA (5 ng/mL), and an extended upper linear range of 280 ng/mL. Intra- and inter-batch coefficients of variation were below 6 %, with spiked serum recovery rates ranging from 99.67 % to 103.48 %. Comparative analysis revealed a 95.54 % agreement with commercial chemiluminescence methods, along with a strong correlation (R 2  = 0.9236). This chitosan hydrogel-based surface modification approach significantly improves ELISA performance and demonstrates promising potential for diagnostic applications.