Improving paper-based ELISA performance through covalent immobilization of antibodies

Abstract

Microfluidic paper-based analytical devices have been widely used for immunoassays, especially in resource-limited settings. Antibodies that have been immobilized on the cellulose surface by physical adsorption become unstable as a result of antibodies’ desorption caused by the washing process and lose their activity. In this paper, we present a new approach for the covalent immobilization of antibodies on the paper surface. More precisely, sodium periodate (NaIO4) was used to activate the cellulose for introducing aldehyde groups. Antibodies were bound to the oxidized cellulose paper surface through imine formation (Schiff base) between the aldehyde groups and the primary amine groups of the antibody. Thus, using α-fetoprotein (AFP) as a model, a new paper-based sandwich ELISA was developed. The linear range of the assay for AFP was from 0.1ng/mL to 11.2ng/mL, the detection limit was 0.04ng/mL, and the recovery rate of AFP spiked into human serum sample ranged from 89.2% to 101.5%. The intra- and inter-assay coefficients of variations obtained were 4.6–7.1% and 5.8–10.3% for standard AFP, respectively. Compared with the conventional paper-based ELISA, the developed assay exhibited superiority in terms of sensitivity, lower detection limit, stability and reliability. This simple and sensitive paper-based sandwich ELISA holds promise in clinical applications.