Fabrication of sensitive immunosensors: the selectione of antibody immobilization strategies and conjugation carriers

Abstract

Immunosensors are typical biosensors using the antibody as the biorecognition element that is immobilized on a suitable transducer to qualitatively determine the antigen-antibody interaction. An ideal immunosensor with combined advantages including the high sensitivity, easy-tooperate and cost-effective, would have attributed to the rational selection of appreciating bioreceptors carriers and site-oriented antibody immobilization strategy. Achieving the sitedirected immobilization only involves the fragment crystallizable (Fc) region would maximize the biofunctionality of tethered antibodies, while it would be a great challenge faced with the heterogeneous distribution of amino residues through the antibody surface. Meanwhile, the challenge still lies with the selection of bioreceptors carriers bearing desirable surface chemistries (antifouling, robustness and stability) for the irreversible conjugation of antibodies. In this dissertation, the objective is to evaluate the analytical performance of fabricated immunosensors, in which antibody immobilization strategies and bioreceptors carrier types are primary considerations mainly in terms of the acceptable sensitivity, storage stability of fabricated immunosensors toward the low abundance of analytes and avoiding the undesirable interference dealing with clinical diagnosis. Antibodies conjugated on the glyoxyl-agarose support results in different orientations by simply manipulating the pH of incubation conditions while the inevitable involvement of antigen-binding fragment (Fab) during the antibody/glyoxyl-agarose interaction caused the deterioration of its biofunctionality. Covalently fixing the uniform and site-directed orientation of coupled antibodies following the pre-affinity adsorption demonstrated the obvious advantage of controlling the interaction regions, over 75 % of preserved biofunctionality was observed with tethered antibodies. Magnetic nanomaterials with adequate dextran coverage have demonstrated the equilibrium between facile magnetic recovery and sufficient colloidal stability, and magnetic nanoparticles bearing the polyaldehyde groups of 139 μmol g-1 nanoparticles were proven a better nanocarrier than carboxylic groups and chelate groups functionalized nanoparticles. In addition, the multifunctionality of polyaldehyde-dextran@nanoparticles was exploited as the nanoprobes and nanocarriers. Over 4-fold higher sensitivity of immunosensors was enhanced when horseradish peroxidase-streptavidin/antibody dual functionalized nanoparticles work as the nanoprobes, and the reliability of nanoparticles as the nanocarriers to detect biomarker concentration in clinical serums was confirmed by its consistency with golden standard-ELISA (enzyme-linked immunosorbent assay) analysis