Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10429597 | Biosensors and Bioelectronics | 2005 | 8 Pages |
Abstract
Prostate-specific antigen (PSA) is a valuable biomarker for prostate cancer screening. We developed a PSA immunoassay on a commercially available surface plasmon resonance biosensor. Our PSA receptor molecule consists of a single domain antigen-binding fragment, cAbPSA-N7, derived from dromedary heavy-chain antibodies and identified after phage display. It binds PSA with a high kon value of 1.9Â ÃÂ 106Â Mâ1Â sâ1, and was covalently immobilised on a gold substrate via a mixed self-assembled monolayer (SAM) of alkanethiols by using carbodiimide-coupling chemistry in 10Â mM acetate buffer pH 5.5 to obtain an optimal pre-concentration. The best performing and optimised mixed SAM consisted of (10%) 16-mercapto-1-hexadecanoic acid (16-MHA) for covalent cAbPSA-N7 immobilisation and (90%) 11-mercapto-1-undecanol (11-MUOH) to minimise non-specific adsorption of the analyte. In this way, two advantages are incorporated in a single coupling layer. Up to 28Â fmol/mm2 of cAbPSA-N7 could be immobilised and 30% of its binding sites participate actively in PSA interaction. In addition, the optimised layer showed also optimal performance to assess physiological samples. Although PSA concentrations as low as 10Â ng/ml could be detected directly, this detection limit could be enhanced to PSA levels in the sub ng/ml range by introducing a sandwich assay involving a biotinylated secondary antibody and streptavidin modified gold nanoparticles. This approach realises the PSA detection at clinical relevant concentrations.
Related Topics
Physical Sciences and Engineering
Chemistry
Analytical Chemistry
Authors
Lieven Huang, Gunter Reekmans, Dirk Saerens, Jean-Michel Friedt, Filip Frederix, Laurent Francis, Serge Muyldermans, Andrew Campitelli, Chris Van Hoof,