title: Prostate-specific antigen immunosensing based on mixed self-assembled monolayers, camel antibodies and colloidal gold enhanced sandwich assays.
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 k(on) value of 1.9x10(6) M-1 s-1, and was covalently immobilised on a gold substrate via a mixed self-assembled monolayer (SAM) of alkanethiols by using carbodiimide-coupling chemistry in 10mM 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 28 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 10 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 realizes the PSA detection at clinical relevant concentrations.
authors.affiliation: IMEC, MCP-BIO, Kapeldreef 75, B-3001 Leuven, Belgium.
authors.forename: Lieven
authors.initials: L
authors.lastname: Huang
authors.forename: Gunter
authors.initials: G
authors.lastname: Reekmans
authors.forename: Dirk
authors.initials: D
authors.lastname: Saerens
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Friedt
authors.forename: Filip
authors.initials: F
authors.lastname: Frederix
authors.forename: Laurent
authors.initials: L
authors.lastname: Francis
authors.forename: Serge
authors.initials: S
authors.lastname: Muyldermans
authors.forename: Andrew
authors.initials: A
authors.lastname: Campitelli
authors.forename: Chris
authors.initials: C
authors.lastname: Van Hoof
mesh_terms: D000327:Adsorption; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D001774:Blood Chemical Analysis / Q000295:instrumentation / Q000379:methods; D002162:Camelus / Q000276:immunology*; D020099:Coated Materials, Biocompatible / Q000737:chemistry; D003460:Crystallization / Q000379:methods; D017979:Gold Colloid / Q000737:chemistry; D006801:Humans; D007118:Immunoassay / Q000295:instrumentation / Q000379:methods*; D017430:Prostate-Specific Antigen / Q000097:blood* / Q000737:chemistry / Q000276:immunology*; D011485:Protein Binding; D020349:Surface Plasmon Resonance / Q000295:instrumentation / Q000379:methods*
chemical_list: D000906:Antibodies; D020099:Coated Materials, Biocompatible; D017979:Gold Colloid; D017430:Prostate-Specific Antigen