title: Aptamer-based bio-barcode assay for the detection of cytochrome-c released from apoptotic cells.
abstract: The recently developed bio-barcode (BBC) assay using polymerase chain reaction (PCR) to generate signals has been shown to be an extraordinarily sensitive method to detect protein targets. The BBC assay involves a magnetic microparticle (with antibody to capture the target of interest) and gold nanoparticle (with recognition antibody and thiolated single-stranded barcode DNAs) to form a sandwich around the target. The concentration of target is determined by the amount of barcode DNA released from the nanoparticles. Here we describe a modification using aptamers to substitute the gold nanoparticles for the BBC assay. In this study, we isolated a 76-mer monoclonal aptamer against cytochrome-c (cyto-c) and this single-stranded DNA in defined 3D structure for cyto-c was used in the BBC assay for both recognition and readout reporting. After magnetic separation, the aptamer was amplified by PCR and this aptamer-based barcode (ABC) assay was sensitive enough to detect the cyto-c in culture medium released from the apoptotic cells after drug treatment at the picomolar level. When compared to the conventional cyto-c detection by Western blot analysis, our ABC assay is sensitive, and time for the detection and quantification with ready-made probes was only 3 h.
authors.affiliation: Department of Biochemistry, The Chinese University of Hong Kong, Shatin, NT, Hong Kong.
authors.forename: Irene P M
authors.initials: IP
authors.lastname: Lau
authors.forename: Erika K S
authors.initials: EK
authors.lastname: Ngan
authors.forename: Jacky
authors.initials: J
authors.lastname: Loo
authors.forename: Y K
authors.initials: YK
authors.lastname: Suen
authors.forename: H P
authors.initials: HP
authors.lastname: Ho
authors.forename: S K
authors.initials: SK
authors.lastname: Kong
mesh_terms: D017209:Apoptosis; D052157:Aptamers, Nucleotide / Q000737:chemistry*; D001483:Base Sequence; D045304:Cytochromes c / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D007118:Immunoassay
chemical_list: D052157:Aptamers, Nucleotide; D045304:Cytochromes c