Aptamer-based bio-barcode assay for the detection of cytochrome-c released from apoptotic cells.
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.
Lau
Irene P M
IP
Department of Biochemistry, The Chinese University of Hong Kong, Shatin, NT, Hong Kong.
Ngan
Erika K S
EK
Loo
Jacky
J
Suen
Y K
YK
Ho
H P
HP
Kong
S K
SK
D017209:Apoptosis; D052157:Aptamers, Nucleotide / Q000737:chemistry*; D001483:Base Sequence; D045304:Cytochromes c / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D007118:Immunoassay
D052157:Aptamers, Nucleotide; D045304:Cytochromes c