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Info. Vol.8 - No.1 (2014.03.20)
Title A rapid and sensitive immunoassay for detection of E. coli O157:H7 using multienzyme - Au nanoparticle complex
Authors Hyun-Soo Kim1 & Byung-Keun Oh1
Institutions 1Department of Chemical & Biomolecular Engineering, Sogang University, #1 Shinsu-dong, Mapo-gu, Seoul 121-742, Korea
Abstract In this study, a novel design of gold nanoparticle (AuNP) probe containing large number of enzymes is reported for ultrasensitive detection of Escherichia coli (E. coli) O157:H7. The AuNP was functionalized with a polyclonal antibody (Pab) that has an affinity for E. coli O157:H7. The digoxigenin (DIG) incorporated oligonucleotides were covalently attached on the AuNP. And then Horseradish peroxidase (HRP) labeled anti-DIG was bound to the DIG incorporated oligonucleotide on the AuNP. Afterwards, magnetic microparticles (MMPs) were functionalized with monoclonal antibody (Mab) to E. coli O157:H7 and used to form a sandwich structure with target E. coli O157:H7 and AuNP probes via immune reaction. A magnetic field was used to effectively remove unbound AuNP probes. Finally, we exploited the reaction of HRP enzyme immobilized on AuNP probes with tetramethylbenzidine (TMB) that is a substrate of HRP, followed by stopping the reaction with 2 M H2SO4. The resulting end products were analyzed by UV-vis spectroscopy. Using this method, we could detect ca. 102 CFU/ mL of E. coli O157:H7 within an hour.
Keyword E. coli O157:H7, Immunoassay, Gold nanoparticle, Horseradish peroxidase, Enzyme-linked immunosorbent assay
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(사)한국바이오칩학회 (The Korean BioChip Society)
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