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A rapid and sensitive immunoassay for detection of E. coli O157:H7 using multienzyme — Au nanoparticle complex

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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.

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Authors and Affiliations

  1. Department of Chemical & Biomolecular Engineering, Sogang University, #1 Shinsu-dong, Mapo-gu, Seoul, 121-742, Korea

    Hyun-Soo Kim & Byung-Keun Oh

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  1. Hyun-Soo Kim
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  2. Byung-Keun Oh
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Correspondence to Byung-Keun Oh.

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Kim, HS., Oh, BK. A rapid and sensitive immunoassay for detection of E. coli O157:H7 using multienzyme — Au nanoparticle complex. BioChip J 8, 1–7 (2014). https://doi.org/10.1007/s13206-014-8101-7

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  • DOI: https://doi.org/10.1007/s13206-014-8101-7

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