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

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 faun 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. 10(2) CFU/ mL of E. coli O157:H7 within an hour.