Abstract
Waterborne bacterial pathogens continue to be a serious threat to human health globally. Rapid and sensitive detection of these pathogens is of paramount importance for the implementation of effective measures to combat the spread of their infections in a timeous manner. With conventional methods being limited in terms of operation time, sensitivity and the need for concentrated, large volume samples among other challenges, the use of nanomaterials especially gold nanoparticles (AuNPs) has been shown to improve techniques for pathogen detection. Gold nanoparticles display crucial characteristics including but not limited to fluorescence enhancing, large surface-area-to-volume ratio and quenching effects. Furthermore, AuNPs can easily be functionalized with antibodies to detect pathogens of interest with great specificity. In this study, a sandwich immunoassay using biologically synthesized AuNPs functionalized with polyclonal specific antibodies against Escherichia coli was developed for E. coli detection in water. Furthermore, the differentiation of E. coli 0157:H7 from other strains of E. coli was performed using fluorescence microscopy. Cell extract of Enterobacter xiangfangensis Pb204 was used to synthesize AuNPs from 1 mM gold (III) chloride trihydrate (HAuCl4.3H2O) as a precursor ion at 37 oC. The resulting AuNPs were characterized by ultraviolet-visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM) in conjunction with energy dispersive X-ray analysis (EDX), and dynamic light scattering (DLS). Streptavidin (1 mg/ml, SA) was used to coat the as-synthesized AuNPs (AuNP-SA) followed by conjugation with biotinylated polyclonal antibodies (specific for E. coli, Ab) in a 1:100 dilution to produce functionalized AuNPs (AuNP-SA-Ab). Functionalization of the AuNPs at every step of binding was monitored by UV-Vis spectra, TEM and DLS...
M.Sc. (Biotechnology)