Development of a novel hand-held immunoassay for the detection of enterohemorrhagic Escherichia coli O157:H7

Escherichia coli O157: H7 is an important human pathogen responsible for numerous outbreaks of hemorrhagic colitis and hemolytic uremic syndrome world-wide. A portable detection device is needed for field testing and point-of-care testing. Poly(dimethylsiloxane) (PDMS) is a solid substrate well suited for adsorption of macromolecules. The purpose of this study was to develop a hand-held enzyme-linked immunosorbent assay (ELISA) for the detection of E. coli O157: H7 antigens. The prototype consisted of three modules: one for loading reagents, a second for immunosensor detection, and a third for discharge of wastes. Reagent delivery was achieved by using 1-ml syringes embedded within the loading module. The detection module was based on a PDMS layer on which varying concentrations of E. coli O157: H7 antigens, and negative controls (Lactobacillus rhamnosus R011 and phosphate-buffered saline) were passively adsorbed. Commercially available goat anti-E. coli O157: H7 antibody was used as a primary antibody, a donkey anti-goat IgG conjugated with horseradish peroxidase was used as a secondary antibody, and a precipitating substrate was employed for colorimetric detection. Results obtained with the prototype were compared to those obtained using a conventional nitrocellulose membrane-based immunoassay. Using the prototype, E. coli O157: H7 antigens, in quantities from 4 to 400 ng, were accurately detected. This detection limit was comparable to that observed using conventional dot-blot assays. The PDMS layer could be re-used without loss of sensitivity. Colorimetric detection could be visualized readily without the need for sophisticated equipment. Furthermore, this device can be adapted to perform diagnostics for other microbial pathogens currently detected using immunoassay methodology.