An evanescent wave fiber optic biosensor for the rapid detection of pathogenic bacteria using Escherichia coli O157:H7 as a model organism for assay development

An evanescent wave fiber optic biosensor was used in an attempt to develop a system for the rapid detection of pathogenic bacteria. The principle of the biosensor is a sandwich immunoassay. Capture antibodies are immobilized on the surface of specially designed silica or polystyrene fiber-optic probes and fluorescent dye-labeled antibodies are used for generation of the specific signal. Fluorescent molecules within approximately 100–1000 run of the fiber surface are excited by the evanescent field and a portion of their emission energy recouples into the fiber, which is then collected and quantitated.; The human pathogen, Escherichia coli O157:H7 was used as a model organism for assay development. Biosensor assays were developed to detect E. coli O157:H7 in pristine buffer, one, ten, and twenty-five grain ground beef samples, and unpasteurized apple juice/cider. A sample preparation scheme for direct detection of E. coli O157:H7 in ground beef and apple/juice cider samples was developed that optimized biosensor signal response. Additionally, a rapid enrichment protocol for amplification and subsequent biosensor detection of extremely low levels (<10 CFU/g) of E. coli O157:H7 in ground beef was designed. The sensitivity and specificity of the biosensor was evaluated with both silica and polystyrene waveguides in all of the sample matrices tested with spiked samples prepared using the developed sample preparation protocols.; The evanescent wave biosensor was found to be a sensitive and specific system for the detection of Escherichia coli O157:H7. Lower limits of detection ranged from 3–30 CFU/mI with the silica waveguides to 100–1000 CFU/ml/g with the polystyrene waveguides. As few as 4 CFU or 0.08 CFU/g/ml could be detected following a 6 hour enrichment. The biosensor was shown to be unique in its ability to achieve sensitive detection in relatively dirty sample homogenates. False positive results generated by non-E. coli O157:H7 organisms were minimal. Only one organism, Escherichia hermanii, showed significant cross-reactivity with the biosensor assay. Calibration curves showed the biosensor to behave as a typical sandwich immunoassay with a linear signal response when plotted on a logarithmic scale.