Novel O157:H7 E. coli Detector Utilizing a Langasite Surface Acoustic Wave Sensor

摘要

The toxigenic E. coli O157:H7 bacterium has been connected with hemorrhagic colitis and hemolytic uremic syndrome, which may be characterized by diarrhea, kidney failure, and death. On average, O157:H7 causes 73,000 illnesses, 2,100 hospitalizations, and 60 deaths annually in the United States alone. There is the need for sensors capable of rapidly detecting dangerous microbes in food and water supplies to limit the exposure of human and animal populations. Previous work by the authors used shear horizontal surface acoustic wave (SH SAW) devices fabricated on langasite (LGS) Euler angles (0 deg, 22 deg, 90 deg) to successfully detect macromolecular protein assemblies. The devices also demonstrated favorable temperature stability, biocompatibility, and low attenuation in liquid environments, suggesting their applicability to bacterial detection. In this paper, a biosensor test setup utilizing a small volume fluid injection system, stable temperature control, and high frequency phase measurement was applied to validate LGS SH SAW biosensors for bacterial detection. The LGS SH SAW delay lines were fabricated and derivatized with a rabbit polyclonal IgG antibody, which selectively binds to E. coli O157:H7, in this case a nontoxigenic test strain. To quantify the effect of nonspecific binding (negative control), an antibody directed against the trinitrophenyl hapten (TNP) was used as a binding layer. Test E. coli bacteria were cultured, fixed with formaldehyde, stained with cell-permeant nucleic acid stain, suspended in phosphate buffered saline, and applied to the antibody-coated sensing surfaces. The biosensor transmission coefficient phase was monitored using a network analyzer. Phase responses of about 14 deg were measured for the E. coli detection, as compared to 2 deg due to non-specific anti-TNP binding. A 30:1 preference for E. coli binding to the anti-O157:H7 layer when compared to the anti-TNP layer was observed with fluorescence microscopy, thus confirming the selectivity of the antibody surface to E. coli.