Collaborative Research: Phage-based nanobiosensors for the rapid detection of food and waterborne pathogens

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Accurately detecting low levels of disease-causing bacteria in a large population of many types of (often similar but non-disease causing) bacteria is challenging, and will be addressed in this project by designing a nanobiosensor that is able to separate low numbers of the bad bacteria from complex samples, and concentrate it into a small volume and finally detect the presence of the disease-causing bacteria. The proposed work consists of creating a genetically engineered and magnetized virus (phage) which can specifically bind, separate, concentrate, and infect target bacteria in liquid samples. The phage is specific to E. coli bacteria and will be genetically engineered to produce large quantities of a reporter enzyme (phosphatase) during the 1-hour infection. Following infection, the E. coli is naturally lysed to release replicated viruses, thereby also releasing the high concentration of reporter enzyme. The engineered enzymes can then be sensitively detected using electrochemistry on a nanostructured electrode. It is proposed that phage can be utilized in combination with magnetic nanoparticles and nanostructured electrodes to promote the rapid recognition, separation, and detection in a pragmatic manner. The following research aims will be pursued: 1) Engineer the phage T4 to carry a gene for an engineered phosphatase enzyme and allow targeted attachment onto magnetic nanoparticles, 2) Use the magnetic nanoprobes to separate and concentrate E. coli from liquid samples, and 3) Electrochemically detect the phosphatase on a nanostructured electrode correlating amperometric signal to initial bacterial concentration. As part of this proposal, the PIs? research groups will develop high-definition computer-generated teaching tools which will allow students to visualize the concepts of genetic engineering and pages. The educational videos will be produced documentary-style and will include photo-realistic animations. The videos will be freely available for download via YouTube and the PI websites.