Mammalian cell-based biosensor for rapid screening of pathogenic bacteria and toxins

Pratik Banerjee, Purdue University

Abstract

Rapid screening and detection of pathogenic bacteria and toxins in foods and beverages is critical to ensure distribution of safe and wholesome food to the end-users. Prevalence of pathogenic organisms or toxins renders huge threat to the food industry with disparaging consequences from both economic and reputation standpoints. In view of this, rapid food diagnostics became a very crucial research area of food science, and the uses of rapid sensing devices such as biosensors are increasing in food sector. The current sate-of art of biosensor-based detection depends mostly on several methods employing nucleic acid or immunological techniques. Although these techniques are fast and specific on target pathogens or toxins, but often fail to provide any information on the biological and physiological activity of the target analyte and identification of unknown hazardous agents. Mammalian cells, when used in cell-based biosensors (CBBs), can screen and detect analytes in a way that is physiologically pertinent and provide information about the biological function of the target. A CBB was developed utilizing a rapid Ped-2E9 cell cytotoxicity-based bioassay, to screen virulent Listeria or Bacillus species and different cytolytic toxins found in foods. However, issues pertaining to biological life of mammalian cells, their preservation and maintenance of viability on a sensor platform for a prolonged duration are some of the key factors precluding wide use of CBBs in real-life applications. To address some of the above-mentioned problems associated with CBBs, key factors affecting the survival and growth of Ped-2E9 cells under conditions mimicking the environment in a sensor device for on-site use were investigated. Majority of Ped-2E9 cells remained viable in reduced serum media in enclosed environment without any replenishment of nutrients or exogenous CO2 supply for 6 days without losing the sensitivity (as measured by cytotoxic responses) to virulent Listeria and Bacillus species and toxins. In order to integrate the Ped-2E9 cell-based cytotoxicity assay to a portable biosensor device, the cells were immobilized and encapsulated in a biocompatible type I collagen matrix and tested with pathogenic Listeria, the toxin listeriolysin O, enterotoxin from Bacillus species, α-hemolysin from Staphylococcus aureus, phospholipase C from Clostridium perfringens, cholera toxin from Vibrio cholerae and cytolysin from Stoichactis helianthus. Pathogenic L. monocytogenes cells and cytolytic or hemolytic toxin preparations from L. monocytogenes, B. cereus and other organisms showed cytotoxicity ranging from 24% to 100% at 2–6 h post infection. On the contrary, a non pathogenic L. innocua (F4247) or a B. subtilis and non-cytolytic cholera toxin induced minimal cytotoxicity ranging only 0.4% to 7.6%. Three different hand-held prototypes, filtration tube-based, micro-slide-based and multi-well plate based, were integrated with collagen encapsulated Ped-2E9 cells for rapid detection of pathogenic bacteria or toxins. The prototypes were tested with either pure bacteria or toxins or with artificially contaminated food and beverage samples. Results indicated 102 CFU/g (initial inoculum) of Listeria cells in meat samples could be detected using these prototypes by 16 h pre-enrichment followed by 6 h on-device enrichment. The limits of detection for different pure and crude cytolytic or hemolytic toxin preparations were found to be in the concentration range of 40 ng/ml – 20.0 µg/ml.

Degree

Ph.D.

Advisors

Bhunia, Purdue University.

Subject Area

Food Science|Cellular biology

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