Optical Immunosensor for On-Site Rapid Detection for Foodborne Pathogens

Non-Technical Summary:<br/>
Recent outbreaks of E. coli-related illnesses in the US and, more recently, in Europe underline the need for more effective monitoring and remediation technologies for bacterial contamination in food. According to the Food and Drug Administration, outbreaks of food-borne illnesses due to consumption of contaminated produce have increased over the past decade. Along with the threat to human health and safety, there is also a major economic impact caused by such outbreaks. According to the United States Department of Agriculture (USDA), the economic impact in the year 2003 associated with E. coli illness totaled 405 million dollars (2008). It is understood that the most effective method for preventing such illnesses is to control points of potential contamination. To most effectively control contamination, such points must be identified accurately and quickly. For the detection of food-borne pathogens, a simple and effective fluorescence-based immunosensor is proposed. Traditional methods for detection of such pathogens rely on sampling and laboratory-based culturing techniques. It often takes days to obtain results using such methods, at which point the food product is already on its way to the consumer. We therefore propose to miniaturize and expedite this process, allowing the presence of bacteria to be accurately identified before the product reaches the consumer. The proposed sensing technique utilizes 4-methylumbelliferyl-?-D-glucuronide (MUG) as a signaling reporter for the presence of the pathogenic bacteria Escherichia coli. When the E. coli bacteria come into contact with MUG, it cleaves the substrate via the enzymatic activity of ?-glucuronidase. The resulting cleavage products include 4-methylumbelliferone, a highly fluorescent species. While this allows the method to be selective for E. coli in general, it is only the pathogenic strains of E. coli that are hazardous to human health. To enhance the selectivity of the MUG-based signaling scheme, fluorescent reporter will be linked with IgG antibodies that are highly specific to pathogenic and dangerous strains of E. coli bacteria. This project will lead to changes in knowledge, actions and conditions. We will add to our knowledge about new simple detection technique of multiple foodborne pathogens. By developing this detection technique, food processing facilities can use this early detection technique and become proactive. Contaminated food can be removed prior of reaching consumers. This user friendly detection technique will also ensure food quality conditions and reduce the spread of contaminated food products by conducting regular testing.
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Approach:<br/>
The research team has a BSL-2 laboratory. This project consists of seven tasks. Each task has a specific method for approaching, data collection and analysis. Task 1: Examine fluorogenic response of MUG to viable E. coli in solution. The first step in development of the proposed detection scheme will be to determine the effectiveness of the MUG-based reporter system. To determine the effectiveness of the MUG reporter, aqueous solutions containing various concentrations of MUG - ranging from 0.01 mM to 0.1 M - will be prepared. To these solutions, purified viable E. coli will be introduced at a concentration of 105 CFU/ml.
<br/>Task 2: Develop an immobilization strategy for binding anti-E. coli IgG antibody onto a glass substrate. The next step in the development of the proposed sensing scheme is to successfully link the E. coli-specific antibody to a suitable substrate. The tip of a glass bar will be cleaned with acetone and then activated using 3-mercaptopropy(trimethoxysilane). This activation step consists of immersion of the tip of the glass bar into a 5% (v/v) solution of MTS in toluene for one hour, and results in a reactive glass surface with exposed -SH functional groups. After activation, the glass bar will be exposed to the heterobifunctional crosslinker 4-maleimidobutyric acid N-hydroxysuccinimide ester (GMBS) at a concentration of 2 mM for 1 hour.
<br/>Task 3: Test binding of E. coli to the immobilized IgG antibody by fluorescent microscopy. To ensure binding of the E. coli antigen to the surface of the antibody-immobilized glass surface, fluorescence microscopy will be used. The modified glass bars immobilized with various concentrations of antibody prepared in Task 2 will be exposed to fluorescently labeled E. coli bacteria.
<br/>Task 4: Study the fluorogenic response of the bound E. coli when exposed to MUG. The glass rod with the bound E. coli will then be immersed in a solution of MUG at the concentration determined from Task 1.
<br/>Task 5: Quantitatively determine performance of the fluorescent sensing scheme. Performance metrics to be examined including sensitivity and lower detection limit, response time, selectivity, repeatability, and longevity. For all performance metrics, data will be analyzed using Graphpad Prism 5. Comparisons will be performed with a confidence interval of 95%. A p-value equal to or less than 0.05 will allow the refection the null hypothesis - that the two means are not significantly different. Sensitivity and time response measurements will also be analyzed using nonlinear regression to obtain information regarding statistical error of dose- and time-response curves, as well as the repeatability and predictivity of the data by means of R2 value.
<br/>Task 6: Validate the performance metrics of the sensing system on contaminated and non-contaminated food samples. Food samples of beef, poultry, and spinach will be contaminated with viable E. coli via innoculating loops from a contaminated soy broth. The research team has enlisted an experienced microbiologist, Dr. Guolu Zheng, to aid in culturing and preparation of the target pathogens.