An Integrated Research and Education Program on Highly Sensitive and Sensitive Biosensors for Food Borne Pathogens and Toxins

NON-TECHNICAL SUMMARY: Detection of pathogens and toxins in food substances. An example is E coli in ground beef. The primary purpose is to detect pathogens in food substances without any enrichment step. The project provides a new tool in pursuit of safe and uncontaminated food.

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APPROACH: Current methods require enrichment and an analysis time of eight hours to measure very low concentration of pathogens and low concentration of toxic substances in food. The proposed work builds upon recent successes in the authors lab with the cantilever sensor, which offers a new sensing modality that has not been exploited previously. We showed that E. coli 0157:H7 can be detected in contaminated beef at 10 cells/mL and SEB at 200 pg/mL. The sensors are constructed in the author's lab using glass and piezoelectric material (lead zirconate titanate) and are electrically actuated. The resonant frequency of the sensor is a function of its mass. The surface of the sensor is immobilized with the desired antibody, and when the target pathogen or toxin binds to it, the resonant frequency changes, and is measured. Graduate students will conduct detection experiments in food products working closely with our research collaborators at EERC-USDA labs.

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PROGRESS: 2007/09 TO 2008/09 <Br>
OUTPUTS: Fabricated cantilever sensors for detecting foodborne pathogens and toxins. Conducted experiments with E. coli O157:H7 and Listeria monocytogenes both in buffer and food matrix Effect of detection sensitivity on food matrix (milk, apple juice, beef wash) was measured experimentally Effects of sensor geometry on sensitivity was measured An new approach to sensor hydrophobic-coating was initiated An rapid approach to extract geonomic DNA of pathogen was developed An undergraduate course that integrates nature of food pathogens and toxins, and methods to detect or measure was taught in spring 2008 A course assessment of how the course improved the student knowledge of food safety issues was measured <Br>PARTICIPANTS: Participants: Raj Mutharasan, Department of Chemical and Biological Engineering, Drexel University, Philadelphia, PA 19104. David Maraldo, PhD student, Department of Chemical and Biological Engineering, Drexel University, Philadelphia, PA 19104. Kishan Rijal, PhD student, Department of Chemical and Biological Engineering,Drexel University, Philadelphia, PA 19104. Harsh Sharma, (new PhD student) Department of Chemical and Biological Engineering, Drexel University, Philadelphia, PA 19104. Shu-I Tu, USDA,REE,ARS,NAA,ERRC, 600 E MERMAID LANE, WYNDMOOR, PA, 19038-8598. - Dr. Tu gave special lecture in course. Professional training of David Maraldo and Kishan Rijal was provided by the project. New student Harsh Sharma is currently doing experiments on pathogen detection in food matrixes. <Br>TARGET AUDIENCES: Main target audiences are: (1) industry that requires rapid measurement of pathogens in food (2)training of PhD students, (3) students to become aware of pathogen measuring methods and biosensors. PROJECT MODIFICATIONS: At this point, we have demonstrated the principles of detecting pathogens (E. coli O157:H7 and Listeria monocytogenes) and toxin (SEB) using antibody immobilized on the sensor. The DNA-based detection that was included last year as an additional objective has given us very good results, and will emphasize more during the third year. With the current method (a manuscript is being prepared) we can detect the presence of ~ 700 copies of a 300 nt gene. If we use several defining gene segments for detection, it may be possible to detect a much lower number of pathogens. The increased emphasis on DNA-based method is driven by this new result.
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IMPACT: 2007/09 TO 2008/09<Br>
Successful detection of Listeria monocytogenes was accomplished at 100 cells/mL in buffer medium Sensors that exhibited sensitivities of ~ 100 attogram/Hz were successfully fabricated Using genomic DNA, successful experiments were conducted to detect the presence of E. coli O157:H7 based on the stx2 gene at a level of ~ 700 cells Twelve undergraduate students benefited from the course that integrated food safety into the course on biochemical engineering principles Student survey showed a five-fold increase ( 16% to 80%) increase in the knowledge of both the nature of foodborne pathogens and methods to detect them
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PROGRESS: 2006/09/15 TO 2007/09/14<Br>
OUTPUTS: The goal of proposed research activity is to apply antibody-immobilized piezoelectric-excited millimeter-sized cantilever sensors (PEMC) for detecting pathogenic agents (PA) and toxins. Specifically we will detect and measure the concentrations of Salmonella enteritidis(SE), Listeria monocytogenes(LM), Escherichia coli 0157:H7(EC), listeriolysin O (LLO), shigatoxin-1 (STX-1), shigatoxin-2 (STX-2) and Staphylococcal Enterotoxin B (SEB) in food products without an enrichment step. PEMC sensors that are mechanically robust and very highly sensitive will find widespread use, and serve as a tool for public health and national security. The proposed program of applied research addresses this issue, building upon on-going research in the PD's laboratory. The five specific objectives listed below address issues that will lead to practical applications of the PEMC sensor. <Br>OBJECTIVE 1. PATHOGEN IN FOOD MATRIXES. Devise and evaluate performance of antibody immobilized piezoelectric-actuated millimeter-sized cantilever sensor for detecting one pathogen (Salmonella enteritidis(SE), Listeria monocytogenes(LM), Escherichia coli 0157:H7(EC) per gram of meat and poultry, and at one pathogen per mL of milk and apple juice using the new cantilever oscillation and measurement modalities. The object here is to validate the procedures we have developed with real world samples, and then to optimize them for improved performance. <Br>OBJECTIVE 2. TOXINS IN FOOD MATRIXES. Devise and evaluate performance of antibody immobilized piezoelectric-actuated millimeter-sized cantilever sensor for detecting toxins (listeriolysin O (LLO), shigatoxin-1 (STX-1), shigatoxin-2 (STX-2) and Staphylococcal Enterotoxin B (SEB)) at 1 pg/mL in meat and poultry, and at 1 pg/mL of milk and apple juice using the new cantilever oscillation and measurement modalities. The general approach for toxins will be same as for pathogens outlined in Objective 1. <Br>OBJECTIVE 3. LARGE VOLUME SAMPLE. Devise and test PEMC sensor for the above list of pathogens and toxins when sample volume is large, 100 - 600 mL. Pathogens and toxins in large sample volumes is best carried out when the sensor surface is exposed to the sample in a flow configuration. The existing flow apparatus will be modified and tested for measurement of 600 mL samples. <Br>OBJECTIVE 4. SIMULTANEOUS DETECTION. Using the results of Objectives one and two, detect all seven antigens SE, LM, EC, LLO, STX-1, STX-2 and SEB simultaneously. The purpose here is to demonstrate that multiple agents can be measured simultaneously. In fact, the sensor is small enough that as many as 20 to 30 can be measured simultaneously. <Br>OBJECTIVE 5. COURSE An important part of the project is to develop a course titled: Biosensors for Food and Bio-Warfare Pathogens and Toxins. The target audience will be seniors and first year graduate students. <Br>PARTICIPANTS: Participants: Raj Mutharasan, Department of Chemical and Biological Engineering, Drexel University, Philadelphia, PA 19104. David Maraldo, Department of Chemical and Biological Engineering, Drexel University, Philadelphia, PA 19104. Kishan Rijal, Department of Chemical and Biological Engineering, Drexel University, Philadelphia, PA 19104. Harsh Sharma, Department of Chemical and Biological Engineering, Drexel University, Philadelphia, PA 19104. Shu-I Tu, USDA,REE,ARS,NAA,ERRC, 600 E MERMAID LANE, WYNDMOOR, PA, 19038-8598. Professional training of David Maraldo and Kishan Rijal was provided by the project. New student Harsh Sharma is currently being trained on pathogen detection in food matrixes. <Br>TARGET AUDIENCES: Main target audiences are: (1) industry that requires rapid measurement of pathogens in food, and (2) training of PhD students, (3) students to become aware of pathogen measuring methods and biosensors. <Br>PROJECT MODIFICATIONS: We will keep to the original objectives with the added research objective of DNA-based detection.
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IMPACT: 2006/09/15 TO 2007/09/14<Br>
1.We have successfully fabricated sensors, immobilized antibody against one model pathogen E. coli O157:H7 and one model toxin (SEB) and conducted successful experiments in ground beef, milk and apple juice. 2.A sensitive method of detection of 10 pathogen in one mL of beef wash, and the toxin at 2.5 femtogram/mL in milk and apple juice was achieved. 3.A course that integrates biosensors was taught to 17 undergraduate students in an upper level elective course. Dr. Shu-I Tu (ARS-USDA) gave a special lecture on food pathogens and sensing methods for food pathogens to the class in Spring, 2007. 4.Initial methods development for DNA based detection of the pathogen has been developed with synthetic 10-mer target with immobilized 15-mer probe on the sensor. Sensor response to hybridization of 2 attomoles of target was successfully measured.