NOVEL 3-D BIOSENSOR FOR RAPID DETECTION AND ACCURATE IDENTIFICATION OF E. COLI O157:H7 IN BEEF

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: EVANS-ALLEN
• Reporting Frequency: Annual
• Accession No.: 0217081
• Project Start Date: Feb 1, 2009
• Project End Date: Jan 31, 2012
• Program Code: [(N/A)]- (N/A)

Project Director
El-Dweik, MA.

Recipient Organization
LINCOLN UNIVERSITY
820 CHESTNUT ST
JEFFERSON CITY,MO 651023537

Performing Department
Agriculture

Non Technical Summary
Escherichia coli O157:H7 is clearly one of the deadliest food borne pathogenic bacteria. It causes an estimated 73,000 cases of infection and 61 human deaths in the United States each year (Centers for Disease Control and Prevention, 2006). This bacterium has been linked to hemolytic uremic syndrome and hemorrhagic colitis. These illnesses may cause diarrhea, seizure, stroke, kidney failure and even death (Food and Drug Administration, 2008). They are often misdiagnosed, resulting in expensive medical testing before they are correctly diagnosed. In addition, E- coli has the potential to cause enormous national and international economical devastation due to medical costs and product recalls, as recently occurred with the recall of tomatoes due to E. coli O157:H7 contamination. It can also be found in vegetables, unpasteurized milk, juice and unchlorinated water. Contamination can have a significant impact on businesses such as the beef -industry. E. coli O157:H7 can be found on most cattle farms and can live in the intestines of healthy cattle. Thus, the meat can become contaminated with E. coli O157:H7 during slaughter. It is important to note that the infectious doses of E-coli O157:H7 is as low as 10 cells (Federal Register, 1990, 1991). Therefore, effective detection techniques are crucial to monitor and control E-coli O157:H7 in food products. Testing for the bacteria requires extensive analysis which has to meet certain challenging criteria. Sensitivity and response time for the analysis are imperative factors related to the usefulness of microbiological testing. An extremely selective detection methodology is also required because low numbers of pathogenic bacteria are often present in a complex biological environment along with many other nonpathogenic organisms. Traditional methods for the detection of bacteria are not available in the time scale desired in a clinical laboratory. In response to this problem, a number of instruments have been developed using various principles of detection, such as flow cytometry polymerase chain reaction, immunomagnetic separations, bioluminescence and mass spectrometry. These methods, however, are still time consuming and expensive. The proposed project will develop a novel 3-dimensional (3-D) interdigitated microelectrode array (IDE) based impedance biosensor. This biosensor will be capable of rapid detection and selective for accurate identification of E. coli O157:H7. This design is unique in the use of a 3-D IDE which increases the surface area compared to a single (2-D) IDE sensor. The increased surface area will enhance the sensitivity of impedance detection. Each IDE biosensor consists of 100 pairs of gold electrode "fingers" with a length of 0.5 mm. The IDE array will be designed with spaces between the interdigitated electrodes nearly the size of the bacteria in order to detect a single or a few bacteria cells.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
402	3330	1060	10%
402	3330	1100	10%
402	3330	2000	10%
402	3330	2010	10%
402	3330	2020	10%
402	4010	1060	10%
402	4010	1100	10%
402	4010	2000	10%
402	4010	2010	10%
402	4010	2020	10%

Knowledge Area
402 - Engineering Systems and Equipment;

Subject Of Investigation
3330 - Other beef cattle products; 4010 - Bacteria;

Field Of Science
2010 - Physics; 2020 - Engineering; 1060 - Biology (whole systems); 1100 - Bacteriology; 2000 - Chemistry;

Keywords

3-d biosensor

rapid detection

e. coli o157:h7 beef

Goals / Objectives
1. Designing and fabricating MEMS based impedance biosensor system. The device will consist of two arrays of 3-D interdigitated electrodes (IDE) and a fluidic channel with an inlet and outlet. Each IDE array will consist of 100 pairs of gold electrode fingers fabricated using surface micromachining and photoresist sacrificial layer. 2. Immobilizing the antibody using the Self-Assembled Multilayer (SAM) process. We will use the Self-Assembled Multilayer (SAM) process to immobilize the antibodies onto the IDE. This stage will provide the binding between bacteria and antibodies due to the high affinity between them. 3. Testing the device using impedance measurements. We will analyze the biosensor for the detection and selective identification of E. coli O157:H7 in beef when used in conjunction with the immobilized antibodies, and determine the magnitude and phase of the impedance of the bacteria effect alone. The effect of frequency on impedance measurements will be monitored and analyzed.

Project Methods
Biosensor Design The proposed E-Coli O157:H7 biosensor will be designed with a 3-D high density cylindrical interdigitated electrode array (IDE). The IDE surface is modified to immobilize antibodies and is used as the sensing surface to detect bacteria concentration in solution. The design is unique in the use of 3-D IDE with a microchannel to increase the surface to volume ratio and decrease the sample volume, resulting in a rapid and high sensitivity impedance biosensor. The biosensor will be exposed to bacteria via a flow channel. When bacteria bind to antibodies, only a region of 2-4 micrometer above the sensor surface is modified. The impedance measurement results will change as the concentration of bacteria bound to the sensor surface changes. The advantages of using 3-D IDE impedance system include a reduction in the sample volume, a more rapid detection time (within several minutes), low resistance, and a high signal to noise ratio. Culture and Coating of Bacteria The next stage is immobilization of the antibodies on to the IDE by forming a Self-Assembled Multilayer (SAM). Between and after these treatments, the electrodes will be rinsed thoroughly with ultra pure water, then rinsed with absolute ethanol and dried in a flow of pure nitrogen. After cleaning, the substrates will be immersed immediately into a mixed solution of biotin thiol and spacer alcohol thiol in 50/50 ethanol/chloroform solution. The electrodes functionalized with mixed SAMs will be removed, rinsed with the corresponding solvent, dried under N2 flow/ and fixed to the supporting prism of the Surface Plasmon Resonance (SPR) instrument (n) 1.61. The mixed SAMs will be stabilized by injecting Phosphate-Buffered Saline (PBS) (pH 7.0). Subsequently, Bovine Serum Albumin (BSA) solution will be added to prevent any further nonspecific adsorption on the mixed SAMs. Next, the same protocol will be used to inject neutravidin followed by adding the biotinylated polyclonal anti-E. coli antibodies. After each step, the electrode will be rinsed by injecting PBS solution (pH 7.0) to remove the unbound biological compounds. Testing and Characterization The magnitude and phase of the impedance across the interdigitated electrode array will be measured using an impedance analyzer. A modulated AC voltage (sine wave) will be applied to the microelectrode array at frequency range from 10 - 10 MHz. The impedance will be measured using two beef samples, one with bacteria and the other without bacteria as described above. The measurement will be performed prior to immobilizing the antibodies on to the surface of the IDE array, after immobilizing the antibodies, and after the exposure of bacteria. This technique will establish the baseline impedance and enable the extraction of the impedance of the bacteria effect alone. In addition, the effect of frequency on impedance measurement will be monitored and analyzed. The total detection time will be measured. The detection time will include immunoreactions, washing, and measurement. A calibration curve for E-coli O157:H7 concentration will be obtained using standard techniques adopted by FDA.

Progress 02/01/09 to 01/31/12

Outputs
OUTPUTS: Activities: The PI has completed a certified BSL2 facility for culturing E.coli O157:H7 and then prepare fresh contaminated samples at different concentrations for testing the Interdigitated Electrodes BIO-MEMS. The co-PI has fabricated open and micro fluidic detection platforms to control the detection upper and lower limits. The immobilization of antibodies was verified by using optical labeling to ensure presence of antibodies on the gold electrodes. A graduate student had his master thesis work was in the fabrication and testing. There are 8 undergraduate students and 4 graduate students. Those students are from both the PI and co-PI laboratory. This has created collaborative environment among students. Events: Results from this project has been presented in three oral presentations and eight poster presentations at different professional societies and universities. There were twelve students received training about various aspects of the project. Services: The expertise gained in the project allowed collaboration with five industrial companies. Products: The PI and co-PI are utilizing this project in Biosensor design course and Biotechnology course. The purpose is to use the project outcomes as educational materials. Dissemination: Students are given electronic copies of materials used in this project. PARTICIPANTS: Majed Dweik is the PI. He set the goals and objectives to be accomplished during the year. The main focus this year was to establish process for making contaminated samples at different concentrations for testing. The second objective is to establish protocol for immobilization of antibodies on gold electrode which are imbedded in a micro-channel. The third objective is to establish protocol and standards for testing the fabricated devices. The fourth objective is to compile instructional manual for all new participants to view project and aspects of project which they will work with. There are nine students working on this project. Aurora Molitoris is over see the process for culturing the bacteria with two other students, Katrina Hays and Joseph Ochoa. The immobilization team consists of Kathryn Wilson and I'Brianna Moore. The tasting team consists of Logan White, Ashrafuldin Mohammad, Shibajyoti Ghosh Dastider, and Ali Sahin. Three of those students are part of the fabrication team in Dr. Almsri's laboratory. Mahmoud Almasri is the co-PI. He is leading the design, fabrication, and characterization of devices. There are five students on this team. Shibajyoti Ghosh Dastider, Ali Sahin, Yifan Wu, and Ashrafuldin Mohammad. TARGET AUDIENCES: There were two high school students participants. They have expressed their interest to work during the summer on this novel device. Shawn Callahan and Sanjay Roberts has joined the sample making and testing teams respectively. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Experimental results indicated that the tested device is very sensitive. This device can detect 103 CFU/ml where other reported devices from the literatures, the lowest detection limit is around 1000 CFU/ml. The performance of this device will provide accurate and real-time measurements for small samples of E.coli O157:H7. FSIS as well as meat processors and handler will be able to identify the contamination and quantify it too. This valuable information will be used to determine elimination of the contaminated meat and save the market from recall which can be very costly. This project is providing valuable design that can be extending to detection of other bacteria. We are exploring the detection of Salmonella. Majed Dweik is the PI. He set the goals and objectives to be accomplished during the year. The main focus this year was to establish process for making contaminated samples at different concentrations for testing. The second objective is to establish protocol for immobilization of antibodies on gold electrode which are imbedded in a micro-channel. The third objective is to establish protocol and standards for testing the fabricated devices. The fourth objective is to compile instructional manual for all new participants to view project and aspects of project which they will work with. There are nine students working on this project. Aurora Molitoris is over see the process for culturing the bacteria with two other students, Katrina Hays and Joseph Ochoa. The immobilization team consists of Kathryn Wilson and I'Brianna Moore. The tasting team consists of Logan White, Ashrafuldin Mohammad, Shibajyoti Ghosh Dastider, and Ali Sahin. Three of those students are part of the fabrication team in Dr. Almsri's laboratory. Mahmoud Almasri is the co-PI. He is leading the design, fabrication, and characterization of devices. There are five students on this team. Shibajyoti Ghosh Dastider, Ali Sahin, Yifan Wu, and Ashrafuldin Mohammad. There were two high school students participants. They have expressed their interest to work during the summer on this novel device. Shawn Callahan and Sanjay Roberts has joined the sample making and testing teams respectively.

Publications

• Majed Dweik, R. Cody Stringer, Shibajyoti Ghosh Dastider, Yifan Wu, Mahmoud Almasri, Syed Barizuddin, Specific and targeted detection of viable Escherichia coli O157:H7 using a sensitive and reusable impedance biosensor with dose and time response studies, Talanta May 2012. Shibajyoti Ghosh Dastider, Syed Barizuddin, Majed Dweik, and Mahmoud F. Almasri, Impedance biosensor based on interdigitated electrode array for detection of E.coli O157:H7 in food products, Proc. of SPIE Vol. 8369 July 2012.

Progress 10/01/10 to 09/30/11

Outputs
OUTPUTS: The PI and co-PI has accomplished the following: 1. E.coli O157:H7 samples for testing are prepared fresh for every experiment run. These samples are made at different concentrations. 2. Bio-MEMS has been supplied by the co-PI on a regular basis. The Bio-MEMS are characterized for by the PI to determine operating conditions. 3. The PI conducted experiments to identify the performance factors such as detection limit and response time for the design submitted by the co-PI. 4. The devices are immobilized with antibodies that are specific to E.coli O157:H7. 5. The immobilization goes through characterization to ensure presence on electrode surface by using fluorescence microscope. 5. The current device is specific for E.coli O157:H7 detection only and the detection range is from 10 4 CFU/ml to 10 7 CFU/ml. 5. There are a team of four undergraduate and four graduate students working on this project. Students from the PI and co-PI lab collaborate and visit both labs. The PI's students went to the co-PI lab to observe and participate in the fabrication of devices. Then the co-PI's students visit the PI lab to learn about preparation of samples, immobilization, and testing. 6. The co-PI still improving the fabrication of the 3-D Bio-MEMS. Four students this year have presented results from this project at three different conferences. They were poster presentations. Also students from both labs have attended training on fabrication and testing.Services: We are working on a different design to improve detection limit. The nano facilities at Washington University will collaborate on the fabrication. Dissemination: This research project is used in Biotechnology course as one of the application of biotechnology to develop testing equipment for food pathogen detection. PARTICIPANTS: Majed Dweik is the PI. He sets the goals and objectives to be accomplished during the year. The main focus this year was to test devices and conduct extensive experiments on characterizing them. The second objective is to test immobilization protocol to ensure device specificity to E.coli O157:H7. The third objective is to work on the 3-D bio-MEMS fabrication.. There are eight students working on this project. Aurora Molitoris oversees the process for culturing the bacteria with two other students, Katrina Hays and Kathryn Wilson . The immobilization team consists of Cody Stringer and Katrina Hays. The tasting team consists of Syed Barizulldeen, Logan White, Shibajyoti Ghosh Dastider, and Ali Sahin. Two of those students are part of the fabrication team in Dr. Almsri's laboratory. Mahmoud Almasri is the co-PI. He is leading the design, fabrication, and characterization of devices. There are four students on this team. Shibajyoti Ghosh Dastider, Ali Sahin, and Yifan Wu. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Currently, the Bio-MEMS design used has provided stable results. The device was tested for its specificity to ensure that it will work only for E.coli-O157:H7. It was tested against Salmonella but it did not show any response. The detection limits are between 10^4 CFU/ml to 10^7 CFU/ml. The 3-D Bio-MEMS will increase the sensitivity by several folds. This technology will allow detection not only qualitatively but also quantitatively which will help the food industries from massive recalls. This technique can be used also for detection of Salmonella. There is one publication ready to submit to scientific journal.

Publications

• No publications reported this period

Progress 10/01/09 to 09/30/10

Outputs
OUTPUTS: Activities: The PI and co-PI have completed the following activities: 1. The PI has a complete setup for culturing E.coli O157:H7. Currently, bacteria are cultured on a weekly basis to keep fresh and colony. 2. Every week, fresh beef contaminated samples are prepared to use in testing the device performance. Beef samples are inoculated with E.coli O157:H7 by using the Stomacher at different concentrations. 3. The other activity is related to the immobilization. Currently, protocol was developed for the immobilization of antibodies on the gold electrode surfaces. This step was needed to make the device specific for the detection of the E.coli O157:H7. 4. The co-PI has been fabricating two dimensional Bio-MEMS devices for testing. The PI has developed a standard protocol for testing and evaluating the devices. 5. There are 8 undergraduate students and 4 graduate students continually working on this project. The collaboration is intense among the students. This interaction includes training of undergraduate students in bacteria culturing, immobilization of antibodies, Bio-MEMS fabrication, testing of devices, and results analysis. 6. The co-PI just completed the fabrication of 3-D device and is currently conducting an evaluation of the fabricated parts. An Events Poster presentation was conducted at the Lincoln University Annual Sciences Symposium. This year eight students received training on various aspects of the project. Services: We are currently working with three companies on collaborative efforts related to this project: 1. Addison Biological Laboratory 2. CEVA 3. Hach Company Products. We are compiling a complete instruction manual for the different aspect of this project. The purpose is to use it as educational material for all new participants. Dissemination: Students are given electronic copies of materials used in this project. PARTICIPANTS: Majed Dweik is the PI. He sets the goals and objectives to be accomplished during the year. The main focus this year was to establish the process for making contaminated samples at different concentrations for testing. The second objective is to establish protocol for immobilization of antibodies on gold electrode which are imbedded in a micro-channel. The third objective is to establish protocol and standards for testing the fabricated devices. The fourth objective is to compile instructional manual for all new participants to view project and aspects of project, with which they will work. There are nine students working on this project. Aurora Molitoris oversees the process for culturing the bacteria with two other students, Katrina Hays and Joseph Ochoa. The immobilization team consists of Kathryn Wilson and I'Brianna Moore. The tasting team consists of Logan White, Ashrafuldin Mohammad, Shibajyoti Ghosh Dastider, and Ali Sahin. Three of those students are part of the fabrication team in Dr. Almsri's laboratory. Mahmoud Almasri is the co-PI. He is leading the design, fabrication, and characterization of devices. There are five students on this team. Shibajyoti Ghosh Dastider, Ali Sahin, Yifan Wu, and Ashrafuldin Mohammad. There were two high school students participants. They have expressed their interest to work during the summer on this novel device. Shawn Callahan and Sanjay Roberts has joined the sample making and testing teams respectively. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Experimental results indicated that the tested device is very sensitive. This device can detect 10-3 CFU/ml where other reported devices from the literature, indicates the lowest detection limit is around 103 CFU/ml. The performance of this device will provide accurate and real-time measurements for small samples of E.coli O157:H7. FSIS as well as meat processors and handlers will be able to identify and quantify the contamination. This valuable information will be used to determine eliminate the contaminated meat and save the market from a costly recall. This project is providing valuable design that can be extended to detection of other bacteria. We are exploring the detection of Salmonella.

Publications

• No publications reported this period

Progress 10/01/08 to 09/30/09

Outputs
OUTPUTS: The PI and the collaborator from University of Missouri Columbia has been meeting regularly to work on design and modeling of the biosensor platform. Incubation of non pathogenic E. Coli has been established in the lab at Lincoln University. Also one student has been hired and waiting for one more graduate student to start. The design and modeling of two dimensional platform has been completed. We are awaiting the fabrication of the two platforms to start the immobilization process. The modeling for the three dimensional platform design has been started. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The design and modeling for two dimensional platform has been completed. This work provided students to learn great deal about design and modeling. Also the collaboration effort is present in this project which will increase the efficiency and productivity.

Publications

• None