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Development of the Nanodetect Integrated Pathogen Detection System

Stelick, Scott
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This Phase I SBIR grant will focus on the integration of a target concentration component into a nucleic acid amplification-based pathogen detection system. The objectives include the design and construction of a microfluidic IMS cartridge for use with the nanoDETECT platform. The IMS cartridge will be a disposable design constructed of PDMS elastomers. The design will optimize the concentration and detection of S. typhiumrium from liquid samples. The final objective is to concentrate and detect S. typhimurium from raw chicken using the microfluidic IMS cartridge and the nanoDETECT.
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NON-TECHNICAL SUMMARY: Sample preparation is extremely important to food borne pathogen detection because food matrices can severely inhibit nucleic acid amplification based detection methods. Samples must typically be pre-enriched to provide adequate numbers of target cells for detection. In addition, current zero-tolerance levels necessitate a very rigorous detection level. IMS is an attractive alternative to preenrichment since it can both remove target cells from complex matrices (such as food samples) and also concentrate target cells for rapid detection. The sample preparation cartridge will complement the existing breadboard instrumentation of Illuminarias nanoDETECT platform for the detection of the target pathogens from contaminated food samples.

APPROACH: The nanoDETECT platform has been proven to rapidly detect pathogenic microorganisms and has high commercialization potential in both the government and non-government sectors. The detection platform automates sample preparation and can detect bacteria by real-time PCR amplification. For this effort, Illuminaria will develop an automated immunomagnetic target concentration cartridge for the separation of the common bacterial food pathogen Salmonella typhimurium. The sample preparation cartridge will complement the existing breadboard instrumentation of Illuminarias nanoDETECT platform for the detection of the target pathogens from contaminated food samples.

PROGRESS: 2006/05 TO 2007/12
OUTPUTS: Products: Invention disclosure filed with the Cornell Center for Technology Transfer. It is currently in the process of being patented. The microfluidic cell separator cartridge consists of a polymeric base that contains microfluidic channels and has a total internal volume of approximately 20ƒÝl. This base was designed to be cast in the silicone elastomer PDMS, using a photolithographically patterned SU8 mold. The microfluidic base is attached to a second PDMS sleeve that can support a 30ml syringe. The syringe is a standard 30ml luer-lock syringe that has had the last 1cm at the tip removed. This leaves the syringe barrel and plunger which can then be inserted into the PDMS sleeve. The syringe plunger was modified by attaching a round, flat-faced piece of PDMS elastomer to the face of the plunger. Additionally, the plunger face is approximately 1-2mm smaller in diameter than the barrel of the syringe, however the rubber seal can be left intact to allow a tight seal with the barrel or removed. Finally, 0.01" and 0.0625" diameter Tygon tubing is glued into the PDMS microfluidic base in order to pump the concentrated magnetic beads and cells out of the device. First, the fluid sample is placed in the barrel of the syringe and allowed to fill the device. After loading, the solution is allowed to incubate so that the cells can attach to the Dynabeads. The plunger is then placed into the barrel of the syringe so that it makes contact with the top of the fluid. A magnet is then applied to the bottom of the concentrator, directly below the microfluidic base. In this way, the Dynabeads and cells can be pulled to the bottom of the concentrator and be removed from the bulk solution. After this concentration step, the plunger is depressed and the bulk solution is pumped out of the 0.0625" tubing into a waste collector. The plunger face is then depressed against the microfluidic channels, creating an upper seal (or cover) for the channels. In this way, the Dynabeads and cells are trapped within the small volume (20 microliters) of the microfluidic channels. Following this step, the magnet can be removed and the concentrated Dynabeads and cells are pumped out of the microfluidic base using the attached tubing.
PARTICIPANTS: Dr. Carl A. Batt, is President and co-founder of Illuminaria. He also serves as Scientific Director of Agave BioSystems. He holds a Ph.D. and M.S. degree from Rutgers University, Department of Food Science and a B.S. from Kansas State University in Microbiology. Dr. Batt is the Liberty Hyde Bailey Professor of Food Science and has been research director of a group at Cornell University for over ten years. During this time, he served as PI on grants and contracts in excess of $25 million. He is also the founding director of the Laboratory for Molecular Typing a fee-for-service facility at Cornell University that provides genetic typing services for microbial identification. Dr. Batt also held a number of research positions in the Department of Applied Biological Sciences and Department of Nutrition and Food Science at the Massachusetts Institute of Technology. Scott J. Stelick is an Optics and Microfabrication Engineer at Illuminaria and holds a Master of Engineering degree from Cornell University and is a co-founder of Illuminaria. Mr. Stelick is responsible for the design of micro-/nanofabricated devices and electro-optical systems. He is currently developing microcontact printed optical diffraction (MiCOD) chips for use in the detection of microorganisms, and fabricating a two-stage microfluidic PCR chip. Prior to joining Illuminaria, Mr. Stelick was a Senior Research Associate with the Alliance for Nanomedical Technologies. Nathaniel C. Cady is currently finishing his doctoral research in microbiology at Cornell University. He is a co-founder of Illuminaria and has spent most of his graduate research on the development of portable biosensors. He is currently working on the development of hand-held PCR based detection systems for a variety of pathogens, including B. anthracis, L. monocytogenes and S. aureus. Nathaniel is a member of the American Society for Microbiology and is a W.M. Keck fellow in Nanobiotechnology.

IMPACT: 2006/05 TO 2007/12
Change in Knowledge The PI, Scott Stelick has a strong engineering background and this project allowed him to work with microbiologists. He was able to learn to grow and maintain Salmonella cells for testing purposes. The results from this proposal was filed for an invention disclosure and is on the way of becoming a patent.

Funding Source
Nat'l. Inst. of Food and Agriculture
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Bacterial Pathogens