Continuous threat of terrorist attacks has rendered the detection of explosive compounds a crucial component of homeland security. The primary goal of the present project is to design, fabricate, and test a flagellar motor biosensor prototype that will provide sensitive, fast, and specific trace-level detection of trinitrotoluene (TNT), one of the most widely used explosive materials. Flagellar motors are nanoscale biological motors commonly found in many bacterial species such as Escherichia coli and Salmonella. Their rotational behavior is extremely sensitive to slight changes in the level of environmental chemical compounds such as nitrate and nitrite, two key components of explosive materials. The proposed biosensor integrates E. coli flagellar motors with a microfluidic system to detect the presence of TNT by monitoring changes in the rotational behavior of a single flagellar filament attached to an immobilized cell body. Based on an in-house preliminary study, the sensitivity level of a non-optimized flagellar motor biosensor is about the same as the existing explosive detection systems, including both the conventional electrochemical and the new biological based designs. However, the proposed biosensor has a much shorter detection time due to the fact that the sensing mechanism is controlled by the fast chemotactic process of the cell' sensory system. The proposed biosensor will be fabricated based on the core flagellar motor assembly techniques developed by the PIs' research groups. The PIs will utilize the one-year EAGER support to develop a complete prototype of the flagellar motor TNT biosensor. The following are the specific objectives of the proposed project.
1. Optimize chemotaxis signaling sequence for TNT sensing. 2. Develop a single-cell tethering technique using PDMS micro sieves. 3. Fabricate biosensor prototype and evaluate performance.
Intellectual Merit of Proposed Activity The proposed flagellar motor biosensor is an unconventional technique for explosive detection. It uniquely combines molecular biology with MEMS microfabrication to realize a hybrid microsystem that can provide fast and sensitive TNT detection in a compact package. Successful development of this project is not only important to the field of explosive detection; it also represents a significant step towards the application of flagellar motors for trace-level detection of a wide variety of chemicals. The proposed project will face many science and engineering challenges, including optimizing the signaling process of bacterial chemotaxis for TNT sensing, single-cell tethering in a microfluidic system, and developing an effective interface for delivering TNT samples into the biosensor. Results of the project are expected to provide a wealth of information for future designs of biological and biomedical microsystems. The proposed research team has extensive experience in the handling and processing of flagellar motors. Their work will be carried out in well-equipped biological processing laboratories and large-scale microfabrication facilities at the University of Arkansas.
Broader Impacts of Proposed Activity The ability to detect trace level TNT is critical to homeland security, as well as forensics science, environmental monitoring, and landmine detection. Successful development of the proposed biosensor will lead to the development of a hand-held tool for on-site TNT detection which can be used in real-time sample analysis so that subsequent remediation, when necessary, can be carried out in a timely manner. The proposed research will generate significant learning opportunities for students at the University of Arkansas. Students supported under this effort will have a rare opportunity to be cross-trained in MEMS microfabrication and biological processing. The fabrication techniques and recipes developed by this project will greatly enhance the technical capability of the High Density Electronics Center (HiDEC), a multi-user microfabrication facility at UA, in the areas of MEMS, BioMEMS, and NEMS.
Intellectual Merit The present project aims at the development of a new rapid and highly sensitive detection system for TNT related explosive materials. Unlike the conventional TNT detection systems, the present design utilizes the nanoscale flagellar motors of nonpathogenic bacterial cells as the sensing unit and combine them with a novel microfluidic system as the sensor platform. During the project period, biochemical protocols were developed to increase the TNT sensitivity of the flagellar motors ten folds and successfully tether live bacterial cells in a microfluidic system. The microfluidic system developed for the cells is manufactured mostly from a soft polymer called PDMS and cosists of individually controlled micro pumps, valves, and mixers for delivering the bacterial cells from external sources to specific locations in the system. Once tethered, the cells in the microfluidic system are capable of sensing trace-level TNT in a liquid sample. Broader Impacts The flagellar motor based TNT detection mechanism developed by the present project demonstrates a new concept for a portable TNT sensor that can be applied to support homeland security. Successful development of the sensor will have an immediate positie impact on the techniques and routines currently used in scanning passenger luggage at the airport. Additionally, by slightly modifying the sensing mechanism and the microfluidic platform, the TNT sensor can also be applied to environmental monitoring by providing sensitive measurements of contaminants in water samples.
