Progress 10/01/03 to 09/30/06
Outputs
Work in the last year of this project has primarily focused on troubleshooting a new circuit design for direct detection of fluorescence lifetime. This circuit is implemented with simple and inexpensive logic gates, a fact which is particularly compelling for applications where rapid multiplexing to many parallel sensors is required. Fluorescence lifetime is a principle that can be used to measure quenching processes related to presence of many molecules of interest, including O2, H+, and CO2, and may also be used to make homogeneous phase sensors for different biomolecules based on conformational or binding interactions. In other words, the technology has potentially wide ranging applications for the simple rapid detection of process variables for biological production processes as well as for diagnostics of pathogens and molecular markers of agricultural importance. A provisional patent application based on this technology was submitted in October 2006. The other
main focus of this project during the report year was on identifying molecular mechanisms related to the herbicide resistance of various populations of the weed goosegrass collected in Hawaii. Degenerate primers were developed to amplify genes suspected of confering the resistance, and protocols were optimized for cloning and sequencing these genes, but thus far clones from the plants have not been sequenced.
Impacts
The biggest impact of the work completed is the potential development of an inexpensive handheld technology which may be adapted to molecular diagnostics for disease and other health indicators important in agriculture.
Publications
- No publications reported this period
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Progress 10/01/04 to 09/30/05
Outputs
In this year of the project, we have initiated research into identifying the mechanism by which resistant biotypes of goosegrass (Eleusine indica) manifest resistance to the herbicide metribuzine. A primer was designed to amplify a fragment of the psbA gene, which is essential in the electron transport for photosynthesis. Previous studies have indicated that mutations in this gene have imparted resistance to metribuzine. Work is underway to clone this fragment into a bacterial vector using TA cloning, and sequence the clones to identify mutations in the gene. For the sensor engineering portion of this project, we have developed a third prototype for the detection of fluorescent lifetimes with durations of microseconds using an inexpensive square wave clock for excitation. Previous prototypes, while simple and inexpensive, suffered a great deal of noise problems. The new prototype is based on a similar principle but is implemented to be more robust to noise. The
performance of the new prototype is currently being tested. We expect to use this prototype to develop an inexpensive system for monitoring dissolved oxygen, which could be used to monitor photosynthetic and other metabolic activity in cell culture in order to screen for bioactive compounds affecting these processes or leading to cell mortality. Sensor technologies investigated during this research have also been used to develop a prototype sensor for soil moisture. In principle, the technology should allow accurate reflection of bioavailable water in the soil under conditions of varying temperature and soil salinity, unlike capacitive type soil moisture sensors which dominate the market for automated irrigation scheduling. Variations in vapor pressure in the soil due to soil moisture changes could not be measured at the expected range due to instabilities in the sensor, so that testing of the sensor was unsuccessful. Future work using the principle may allow this system to become
practical, however. Ongoing efforts to develop a suspended callus culture have thus far failed, despite success at developing callous culture in solid media.
Impacts
The technology being developed has applications for developing inexpensive sensors for dissolved gases such as oxygen, as well as for detecting binding of certain proteins and nucleic acids through fluorescence quenching. These efforts may allow the development of screening systems to assess the bioactivity of different compounds related to cell metabolism and mortality, sensors to detect plant diseases in the field, and sensors to improve bioprocess control for production of biological compounds.
Publications
- Krishnan, A., Jenkins, D.M. and Fares, A. 2005. A new psychrometric sensor for soil moisture. ASAE Paper #052181.
- Jenkins, D.M., Zhu, C. and Su, W. 2005. Comparison of prototype circuits for direct measurement of fluorescence lifetime. ASAE Paper #053036.
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