Superfund Research Program
Sensing Superfund Chemicals with Recombinant Systems
Project Leaders: Sylvia Daunert (University of Miami), Sylvia Daunert (University of Miami)
Co-Investigators: Leonidas G. Bachas (University of Miami), Leonidas G. Bachas (University of Miami)
Grant Number: P42ES007380
Funding Period: 1997-2014
Project-Specific Links
Final Progress Reports
PublicationsConnect with the Grant Recipients
Final Progress Reports
The main goal of Dr. Sylvia Daunerts research is to develop biosensing systems for the detection of Superfund chemicals, which are sensitive, selective, rapid, easy-to-use, and amenable to field applications. Daunert's strategies involve the design and preparation of biosensing systems based on bacterial whole cells and proteins that are genetically engineered to respond to the analytes of interest with the production of a detectable signal (e.g., fluorescence, bioluminescence, chemiluminescence). This work also includes the development of enabling technologies for on-site studies, which aim at the long-term preservation and storage of these biosensing systems and their incorporation into portable miniaturized analytical devices.
To that end, Daunert developed whole-cell sensing systems for chlorocatechols, arsenic, biphenyls, hydroxylated PCBs, and dihydroxylated PCBs. Recently, she were able to detect hydroxylated PCBs in trace amounts in whole serum samples, directly, without sample pre-treatment. The research team envisions that the method developed can be employed as a rapid and sensitive tool that monitors OH-PCBs in laboratory toxicological studies and evaluates the presence of bioavailable OH-PCBs in natural environments. The potential of whole-cell sensing systems could be further enhanced if they could be used effectively in the field. For that, Daunert developed an inexpensive and simple method of preservation, storage and transport of these sensing systems, which is based on the use of spore-forming bacteria, such as Bacillus species. Spores are known to be very resistant to environmental conditions and shocks; they are easily germinated to generate metabolically active cells. During the current funding period, Daunert's investigative group proved the viability and wide-range applicability of this approach by developing several spore-based whole-cell sensing systems for various analytes, such as arsenic and zinc, and demonstrating their stability in terms of analytical performance after three cycles of sporulation/germination, as well as after long-term storage (6-8 months) at room temperature.
In order to further enhance the on-site employment of spore-based whole-cell sensing systems, Daunert incorporated them into a microcentrifugal microfluidic platform made of poly (methyl methacrylate) (PMMA) in the shape of a compact disk (CD). This platform can be easily transported and operated in situ using a typical CD reader. Upon previous demonstration of fast spore germination and bacterial cell growth on the CD miniaturized microfluidic platform, sensing was successfully performed in the CD. This allowed for the rapid (10 min) detection of zinc at low µM levels in low µL-volume samples. A different and novel approach to the development of biosensing systems takes advantage of the conformational changes that regulatory proteins undergo upon binding their specific ligands. A series of proteins that bind hydroxylated PCBs have been expressed and purified to develop protein-based sensing systems for hydroxylated PCBs. These proteins are either labeled with various fluorophores or genetically fused to fluorescent proteins and expressed as fusion proteins. Preliminary results obtained with protein-based sensing systems showed faster response and better sensitivity when compared to whole-cell biosensors. Proteins are also amenable to incorporation into microfluidic platforms.