Progress 01/15/06 to 01/14/11

Outputs
OUTPUTS: We have made significant advances on several fronts for final goal of "fieldable" inexpensive instruments. (1). Have developed a protocol for stabilization of PNA bound gold nanoparticles. Stabilization of gold nanoparticles is a significant finding and will enhance future instrument development. These can be used as a capture system for target as well as a delivery system of target to microinstruments. (2) Have tested probes on SPIRIT instrument with excellent results. SPR instrument can detect a signal within approximately one (1) minute of sample injection. Ssensor surface has been reused for over 40 times without significant reduction in signal strength and over 120 times before it requires recoating. Aability to reuse each sensor multiple times further reduces running costs. Limit of detection is equivalent to about 200 cells without any amplification of signal. Target molecule currently used is a synthetic oligonucleotide and final target will be ribosomal RNA molecules. Because RNA molecule is significantly longer than current synthetic oligonucleotide we expect a much lower limit of detection with field samples. Further, SPIRIT outperformed industry standard laboratory based BiaCore instrument with increased detection limit. (3) Hhave developed a protocol for efficient disruption of very tough resting spores of Potato Wart fungus from soil samples using a Gunaidinium process and 5 minute incubation. (4) Have developed a rapid and specific colorimetric assay using PNA probes and symmetric cyanine dye 3,3′-diethylthiadicarbocyanine iodide (DiSC2(5)). Short (16-base) PNA probes are used to capture target sequences. Addition of DiSC2(5) to a solution with PNA-containing hybrids causes dye to change color from blue to purple, due to formation of higher-order dye aggregates in minor grooves of duplexes. This change can be quantitatively monitored as an increase in solution absorbance at 540 nm (from dye aggregates) at the expense of dye monomer peak at 650 nm, and "hybridization signal" is calculated as ratio A540/A650. Assay has been tested with synthetic DNA and RNA oligonucleotide sequences, and addition of a short micrococcal nuclease (MNase) digestion step yields single-base mismatch resolution at room temperature (25 degrees C) within 10 minutes, with an unoptimized detection limit of 100 nM (0.4 fmole). (5) A field-compatible two-wavelength colorimeter designed to quantify target molecules using this assay is currently in development. Hybridization signal is calculated as ratio of absorbance values from dye aggregate (green) and dye monomer (red) wavelengths (Agreen/Ared), which approximates value obtained from total absorbance spectrum(A540/A650). Preliminary testing of dye assay using this setup has yielded hybridization signals comparable to those obtained using a benchtop spectrophotometer. (6) Have participated in filed work at Canadian Food Inspection Agency potato filed site near St. Johns Newfoundland. Our collaborators in Canada have supplied us with spores and infected soil. Have presented our work in numerous meetings meant to reach those involved in potato research. PARTICIPANTS: The ultimate goal of this project is to realize a field deployable instrument that will be able to detect and identify pathogens from environmental samples. To attain these goals, tools are needed to identity these microorganisms directly from field collected materials in a rapid, inexpensive and easy to use format. Environmental samples are far more challenging for biological sensor development then clinical sensors because of the extreme biological complexity of the samples and the broad range of chemical components encountered. The focus of the proposed project is to investigate the application of peptide nucleic acids (PNAs) as the selective, functional elements for target RNA mediated aggregation of nanoparticles, and the engineering of a dielectrophoresis microinstrument to capture and detect the aggregates as they form. This approach provides a means to collect and concentrate the target RNA in a highly selective manner, and a means to detect their presence that is relatively low cost, relatively fast (a few minutes), miniaturizable, and simple to implement with existing soil sample preparation techniques. We have developed two instruments for field use (1) a surface plasmon resonance (SPR) based instrument SPIRIT and (2) a dye based protocol that uses a simple and inexpensive colorimetric detection. These instruments were developed with the intention of expansion to other target organisms as needed. Model organism: Potato wart disease is caused by the fungal pathogen Synchytrium endobioticum and has been added to the "Select Agent" list by the US Department of Agriculture. This is a list of agents and toxins deemed to pose a severe threat to animal or plant health or products (http://www.aphis.usda.gov/programs/ag_selectagent/index.html). Animal and Plant Health Service (APHIS) considers it a high-risk pathogen, capable of causing great economic harm to US agriculture. The European Plant Protection Organization (EPPO) deems this pathogen so important that they have enforced strict quarantine legislations for well over half a century. Synchytrium endobioticum has spread throughout Europe, North America and Asia over the last 100 years primarily through transport of tubers or infected materials TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The significance of the findings to plant biosecurity is for rapid detection of pathogens, in this case at Customs or border security. More broadly, these instruments can be converted as platforms for detection of other organisms, or even genes in animals that are appropriate for biosecurity screening. We have had two graduate students, and several undergraduate students working on this project, all are young women. The project has a heavy engineering component and the training of women in engineering fields is of particular interest to over all science education in the US. Further we have established a collaboration with two Universities in Ireland, Queens University, Belfast (QUB) and Dublin City University (DCU). We completed a student exchange with QUB for training and research using the SPR techniques.

Publications

• Duy J and Connell LB. (2009) Field-deployable bioassay using gold nanoparticles and peptide nucleic acid probes for the detection of potato wart fungus, Synchytrium endobioticum. In 36th Maine Biological and Medical Sciences Symposium. Mount Desert Island Biological Laboratory, Salisbury Cove, ME,: MDIBL.
• Duy, J., L. B. Connell, W. Eck, S. D. Collins and R. L. Smith (2010). Preparation of surfactant-stabilized gold nanoparticle peptide nucleic acid conjugates. Journal of Nanoparticle Research on line doi:10.1007/s11051-010-9996-0
• Duy J, Connell LB, and Smith RL. (2008) Direct detection method for potato wart fungus, Synchytrium endobioticum. In Northeast Potato Technology Forum 2008. Fredericton, NB, Canada: Northeast Potato Technology Forum.73.
• Duy J, Smith RL, Collins SD, and Connell LB. (2009) Colorimetric bioassay for field-based detection of red tide organisms using gold nanoparticle aggregation. In NSF 2009 IGERT Principal Investigators Meeting. Alexandria, VA.
• Bratcher AR and Connell LB. (2009) Direct detection method for potato wart fungus, Synchytrium endobioticum, using surface plasmon resonance and molecular probes. In 93rd Annual Meeting of The Potato Association of America. Fredrickton NB.
• Bratcher AR, Connell LB, and Smith RL. (2009) Development of a direct detection method for Alexandrium spp. using surface plasmon resonance and peptide nucleic acid probes. In IEEE Sensors 2009. Christchurch, Canterbury, NZ. in press
• Connell LB, Barrett A, and Iszard M. (2008) The cytochrome B (cob) and ribosomal large subunit (LSU) genes as probe targets for the potato wart fungus, Synchytrium endobioticum. In Northeast Potato Technology Forum 2008. Fredericton, NB, Canada: Northeast Potato Technology Forum 74-77. Connell, L. B., J. Duy and A. R. Bratcher (2009). Development of rapid field-based detection methods for Synchytrium endobioticum. Phytopathology
• Duy J and Connell LB, (2008) Surface plasmon resonance (SPR) detection of potato wart fungus. Phytopathology. 98(6): S49.
• Duy J and Connell LB. (2009) Development of a field-deployable bioassay based on gold nanoparticle aggregation for the detection of potato wart fungus, Synchytrium endobioticum. In 93rd Annual Meeting of The Potato Association of America. Fredricton. NB.

Progress 01/15/06 to 01/14/07

Outputs
OUTPUTS: The purpose of this project is to develop an inexpensive (per test) rapid (seconds to minutes) test that can be used by non-scientific personnel. This instrument must be field ready with no additional reagents (kit form) and long shelf-life for all components. Potato wart fungus is our target system but the instrument development should be applicable to many other pathogens. To date, we have developed a protocol for binding our PNA probes to gold nanoparticles and flat gold surfaces. Both of these surfaces will be used in the final instrument. Stabilization of the gold nanoparticles is a significant finding and will enhance future instrument development. We have tested our initial probes on one prototype instrument (Surface Plasmon Resonance or SPR) with excellent results. The SPR instrument can detect a signal within approximately one (1) minute of sample injection. Our sensor surface has been reused for over 40 times without a significant reduction in signal strength and over 120 times before it requires recoating. The ability to reuse each sensor multiple times further reduces running costs. Our limit of detection is equivalent to about 200 cells without any amplification of signal. The target molecule currently used is a synthetic oligonucleotide and the final target will be ribosomal RNA molecules. Because the RNA molecule is significantly longer than the current synthetic oligonucleotide we expect a much lower limit of detection with field samples. We are in the process of developing additional sequence to the COB gene of related fungi for specific probe development. We are developing a protocol for efficient disruption of the very tough resting spores of the Potato Wart fungus from soil samples. We have participated in filed work at the Canadian Food Inspection Agency potato filed site near St. Johns Newfoundland. Our collaborators in Canada have supplied us with spores and infected soil. We have presented our work in numerous meetings meant to reach those involved in potato research. PARTICIPANTS: Laurie Connell PI- direct all research in the project, coordinate all labs and collaborator. Primary research work is biological Rosemary Smith- co-PI- direct research related to microfluidics and gold surfaces Janice Duy- graduate student- project involves PNA attachment to gold Amber Bratcher- graduate student- project involves SPR detection Steve Wood- Canadian food Inspection Agency- director of potato pathogen laboratory in St. Johns Newfoundland. collaborator for field work. Partner organizations Canadian Food Inspection Agency- collaboration by in-kind support and collaborate on research project Seattle Sensors- collaborate by supplying know-how and technical assistance for SPR instrument Additional Collaborators and Contacts Kurt Zeller- USDA MD. non-formal collaboration for potato wart detection TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The significance of the findings to plant biosecurity is for rapid detection of pathogens, in this case at Customs or border security. More broadly, these instruments can be converted as platforms for detection of other organisms, or even genes in animals that are appropriate for biosecurity screening. We have two graduate students, and several undergraduate students working on this project, all are young women. The project has a heavy engineering component and the training of women in engineering fields is of particular interest to over all science education in the US. Further we have begun a collaboration with two Universities in Ireland and our students will be traveling to Belfast for additional training and research using the SPR techniques.

Publications

• Bratcher AR and Connell LB. (2009) Direct detection method for potato wart fungus, Synchytrium endobioticum, using surface plasmon resonance and molecular probes. In 93rd Annual Meeting of The Potato Association of America. Fredrickton NB.
• Bratcher AR, Connell LB, and Smith RL. (2009) Development of a direct detection method for Alexandrium spp. using surface plasmon resonance and peptide nucleic acid probes. In IEEE Sensors 2009. Christchurch, Canterbury, NZ. in press
• Connell LB, Barrett A, and Iszard M. (2008) The cytochrome B (cob) and ribosomal large subunit (LSU) genes as probe targets for the potato wart fungus, Synchytrium endobioticum. In Northeast Potato Technology Forum 2008. Fredericton, NB, Canada: Northeast Potato Technology Forum 74-77.
• Duy J and Connell LB, (2008) Surface plasmon resonance (SPR) detection of potato wart fungus. Phytopathology. 98(6): S49.
• Duy J and Connell LB. (2009) Development of a field-deployable bioassay based on gold nanoparticle aggregation for the detection of potato wart fungus, Synchytrium endobioticum. In 93rd Annual Meeting of The Potato Association of America. Fredricton. NB.
• Duy J and Connell LB. (2009) Field-deployable bioassay using gold nanoparticles and peptide nucleic acid probes for the detection of potato wart fungus, Synchytrium endobioticum. In 36th Maine Biological and Medical Sciences Symposium. Mount Desert Island Biological Laboratory, Salisbury Cove, ME,: MDIBL.
• Duy J, Connell LB, Eck W, Collins SD, and Smith RL, (2009) Surfactant-stabilized gold nanoparticle-peptide nucleic acid conjugates. Bioconjugates. in review.
• Duy J, Connell LB, and Smith RL. (2008) Direct detection method for potato wart fungus, Synchytrium endobioticum. In Northeast Potato Technology Forum 2008. Fredericton, NB, Canada: Northeast Potato Technology Forum.73.
• Duy J, Smith RL, Collins SD, and Connell LB. (2009) Colorimetric bioassay for field-based detection of red tide organisms using gold nanoparticle aggregation. In NSF 2009 IGERT Principal Investigators Meeting. Alexandria, VA.