CICEET Progress Report for the period 3/01/09 Through 8/31/09

Project Title: Validation of a Real-Time, Field Deployable Biosensor, for the Detection and Quantification of Polycyclic Aromatic Hydrocarbons (PAHs) in Aquatic Systems
Principal Investigator(s): Michael A. Unger
Additional Investigator(s): Stephen L. Kaattari, Erin S. Bromage
Project Start Date: September 1, 2007
Report compiled by: Michael A. Unger
Contributing team members and their role in the project:
Candace Spier, Doctoral student and researcher; development and evaluation of the Biosensor
Joe Rieger, Director of Watershed Restoration, The Elizabeth River Project; Helping to coordinate field testing of the sensor and evaluate the utility of Biosensor data as an end user.

Figures

Figure 1

A. Project on Objectives for This Reporting Period
Please describe each technical and non-technical objective and related task for this period, and provide an update on your progress on each. It may be appropriate for different members of the project team to address technical and non-technical objectives.

a.) Improve precision and accuracy of Biosensor instrument including evaluation of filtration techniques.

A problem with background fluorescence from particulate material in the Elizabeth River during a field study highlighted the need for an efficient filtration technique for sample collection and processing prior to biosensor analysis. Evaluation of filtering techniques demonstrated that Teflon® filters (1”) could be used in the field to quickly process samples with a syringe prior to biosensor analysis and they could also be used in the laboratory (47mm) to prefilter samples before extraction and GC-MS analysis. This step has increased the precision and accuracy of the biosensor technique and evaluation of aliquots by both the biosensor and GC-MS confirmed satisfactory performance.

Conduct field validation trials of the sensor:

1. In the Elizabeth River
2. At New Bedford, Mass
3. Monitor a laboratory toxicity experiment
4. Monitor a creosote Superfund site.

We have now completed validation trials of the sensor; in the Elizabeth River, New Bedford Harbor, during a toxicity exposure experiment, and at a creosote contaminated Superfund site. Short reports have been produced on the Elizabeth River dredging monitoring at Money Point and the monitoring of PAH contaminated groundwater at the LA Clarke & Son Superfund site in Spotsylvania, VA to disseminate the results from these studies to end user collaborators.

b.) Develop additional antibodies with new patterns of compound specificity.

In our efforts to make antibodies to specific PAH molecules, there will inherently be some amount of cross-reactivity, or a lack of molecular specificity.  As the first antibody we developed recognizes 3-5 ring PAHs, our newest antibody in development recognizes 2-3 ring PAHs (Fig 1).  At this point, the plot below demonstrates that supernatant from a hybridoma cell line (not yet monoclonal) has a higher specificity for the 2-3 ring PAHs (biphenyl, naphthalene, phenanthrene and anthracene) than it does for the 4 ring PAH, pyrene.  Cloning of this cell line is still underway, but we are hopeful that we have secured an antibody that has the ability to recognize the smaller molecular weight 2-3 ring PAHs.  When used in conjunction with the currently used anti-3-5 ring PAH MAB, we will be able to quantify a larger sum of PAHs present in environmental samples and better resolve the complexity of PAH mixtures.

c.) Further develop multi-compound detection with the biosensor using multiple antibodies for simultaneous detection of mixtures.

This work is delayed until better new monoclonal antibodies are developed and available for testing.

d.) Disseminate results

We continue to work closely with end users of the technology through collaboration while field testing of the biosensor, through presentations at meetings and development of handouts reporting on the biosensor testing.

Please include information about:

• Plan/process for project managementand team collaboration:
  The PI’s work in continuous close contact and discussion for all lab portions of this research as well as field-testing of the biosensor. Candace Spier, whose dissertation research is central to the grant objectives, has two of the PIs (Kaattari, Unger) as co-mentors for her PhD and the third PI (Bromage) is on her dissertation committee. Team collaboration and discussion is weekly if not daily. Our end user collaborators have been involved in planning and evaluating the field-testing the sensor and have been giving feedback through meetings.
  

• Development, refinement or demonstration of the technology or approach:
  The biosensor has been continuously refined throughout the field-testing portions of this project. Temperature limits and filtration methods have been established and close work with the instrument manufacturer (Sapidyne Instruments inc., Biose, ID) has resulted in multiple software upgrades. Batteries and an inverter power sources have been evaluated in the laboratory and the field and have been shown to provide a reliable power source.

• Project evaluation:
  The successful field validation tests (reports appended) have allowed the real world evaluation of the near real-time measurement of PAH by the biosensor technology. Working closely with end users from government agencies, consulting firms and a river restoration group has allowed evaluation of the effectiveness of the instrument. The recent documentation of a PAH plume during dredging in the Elizabeth River has demonstrated the success and the potential of this new technology.

• Key findings:
  The biosensor is sensitive to temperature extremes in the field (<40oF or >90oF) but has been shown to produce useful data on dissolved PAH concentration in minutes at concentrations down to the low ppb level.

• Unanticipated issues:
  Difficulties with new antibody development have delayed multi-compound detection development. A six month no-cost extension was requested and granted to continue the antibody development portion of this research.

  Leaking in the fluidics of the instrument at temperature extremes (>90oF) and stability issues at these extreme temperatures has been resolved by warming the instrument when cold, syringe upgrades and by icing down reagents when in extreme warm temperatures in the field.

B. Knowledge dissemination activities during this reporting period:
For example, activities could include:

• Workshops or trainings coordinated/conducted by your project team
• Publications, manuals, protocols
• Patent, copyright, invention disclosure activity
• Outreach activities

The PI (Unger) gave two presentations at meetings with potential end users of the technology:

1) "An update on CICEET funded biosensor research at VIMS: working with industry and government partners" (Unger et al.) VIMS/William and Mary/Industry Partnership Meeting, VIMS. July, 24, 2009. Approximately 40 individuals from academia, industry and federal agencies were present.

2) “Using a Biosensor to measure PAH Concentrations in Near Real-Time at the Money Point Dredging Site in the Elizabeth River, VA. “ (Unger et al.) August 5, 2009. The Elizabeth River Project, Board of Directors Program Committee meeting, Portsmouth, VA.

Spier, C. R., E. S. Bromage, T. M. Harris, M. A. Unger and S. L. Kaattari. 2009 The development and evaluation of monoclonal antibodies for the detection of polycyclic aromatic hydrocarbons. Analytical Biochemistry, 387, 287-293.

Our end user collaborators have requested short reports as handouts to document the field-testing of the PAH biosensor. Those are completed and appended to this progress report.

Using a biosensor to measure PAH concentrations in near real-time at the Money Point dredging site in the Elizabeth River, VA. M. Unger, C. Spier, G. Vadas and S. Kaattari. 7 pp.

A field validation study of a PAH biosensor. C. Spier, M. Unger, G. Vadas and S. Kaattari. 4 pp.

C. Have the results/data gathered during this reporting period indicated that a change to your original approach is necessary? If so, who was involved in the decision-making process? Please explain.
No major changes to the original approach.

D. Please describe collaboration activities with target stakeholders during this period. Has interaction with stakeholders during this period brought about any changes to the project? Have the stakeholders confirmed the relevance of the technology or approach you are working on?
Collaboration has included, field testing, meeting presentations and preparation of reports. No major changes to date. Feedback from stakeholders on the field-testing reports will be incorporated into the next reporting period.

E. Please describe technical and non-technical objectives for the next reporting period and outline your work plan to meet identified objectives.

Objectives:

1. Improve precision and accuracy of Biosensor instrument including evaluation of physical parameters on PAH measurement.
2. Conduct additional field validation trials of the sensor.
3. Develop additional antibodies with new patterns of compound specificity.
4. Further develop multi-compound detection with the biosensor using multiple antibodies for simultaneous detection of mixtures.
5. Disseminate results.

Work Plan to Meet Objectives:

1. Improve precision and accuracy of Biosensor instrument including evaluation of physical parameters on PAH measurement. We will evaluate the effects of dissolved organic carbon and other physical characteristics on the precision and accuracy of PAH measurement by the Biosensor.
2. Conduct additional field validation trials of the sensor. We plan to conduct additional field trails in the Elizabeth River, VA and other locations (TBD) to further evaluate the limits of the PAH Biosensor technology.
3. Develop additional antibodies with new patterns of compound specificity. New antibody development and testing is underway and presented in this progress report. This work will continue.
4. Further develop multi-compound detection with the biosensor using multiple antibodies for simultaneous detection of mixtures. These techniques will be further developed and evaluated as new antibodies become available.
5. Disseminate results. A presentation by C. Spier at the SETAC conference has been accepted and will occur in November 2009. New publications describing field validation are planned for submission in 2009-2010. Field validations will be performed in collaboration with potential end-users of the technology.

Caption:
Figure 1. Response to a suite of PAH molecules by a new antibody under development for the biosensor