CICEET Progress Report for the period 9/16/06 Through 3/15/07

Project Title: Development of Microfluidic Technologies for the Detection and Quantification of Toxins from Harmful Algal Blooms
Principal Investigator(s): Todd Lane and Victoria VanderNoot, Sandia National Labs
Additional Investigator(s): Don Anderson Woods Hole Oceanographic Institute.
Project Start Date: 9/01/2006

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Figure 1

The objectives for this reporting period were four-fold:
1. Document the safety procedures for the project
2. Obtain purified phytotoxins, derivatizing agents and toxigenic phytoplankton
3. Establish a basis for the transfer of funds to co-worker Donald Anderson at WHOI and arrange for the transfer of field samples to Sandia National Labs
4. Redesign the microfluidic chip to give better performance during CZE.

Tasks to meet objectives
We have documented safety procedures for the handling of saxitoxins, domoic acid and toxic phytoplankton and have received approval for the project from the Sandia National Labs, California Institutional Biosafety Committee.

We have obtained Certified Reference Material for saxitoxin and domoic acid. We have obtained and initiated culture of commercially available toxic phytoplankton: Pseudo-nitzschia multiseries Alexandrium affine and Alexandrium minutum

We have developed a statement of work and budget for the work to be carried out at WHOI and are arranging for a transfer of funds.

We have redesigned the CZE chip. The chip redesign was essential to carry out CZE. The original design was effective for rapid filling of the chip with a viscous sieving medium appropriate for molecular weight separations of proteins. For this work, a variety of CZE based methods will be evaluated but they will not employ highly viscous separation media. Additional pressure restriction is needed to prevent swamping of the separation channel on manual injection of a sample into the sample loop. The new design (Figure 1) shows the incorporation of 2 micron shallow etch channels that act as pressure restrictions. The chip was fabricated by Caliper Life Sciences (Hopkinton, MA) in fused silica. Preliminary testing of the chip revealed that the chip performed as expected: 1) it was possible to fully hydrate the chip through the flush port in spite of the severe pressure restrictions and 2) sample overloading of the chip was prevented during manual injection. Preliminary separations carried out with non-toxin standards showed that the chip gave good performance overall as evidenced by peak shape and separation times.

Progress on Tasks
We have accomplished all tasks for this reporting period and are poised to make rapid technical progress during the second reporting period.

Have the results/data gathered during this reporting period changed the project objectives when compared to your original proposal?
The results from this reporting period have not altered the project objectives

Dissemination activities during this reporting period
None in this reporting period

Difficulties
No difficulties encountered.

Data Generated to date
None.

Project Objectives for Next Reporting Period

Objectives
Optimize sample preparation techniques; steps to include dewatering, lysis, solid phase extraction and fluorescent labeling schemes

Develop CZE separation methods in capillaries and/or microfluidic chips for biotoxin analysis using UV-absorbance and/or laser-induced fluorescence detection

Obtain cultured, natural, and spiked samples of toxigenic phytoplankton (Dr. Anderson) or toxin spiked seawater samples and analyze for marine biotoxins in capillaries and/or microchips

Compare detection limits, speed of assay, and multianalyte detection capability with conventional HPLC-based methods (carried out in Dr. Anderson’s lab)

Work plan to Meet Objectives
Initially for the methods development work related to electrophoretic separations, pure samples of toxins will be used, diluted in clean buffers. Once effective separation methods have been developed, we will validate methods by progressing from purified toxins to spiked monocultures to natural samples. For this effort, the single separation method (CZE) will be investigated, but future research programs will include additional separation methods for the unambiguous identification of toxins. We have demonstrated the ability of multiple separation methods to aid in the identification of protein toxins. This approach will be highly advantageous for the problem of imposter toxins.

Methods development related to sample preparation protocols will begin with monocultures of both diatoms and dinoflagellates. Once successful lysis has been demonstrated, spiked and/or natural samples will be evaluated to optimize the release/extraction of marine biotoxins. The progression from purified toxins to natural matrices is essential as they represent a complex and constantly changing background that needs to be addressed for any system to have any real utility.

Dissemination Objectives for next reporting period
We will be meeting with End User Advisor Langlois in early March.

We plan attending and potentially presenting a poster at the Gordon Conference on Mycotoxins and Phycotoxins this summer

Overall Project Timeline Update
No changes; we anticipate that all work will be completed by the end of the one year project life.

Expenditures
We have spent 25% of total project budget during this reporting period. This leaves 75% of the budget to spend in the second and final reporting period of this project. We anticipated carrying out the majority of the technical work during the second 6 months of this project and the pattern of spending supports that plan.

End User Advisor Feedback
Gregg Langlois
Organization: Chief of the Marine Biotoxin Monitoring and Control Program (MBMCP) of the State of California Department of Health Services
Location: California
Phone number: (510) 412-4635
E-mail: glangloi@dhs.ca.gov

At this stage, what are the potential applications for this research? Please discuss how you and others could potentially use the technology.
As reported above, this initial stage has involved project start-up tasks, with the substantive work to be conducted over the next six months. These time lines are quite reasonable given the complexity of the project. The recent accomplishment of CZE chip redesign is noteworthy.

If the investigators are successful in demonstrating the suitability of this technology for detection of low concentrations of domoic acid and the paralytic shellfish poisoning (PSP) toxins, then numerous applications will evolve. The availability of an instrument that can provide field-based quantitative data on toxin concentrations in seafood items would greatly enhance existing monitoring programs and research activities.

What are the key challenges to application of this technology? Please consider the technology itself as well as issues related to regulation, politics, socio-economic pressures, trends in the field etc.
The complexity of the PSP toxins, with different subsets produced by different clonal populations of dinoflagellates, presents numerous challenges. Developing a reliable and non-burdensome analytical protocol (sample preparation, toxin extraction, sample clean-up) and instrumentation capable of detecting all of the possible forms has been a challenge at the bench level. It is an even greater challenge to accomplish this in a miniature format suitable for field application.

Rigorous method validation work will be necessary, involving comparative data from other methods. The current work is rightly focusing on a simple sample matrix for method development, but ultimately more complex matrices (i.e., assorted seafood species capable of accumulating these toxins ­ mussels, crustaceans, small finfish) will need to be investigated. This work will require comparison with existing regulatory methods to demonstrate equivalency. Although the latter work may be outside of the current scope of work, it would be helpful if the investigators could provide some projection of timelines and milestones to this end in the final report.

With government resources shrinking it is imperative that new, reliable methods be developed for marine biotoxin detection in the field. This decentralization trend has already begun with the development of field test kits that can be used at seafood plants or at the harvest sites and the use of volunteers for detecting and tracking toxic blooms. What is currently lacking is a simple, portable (and economical) assay or analytical instrument capable of producing quantitative results in the field. If government monitoring support declines then there will be a financial incentive within the industry to have these tools available for ensuring that the public can be provided safe seafood.

Has anything changed about this project's potential applicability since the last reporting period (not applicable to the first Progress Report)?
n/a

Questions/comments/ suggestions for the researchers?
Will the remaining work over the next six months allow the investigators to determine both the instrument detection limit and quantitation limit for domoic acid and the PSP toxins?
Will the investigators be able to relate the toxin levels identified in natural phytoplankton samples to expected or measured levels in seafood (e.g., mussels)?
Can the investigators clarify the alternative methods that will be used for generating comparative toxin data with the microfluidic instrument being developed (for both domoic acid and the PSP toxins)?

End User Advisor: Dr. Darcie A. Couture
Organization: Director of Biotoxin monitoring for the Department of Marine Resources for the State of Maine
Location: Boothbay Harbor, Maine
Phone number: (207) 633-9570
E-mail: Darcie.Couture@maine.gov

At this stage, what are the potential applications for this research? Please discuss how you and others could potentially use the technology.
We continue to view any advances in HAB detection as potentially important to assisting our small agency in protecting public health, while minimizing economic losses to our shellfish industry, especially after the devastating impacts of the 2005 Alexandrium HAB event.

What are the key challenges to application of this technology? Please consider the technology itself as well as issues related to regulation, politics, socio-economic pressures, trends in the field etc.
The most apparent challenge to the application of this new technology would be the costs associated with the actual use. It is difficult to justify the maintenance of our current funding levels, and would be even more difficult to request additional funding for new technology; our most likely option would be to seek outside funding (grants) to support the use of the technology.

Has anything changed about this project's potential applicability since the last reporting period (not applicable to the first Progress Report)? Questions/comments/ suggestions for the researchers?
At the end of 2006, the state of Maine was granted $2 million in federal relief monies, based on the economic disaster caused by the 2005 Alexandrium HAB event. Based on overwhelming public comments, the Governor determined that the majority of this money would go directly to the shellfish industry as cash relief, while a small portion (~ $390,000) would be used to augment testing and research by the Department of Marine Resources (DMR). This money has been budgeted over the next two years to study the feasibility of commercial depuration of biotoxins, as well as to expand the existing HAB monitoring program from a mostly land-based program, to one that will include near-shore monitoring of sentinel buoys with mussel bags, and phytoplankton samples, by small boat. If we are able to demonstrate a tangible increase in the response to a HAB event with this augmented program, we hope to obtain funding to continue the program beyond two years, and possibly work toward transitioning over completely to a boat-based program in the near future. New technology which would compliment our efforts to work toward a better “warning system”, by detecting HABs and HAB toxicity further offshore, rather than already in the shellfish beds, would fit well into the direction in which we hope to see our overall state program going.

PI Response to End User Advisor Feedback
Will the remaining work over the next six months allow the investigators to determine both the instrument detection limit and quantitation limit for domoic acid and the PSP toxins?
The work over the next 6 months will indeed generate detection and quantitation limits for analysis. We will be able to perform concentration series in standards and spiked samples and determine what we can detect/quantify based on the resultant peaks. We can make estimates of what that might translate to in terms of levels in a toxic HAB or shellfish sample, based on a few assumptions of how we will ultimately carry out the sample preparation. However, the "instrument" detection/quantitation limits can not be determined at this stage since we are not building an instrument. This is a proof of concept effort.

Will the investigators be able to relate the toxin levels identified in natural phytoplankton samples to expected or measured levels in seafood (e.g., mussels)?
The answer to this question depends on the type and quantity of samples that we will be able to receive from outside sources such as the California Marine Biotoxin Monitoring program. If we can obtain matched samples of phytoplankton and shellfish from the same toxic event we may be able to start to correlate phytoplankton toxin levels to those in shellfish. This is not a stated goal of this research program and a definitive answer may require additional research efforts.

Can the investigators clarify the alternative methods that will be used for generating comparative toxin data with the microfluidic instrument being developed (for both domoic acid and the PSP toxins)?
We will be comparing our results to standard HPLC analysis for saxitoxins and immunoassay from Biosense for DA analysis. We also will obtain samples from the California Marine Biotoxin Monitoring program that have been analyzed by their own techniques.