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

Project Title: A fiber optic microarray technology for the detection and enumeration of harmful algal bloom (HAB) species
Principal Investigator(s): Donald M. Anderson and David Walt
Additional Investigator(s): none
Project Start Date: 9/01/05

Figures

Figure 1

Tasks to meet objectives
1 ­ Design a second probe set for the toxic dinoflagellates Alexandrium fundyense and Alexandrium ostenfeldii
2 ­ Design probes targeting toxic Pseudo-nitzschia from the northeastern U.S.
3 ­ Refine assay conditions to reduce processing times

Progress on Tasks
A major focus of work during this six-month period was the design of additional probes for the fiber optic arrays. This included the design of a second set of capture and signal probes for our two dinoflagellate species, Alexandrium fundyense and Alexandrium ostenfeldii. The incorporation of a second sandwich hybridization probe pair for each target will make the measurements more reliable, by reducing the potential for both false negative and false positive detections in environmental samples. A positive or negative signal determination is made only when signal coincidence from several probe types occurs. After analysis of several different regions of the ribosomal RNA operon, we have designed a second capture probe that targets the 5.8 rDNA gene for each of our dinoflagellates. These probes will be incorporated into the array and tested against cultured cells in the upcoming months.

In addition to the design of redundant probe sets for the dinoflagellates, we have also been working towards developing probe sets for toxic Pseudo-nitzschia diatoms. Our original fiber optic array used probes targeting west coast Pseudo-nitzschia, which were suitable for proof-of-concept studies using cultured organisms. However, the west coast probes may or may not match the Pseudo-nitzschia found in the northeastern U.S., depending upon the extent and location of ribosomal sequence differences between west and east coast strains. Thus, the design of Pseudo-nitzschia probes requires a significant amount of work and effort, as we first have to collect sequence information on Pseudo-nitzschia from the northeastern region.

During this reporting period, we have been collecting water samples to find Pseudo-nitzschia cells and establish cultures for sequence analysis. These organisms can be slow growing, and it takes weeks to months to get the cultures established. Nonetheless, we have managed to isolate more than 90 cultures this summer, comprising three different gross morphological types (as observed by light microscopy). To date, the DNA sequence of a portion of the LSU rDNA gene has been obtained from 63 isolates, representing four different ribotypes: three that most closely match sequences of Pseudo-nitzschia delicatissima/pseudodelicatissima and one that most closely matches sequences of Pseudo-nitzschia fraudulenta/subfraudulenta. The sequences of the northeastern strains were compared with the sequences of existing LSU-targeted probes developed by Chris Scholin of the Monterey Bay Aquarium Research Institute. The strains from the Gulf of Maine show one base pair changes and/or indels in the region of the probe sequences, indicating that we will need to tailor detection probes to the specific sequences of Pseudo-nitzschia from the northeastern US.

Another major task was the modification of our fiber optic assay conditions to reduce the per sample processing time. In the proposal, we anticipated that this would be achieved by optimizing the various aspects of the assay protocol (e.g. hybridization and wash temperatures). We have opted, instead, to approach this problem by modifying the hardware components of the fiber optic apparatus. Specifically, a microfluidic sample handling system was incorporated and tested. Prior research in the Walt lab has demonstrated that flowing a target solution across the surface of a sensor array reduces both the limit of detection and required exposure time. Through collaboration with Haim Bau at the University of Pennsylvania, we have access to small-scale design and production of polycarbonate microfluidics cassettes. Our current CICEET prototype is shown below.

The disposable chips consist of three layers of polycarbonate, machined to produce channels and hot-pressed. Small volumes (~50µL) of cell lysate are introduced, and then the chip is alternately heated and cooled to move the solution over the surface of the fiber in an oscillating “push-pull” fashion. This system will be ideal for future instrumentation due to low cost, limited power requirement (<10V), and the ability to incorporate lysis chamber modules. We have tested the microfluidics chips with cultured A. fundyense samples that had been previously analyzed with the original instrumentation. The initial results have been promising, but are complicated by some difficulties with biofouling and incompatibility between the chips and detergents in the current lysis buffer. The fouling results from adsorption of nucleic acids, which is a frequent problem for microfluidic devices. The presence of detergents such as 5% N-lauryl sarcosine and 0.5% sodium dodecyl sulfate (SDS) in the lysis buffer dramatically aids lysis efficiency but causes bubbling in the narrow channels of the chips, impeding proper flow and limiting the effective RNA concentration at the sensor surface. With input from the Bau group, we have begun testing chip pre-treatment with protein blocking solutions containing casein and bovine serum albumin (BSA) to prevent nucleic acid absorption. To address the detergent bubbling, we are investigating both modifications to the chip design and alternative lysis methods (mechanical breakage, enzymatic, extraction columns).

Have the results/data gathered during this reporting period changed the project objectives when compared to your original proposal?
The results and data gathered during this reporting period have not changed the overall objectives of the project. The specific tasks needed to accomplish objective 5 (reducing the assay time) have changed somewhat. Instead of optimizing the assay conditions to reduce the per-sample assay time, we have decided instead to modify the system hardware by incorporating a microfluidic chip for sample handling. We feel that the microfluidics system will both achieve the objective of reducing the assay time and bring us closer to a system design that will be most suitable for instrumentation.

Dissemination activities during this reporting period
Workshops/ conferences/ trainings coordinated/conducted by your team: none
Project related presentations/poster sessions at workshops/conference: none
Manuscripts published or submitted for publication: none
Manuals, Protocols (submitted and in progress): none
Outreach Activities: none
Contact with End Users: Feedback was solicited for the current project report. As detailed above, more extensive discussions with end users are planned for next project period.
Patent, Copyright, Invention Disclosure Activity: none
Student activity (e.g. theses, dissertations, etc.) on the project (please identify students as graduate or undergraduate): Ryan Hayman, a Ph.D. student in the Walt laboratory at Tufts, is working on the microfluidics chips and HAB array system as part of his thesis research.

Difficulties
The primary difficulties that we have encountered are the sample and lysis buffer incompatibilities with the microfluidics chips. However, we feel that these issues can be readily addressed by changes in the chemical formulation of the buffers, and we are actively investigating alternative formulations that would prevent these problems from occurring.

Data Generated to date
To date, we have generated sequence and probe data for three species of toxic algae: Alexandrium fundyense, Alexandrium ostenfeldii and Pseudo-nitzschia.

Project Objectives for Next Reporting Period

Objectives
During the next reporting period, work will focus on the following project objectives:

Objective 2: Design and test probes for toxic Pseudo-nitzschia spp. from the Gulf of Maine;
Objective 3: Design and test a second probe pair for each species, to incorporate redundancy into the array;
Objective 4: Work with coastal managers to determine desired detection limits, precision, and other operational characteristics for the assay and expected instrumentation; and
Objective 5: Refine assay conditions to reduce processing time.

Work plan to Meet Objectives
The probe development for Pseudo-nitzschia species requires additional work before we get to the design and testing stage. Specifically, we need to determine which of our strains are actually toxic, by measuring domoic acid levels in the cultures. Once we establish the toxigenicity of the cultured isolates, we can then design probes that should bind to rRNA from the toxic cells, but not the nontoxic cells.

Likewise, the problem of incompatibility of the lysis buffer with the microfluidic chips will have to be resolved before we can move on to probe testing. Unfortunately, the detergents that do not work well in the microfluidics system are necessary for adequate lysis of the dinoflagellate cells, which are very difficult to break open. The first option that we will investigate is the use of RNA purification for preparing the rRNA samples. This would allow us to use harsh reagents to lyse the cells, but then exchange those chemicals for less aggressive ones during the purification step. RNA purification would also provide a “cleaner” sample, which is generally better for molecular detection methods such as the fiber optic array. There is also a precedent for using RNA purification on remote instruments; the Autonomous Microbial Genosensor being developed by Dr. John Paul’s laboratory at the University of South Florida incorporates partial RNA purification.

When the buffer compatibility issues have been resolved, we will proceed to test the new probe sets. The first step is to test the probes individually, against different numbers of cultured target cells. Next, the probes will be combined into a multi-species array, which will be tested against lysates from different combinations of cultured target cells (2-3 species per lysate). The final test will use cultured cells “spiked” into a natural seawater sample, to determine any effects of the complex natural background on the results.

During this time we also want to work with our end user and NERR partners to discuss the operational and technical needs for this fiber optic assay. This next reporting period will be a time of intense testing and assay optimization, which is an area that would benefit from the input from our end users. Our efforts will focus on identifying the most effective tools for communicating our project goals and progress to our partners, and the most efficient means of generating feedback from them regarding their needs and desires for cell detection assays and instrumentation.

Dissemination Objectives for next reporting period
As described above, one of our goals is to solicit the involvement of our end users in communicating their needs and desires for instrumentation for HAB monitoring. We also plan to present the test results with the updated fiber optic array system at one or more international meetings (e.g. Pittcon, American Society for Limnology and Oceanography).

Overall Project Timeline Update
There are no changes to the original project timeline.

Expenditures
Expenditures are in the range anticipated for the work accomplished to date.

End User Advisor Feedback
We distributed this report to our three end user advisors:

End User Advisor: Michelle Dionne
Organization: Wells National Estuarine Research Reserve
Location: Wells, Maine
Phone number: 207.646.1555x136
E-mail: dionne@wellsnerrcec.lib.me.us

End User Advisor: Darcie A. Couture
Organization: Maine Department of Marine Resources
Location: West Boothbay Harbor, Maine
Phone number: 207.633.9570
E-mail: Darcie.Couture@maine.gov

End User Advisor: Michael Hickey
Organization: Massachusetts Department of Marine Resources
Location: New Bedford, Massachusetts
Phone number: 508.990.2860x122
E-mail: Michael.Hickey@state.ma.us

At this point, we have received replies from two of the three end user advisors. Because we do not want to delay the submission of this report any longer, we are including the replies of the two end users below. We will continue to actively solicit feedback from our third end user, at which point we will submit an updated project report.

At this stage, what are the potential applications for this research? Please discuss how you and others could potentially use the technology.
Dionne: The HAB probes being developed would add an important dimension to our current estuarine water quality monitoring program. Short-term variation and long term trends in HABS are important ecosystem signals that should be measured and related to other parameters already being measured by the NERR system, including weather and land use. At the Wells NERR, and at the Great Bay NERR, the HAB data could also be interpreted in light of estuarine circulation, using the LaGrangian models developed by Dan Lynch's group at Dartmouth.

Couture: After another serious Alexandrium HAB event, it remains our priority to maintain a strong, reliable biotoxin monitoring program within the Department of Marine Resources. After a small but successful pilot study in the Casco Bay area involving sentinel buoys deployed at every major Mya arenaria bed and fine scale nutrient and phytoplankton sampling, we are currently seeking funding to expand this program to other major bays in Maine. These probes could serve as a permanent part of the monitoring program for the state of Maine, providing us with an early warning for toxic species, assisting us in developing a better sense of HAB dynamics, and allowing us to focus our limited resources most effectively.

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.
Dionne: I suspect the only real challenge will be the cost of the equipment, supplies, and time required for implementation.

Couture: I was concerned about the successful coordination in collecting the target species in a timely manner, but this seems to have been accomplished quite well, and is already providing information important to move the project along. The groups seems to be addressing challenges as they come up, and I have no further concerns at this time.

Has anything changed about this project's potential applicability since the last reporting period (not applicable to the first Progress Report)?
Dionne: With the addition of new probes, the project has increased its potential applicability.

Couture: The only other screening tool available to managers for monitoring for domoic acid had been a small kit produced by the Jellett company in Canada; this past year, some serious internal problems at the company have both interrupted the ready availability of these kits, as well as brought into question the integrity of any kits which are currently in production. As a manager, I have made a quality assurance decision to refrain from using any of the Jellett kits in my program, so the need for an alternative method of testing is greater than ever.

Questions/comments/ suggestions for the researchers?
Dionne: none given

Couture: none given

PI Response to End User Advisor Feedback
We are pleased that our end users consider this technology to be a welcome addition to their suite of tools for ecosystem and public health monitoring. The fiber optic analysis approach can provide the flexibility (multi-species detection) and ease of use that is needed for monitoring applications. We will continue to solicit their feedback to ensure that our efforts focus on developing an instrument that is well suited to the needs of coastal managers.