The primary goal of the proposed research is to determine the feasibility of using an Lateral Field Excited (LFE)sensor platform for the detection of saxitoxin (STX) in real-time. Specifically the following objectives will be accomplished: 1. Optimization of the STX LFE sensor element The chemically selective film used for the proposed STX sensor element will be the 18-crown-6 ether (CE) film. The CE film will be deposited on an LFE sensor platform, fabricated in the clean room of the Laboratory for Surface Science and Technology (LASST) at UMaine, in a monolayer to ensure proper operation. This monolayer deposition process has been well established by Mainely Sensors in its preliminary research. However, the film deposition procedure will be optimized to maximize the element's sensitivity, response time, selectivity, and reproducibility while not arbitrarily increasing the time required to deposit CE on an LFE sensor platform. Verification of the deposition of a monolayer will be accomplished with ellipsometry. 2. Testing of LFE STX sensor elements in water Each LFE sensor element with a CE film deposited on its sensing surface will be exposed to varying concentrations of STX in deionized water to determine the doseresponse relationship of the sensor response and STX concentration. This will be followed by exposing the LFE sensor elements to STX in different media such as a lab made NaCl (saline) solution and sea water. Throughout these tests the sensitivity, response time, reproducibility, reusability and selectivity of the CE-coated LFE sensor elements will be evaluated. 3. Testing of LFE STX sensor elements to detect STX in shellfish meat Once the LFE STX sensor element is shown to be capable of detecting STX in sea water the sensing elements will be exposed to liquefied shellfish meat which has been treated with STX and an extraction solution. The sensitivity, response time, reproducibility, and selectivity of the CE-coated LFE sensor elements will be evaluated. These tests will determine the feasibility of detecting STX in shellfish meat and the possibility of the LFE sensing element to replace the current mouse bioassay test. Mainely Sensors will use Phase I of the proposed work to demonstrate the feasibility of using an LFE sensor element to measure levels of saxitoxin in an aqueous (ocean) environment. The proposed work will determine if i) the LFE sensor element provides adequate sensitivity to detect STX in situ, ii) the LFE sensor element exhibits reproducible and predictable responses to STX exposure, iii) the LFE sensor element is selective enough to operate in the presence of interferents found in an ocean environment, and iv) the LFE sensor element has a quick response time on the order of minutes. Successful demonstration of the proposed STX sensor element will result in Mainely Sensors moving toward the development of a prototype LFE saxitoxin sensor system in Phase II.
NON-TECHNICAL SUMMARY: This Small Business Innovation Research Phase I grant will demonstrate the feasibility of using a Lateral Field Excited (LFE) acoustic wave sensor platform coated with a selective chemical film for the rapid in situ detection of saxitoxin (STX). Shellfish containing STX, a product of a Harmful Algal Bloom (HAB) such as Red Tide, is one of the primary causes of Paralytic Shellfish Poisoning (PSP) in humans. Quick and effective methods for detecting STX are necessary to protect the public health as well as soften the economic burden of HAB events on the shellfish industry. The current Association of Official Analytical Chemists (AOAC) accepted method to detect STX is the mouse bioassay. This method is a long lab-based test with both technical and ethical limitations. The objectives of this research will be to i) optimize the STX-sensitive chemical film deposited on the LFE sensor platform surface, ii) obtain the LFE STX sensor element response in deionized water, NaCl (saline) solution, and sea water, and iii) obtain the LFE STX sensor element response to shellfish meat. The anticipated outcome of this Phase I effort will be the demonstration of a rapid, sensitive in situ STX sensor element. This sensor element will be integrated into a total sensor system in Phase II work for deployment in the ocean or as a direct, single shift lab-based replacement for the lab-based mouse bioassay.
APPROACH: The objective of the proposed research is to show the feasibility of using the LFE sensor platform to detect STX in water and shellfish meat. To this end, a proven STX sensitive crown ether (CE) film will be optimized and attached to LFE platforms. The resulting sensing element will be operated in an ideal deionized (DI) water environment to characterize the sensor's performance in ideal conditions where STX will be the only chemical present in the medium. This will prove that the CE film captures STX and the LFE platform has a predictable resonant frequency response as STX concentration is varied. Additionally, the ideal lower detection limit of the LFE STX sensing element will be established. This initial test is not expected to be a practical demonstration of the sensor's performance in a commercial product as it is unreasonable to assume that samples can be purified to such an extent. Testing will continue by introducing additional interferents such as salt or other compounds which can be found in seawater. Once again the level of STX in the non-ideal samples will be varied so that a relationship between the LFE STX sensing element's resonant frequency response and STX concentration can be established. The data that will be extracted from these tests will be the LFE sensing element'a lower detection limit, the reproducibility of sensor responses, and the effect of interferents on the sensor's performance. The lower detection limit will be established by exposing LFE STX sensing element's to decreasing concentrations of STX until the sensor response is indistinguishable from the signal noise variance. Reproducibility of sensor responses will be evaluated by ensuring that resonant frequency changes of the LFE STX sensing element are identical for identical concentrations of introduced STX. There is expected to be some variance due to experimental error. However, it is expected that frequency responses, if reproducible, will not differ by more than a few percent. The effect of the interferents on the sensor's performance will be evaluated by comparing the sensor responses in non-ideal environments to those obtained in an ideal environment. Finally, the LFE STX sensing elements will be tested with shellfish extracts prepared identically as they are for a mouse bioassay. The performance of the sensor will again be evaluated as above. However, the successful outcome of these tests will show a sensing element capable of detecting STX in shellfish extracts. The impact on the target audience from a technology point of view will be profound as the use of mice could be reduced or eliminated without a significant reduction in the confidence of results. Conversely, the impact from the point of view of deployment, integration, and adoption of the new technology would be minimal as existing protocols will be used to collect and prepare samples for testing with the only change being that samples will be introduced to an easy-to-operate sensing system (to be developed in Phase II work) containing an LFE STX sensing element instead of a mouse.
PROGRESS: 2008/05 TO 2009/04 <BR>
OUTPUTS: Some of the results of the project were reported at the 2008 IEEE Sensors Conference, Lecce, Italy (October 2008). The paper was entitled "Novel Transducer Configurations for Bulk Acoustic Wave Sensors" and the authors were D. McCann, M. Wark, L. French, J. Vetelino. Results of the project were also presented at the 2008 IEEE Ultrasonics Symposium, Beijing, China (November 2008). The paper was entitled "The Detection of Chemical and Biological Analytes Using a Monolithic Spiral Coil Acoustic Transduction Sensor" and the authors were D. McCann, M. Wark, P. Millard, D. Neivandt, J.F. Vetelino. <BR> PARTICIPANTS: The principal investigator on the project, Mr. Donald McCann, has overseen all the work in reaching the objectives of the proposal and direct the work conducted by the project consultants. Mr. McCann has also performed many of the experiments to characterize the LFE devices, analyzed experimental data, and evaluated the LFE sensors. He has also explored the use of a Monolithic Spiral Coil Acoustic Transduction (MSCAT) Bulk Acoustic Wave (BAW) sensor platform for use in the project in order to obtain greater sensor sensitivity. Mr. Mitchell Wark, Mainely Sensors research engineer, has assisted Mr. McCann with the laboratory work associated with the development of the LFE STX sensor and oversaw the testing and characterization activities related to the sensor and sensing film. He also supervised the manufacture of all of the LFE sensor elements for this project. Mr. Wark worked with the project consultants to develop alternative STX selective films for the project. Ms. Sarah Hanselman, undergraduate intern, performed several of the tests involving the CE film. Ms. Hanselman tested the sensor in DI, salt, and sea water and found that the CE detected any cations in the solution. She also assisted Mr. Wark in exploring alternative films that simulated a sodium channel to detect STX. Mainely Sensors has utilized personnel and resources at the University of Maine, Orono, ME during the project. Two consultants, Dr. Laurie Connell (Assistant Professor of Marine Sciences) and Dr. David Neivandt (Assistant Professor of Chemical and Biological Engineering) assisted on the project. In addition, Mainely Sensors utilized the cleanroom facilities at the Laboratory for Surface Science and Technology (LASST) to fabricate the sensor platforms for the project. Mainely Sensors has also collaborated with Dr. Titan Fan, President of Beacon Analytical Systems, Portland, ME, to utilize highly selective STX antibodies for the project. <BR> TARGET AUDIENCES: Both Mr. McCann and Mr. Wark are National Science Foundation (NSF) GK-12 Sensors! fellows at the University of Maine. The NSF GK-12 Sensors! program is part of a national initiative to increase the education and vision of young people in science, engineering and math. GK-12 Sensors! brings cutting-edge research in sensors to Maine secondary-school students. As fellows in the GK-12 Sensors! program Mr. McCann and Mr. Wark work to integrate sensors into local middle school and high school classrooms. Both Mr. McCann and Mr. Wark have used their experiences on this project to teach students about the cutting edge research being performed in the State of Maine and to encourage students to consider science, engineering, and math careers. <BR> PROJECT MODIFICATIONS: A selective film based on CE molecule that is selective to saxitoxin (STX) was developed. A procedure to attach CE molecules to LFE sensing platforms using a carbodiimide coupling of a carboxylate-substituted CE reagent to primary amine groups generated by a silanization process on the LFE sensing surface was developed. The coupling of the CE molecule to the LFE surface resulted in a sensitive sensing element for STX in deionized water with a sensitivity of 1 ìM. It was found that the electrical sensitivity of the LFE caused the STX sensing element to confuse STX with electrical changes in the sample environment. An amine-modified null LFE was used in a differential sensing scheme with a CE-functionalized LFE to realize a STX sensing scheme which was not sensitive to changing electrical conditions. The differential LFE STX sensing element was tested in salt water and simulated ocean water. It was discovered that the LFE STX sensing element was sensitive to most positively charged ions in the solutions, indicating that the current sensing scheme would be ineffective in a environment such as seawater or shellfish meat which contains a number of ions and interferrents. This was an unexpected result as the background research performed by Gawley indicated that the CE molecule, when integrated into a ODAC structure, was selective to STX at the exclusion of common ions. Mainely Sensors discovered that the aromatic rings of the modified CE in the ODAC molecule, in addition to providing fluorescent indication of STX binding, was responsible for the selectivity of the CE in Gawley's work. Mainely Sensors has assessed the possibility of producing the ODAC molecule and attaching it to a LFE sensor but the synthesis requires significant time (weeks) and is too complex to be practical. Therefore, new sensing films were assessed. The first approach for the selective detection of STX utilized a phospholipid bilayer with gramicidin ion channels. Mainely Sensors engineered lipid bilayer films for use with LFE sensors. Unfortunately the stability of the films were found to be very poor and the Langmuir-Blodgett deposition techniques require procedures which were not feasible to produce commercially. Therefore a new strategy is being explored. This approach utilizes the highly specific recognition of mammalian antibodies for antigens. Traditionally, antibodies have been employed in ELISA tests for bacteria, viruses, or serum proteins and immunoglobulin. However, a hapten like STX can be conjugated to a protein to elicit a mammalian immune system response and antibody generation. The antibodies (generated by a proprietary process at Beacon Analytical Systems, Portland, ME), which are very specific to the STX molecule, are immobilized on the sensor's surface and the binding of many STX molecules to the antibody-coated sensor surface results in a mechanical and electrical resonant frequency response to the STX. Antibodies have been immobilized on the sensor surfaces by using gluataraldehyde to covalently couple primary amine groups on the Fc tail of the STX antibodies with primary amine groups generated by a liquid silanization process on the sensor surface.
IMPACT: 2008/05 TO 2009/04<BR>
To date Mainely Sensors has developed a chemi-selective film based on an 18-crown-6 ether (CE) molecule that is selective to saxitoxin (STX). We have developed a functionilization procedure to attach the CE molecules to our Lateral Field Excited (LFE) Bulk Acoustic Wave (BAW) sensing platforms using a carbodiimide coupling of a carboxylate-substituted CE reagent to primary amine groups generated by a liquid silanization process on the quartz LFE sensing surface. The coupling of the CE molecule to the LFE surface resulted in a very sensitive sensing element for STX in deionized water showing sensitivity to concentrations as low as 1 ìM. An amine-modified null LFE sensing element was employed in a differential sensing scheme with a CE-functionalized LFE sensing element to realize a STX sensing scheme which was not sensitive to changing electrical conditions in the sensing environment. Mainely Sensors discovered that the aromatic rings of the modified CE in the ODAC molecule, in addition to providing fluorescent indication of STX binding, was responsible for the selectivity of the CE in Gawley's work. Therefore, new sensing film approaches were assessed. Further details are provided in the Major Changes section of this report.